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Chronic prostatitis is one of the common diseases in male urology. The disease of type Ⅲ prostatitis(chronic prostatitis/chronic pelvic pain syndrome) in the classification of prostatitis in the National Institutes of Health(NIH) cannot reflect the nature of the disease, such as unclear etiology and pathogenesis, diverse clinical manifestations, insufficient diagnostic basis, etc. The concept of type Ⅲ prostatitis does not regard the pelvic floor and lower urinary tract as a functional whole. Therefore, the China Chronic Prostatitis Diagnostic Criteria and Efficacy Evaluation Collaborative Group proposed to change the name of type Ⅲ prostatitis to prostatic pelvic syndrome and explore the establishment of symptom-based diagnostic and efficacy evaluation criteria to reflect the nature of the disease and meet the treatment objectives of type Ⅲ prostatitis patients to improve symptoms and improve quality of life. The proposal of new theories and the development of multi-center clinical trials will help urologists deepen their understanding of the prostatic pelvic syndrome and help them to correctly diagnose and treat the syndrome.

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Objective To establish a Tohoku hospital pediatrics-1(THP-1) cell line with G protein-coupled receptor108(GPR108) deletion and explore its functions. Methods According to the requirements of the clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system, two single guide RNAs(sgRNA1 and sgRNA2) paring to the flanking fragments of human GPR108 gene were designed and synthesized. The two oligonucleotides were inserted in the pL-CRISPR.EFS.GFP vector to generate the new recombinant vectors(pL-CRISPR.EFS.GFP-sgRNA1 and pL-CRISPR.EFS.GFP-sgRNA2). The recombinant vectors and packaging plasmids(pMD2.G and psPAX2), were co-transfected into 293T cells to generate virus for infecting THP-1 cells. The GFP+cells were screened and isolated in 96-well culture plates by flow cytometry to obtain single-cell clones. PCR and Western blot were used to detect whether GPR108 was successfully knocked out in THP-1 cells. Both GPR108+/+and GPR108~(-/-) THP-1 cells were treated with lipopolysaccharide(LPS). Interleukin 8(IL-8) derived from the THP-1 cells, which were treated by LPS, was detected with Western blot and cytometric bead array(CBA) analysis. Results The recombinant lentiviral vector pL-CRISPR.EFS.GFP-sgRNA was successfully constructed and single-cell clone F9 was obtained by flow cytometric sorting after transfection of THP-1 cells. PCR and Western blot both confirmed that F9 was a GPR108~(-/-) THP-1 single-cell clone. LPS stimulated GPR108~(-/-) and GPR108+/+THP-1 cells, both Western blot and CBA results showed a significant decrease in IL-8 synthesis and secretion in GPR108~(-/-) THP-1 cells. Conclusion The GPR108~(-/-) THP-1 cell clone is successfully obtained based on the CRISPR/Cas9 system. GPR108 deletion in THP-1 cells treated by LPS leads to a decrease of IL-8 expression and secretion. It lays the foundation for further research on the molecular mechanisms of GPR108 in the immune inflammatory response.

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Objective To establish an inducible macrophage-specific knockout G protein-coupled receptor kinase 2(GRK2) gene(GRK2flox/floxLyz2-CreERT+) mice model. Methods GRK2flox/floxLyz2-CreERT+mice were constructed based on Cre/LoxP system. The genotypes of GRK2flox/floxLyz2-CreERT+mice were identified by PCR amplification and agarose gel electrophoresis. After the mice were sacrificed by carbon dioxide method, the expression of GRK2 in bone marrow-derived macrophages(BMDMs) and peritoneal macrophages(PMs) was detected by Western blot. Immunofluorescence was used to detect GRK2 expression in mouse brain, heart and spleen macrophages. The M1/M2 ratio in PMs induced by prostaglandin E2(PGE2) was analyzed by flow cytometry. Results The results of genotype identification showed that the mice with a band at 355 bp in the length of the flox amplification product and a band at 355 bp in the length of the Cre amplification product were GRK2flox/floxLyz2-CreERT+mice. Western blot results showed that GRK2 expression in BMDMs and PMs of GRK2flox/floxLyz2-CreERT+mice decreased compared with GRK2flox/floxmice(P<0.01). Immunofluorescence results showed that GRK2 expression decreased in the brain, heart and spleen of GRK2flox/floxLyz2-CreERT+mice compared with GRK2flox/floxmice(P<0.01). Flow cytometry showed that compared with GRK2flox/floxmice, there was no significant difference in the proportion of CD86/CD206 in the PMs of GRK2flox/floxLyz2-CreERT+mice. Under PGE2(10 μmol/L) stimulation, the proportion of CD86/CD206 in GRK2flox/floxLyz2-CreERT+mice PMs increased(P<0.01). The proportion of CD86/CD206 in the PMs of GRK2flox/floxmice was higher than that of GRK2flox/floxLyz2-CreERT+mice(P<0.01). Conclusion In this study, GRK2flox/floxLyz2-CreERT+mice model was successfully constructed, and the mice promoted PGE2-induced polarization of PMs to M2-type macrophages compared with control mice.

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Objective To explore the activity of auranofin against ovarian cancer cells and its possible molecular mechanism. Methods The dose-response survival curve and IC50of auranofin on ovarian cancer cell lines, SKOV3, Caov3 and SW626 cells and immortalized normal human embryonic kidney HEK-293T cells were determined by CCK-8 method. Cell cycle was determined by flow cytometry. The levels of total glutathione(GSH), reduced GSH and glutathione disulfide(GSSG), thioredoxin reductase(TrxR) and reactive oxygen species(ROS) in cells were determined by microplate reader, and the reduced GSH/GSSG ratio was calculated. Western blot was used to determine the expression of cyclin dependent kinases(CDK)4, CDK6, Cyclin D1, P53, p-P53 and MDM2 in SKOV3 and Caov3 ovarian cancer cells. Results Compared with HEK-293T cells, the dose-response survival curves and IC50values of SKOV3, Caov3 and SW626 cells showed that ovarian cancer cells were more sensitive to auranofin(P<0.05). After SKOV3 and Caov3 cells were treated with the dose of respective IC50concentrations of auranofin, compared with the untreated cells group, the Auranofin IC50group cells′ intracellular levels of GSH, the ratio of reduced GSH/GSSG and the activity of TrxR decreased(t=25.11/31.18, 14.72/19.92, 43.30/10.74, allP<0.05), and the levels of ROS increased(t=23.82/27.71,P<0.05); cells number at G0/G1phases increased, with cells number at S and G2phases decreased(P<0.05); and the expression levels of cell cycle-related proteins CDK4, CDK6, Cyclin D1 and the P53-specific E3 ubiquitin ligase MDM2 were down-regulated(t=7.51/15.59, 17.32/11.26, 20.78/20.78, 24.25/17.32, allP<0.05), while the expression levels of P53 and p-P53 were up-regulated(t=17.32/24.25,12.12/10.39,allP<0.05). Conclusion Auranofin causes oxidative stress in ovarian cancer cells by inhibiting TrxR activity, and by partially degrading MDM2 to stabilize and activate P53, so as to block the cancer cells in G0/G1phase, and exert anti-ovarian cancer activities.

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Objective To investigate the effect of co-culture of adipose-derived stem cells(ADSC) and endothelial progenitor cells(EPC) on the activity of EPC and its related mechanism. Methods Rat ADSC and EPC were isolated, cultured, expanded and identifiedin vitro. The experiment was divided into three groups: EPC group, EPC+ADSC co-culture group, and EPC+ADSC+PI3K-inhibitor group. After 48 hours of co-culture, the cells of the three groups were treated with Transwell. The effects of ADSC and EPC co-culture and PI3K/AKT pathway on EPC activity were evaluated by CCK-8 assay, scratch assay and angiogenesis assay, respectively. Western blot was used to detect vascular endothelial growth factor A(VEGFA) and endothelial nitric oxide synthase(eNOS),vascular endothelial-cadherin(VE-cadherin), CD133, phospho-phosphatidylinositol 3-kinase(phospho-phosphatidylinositol 3-kinase(p-PI3K) and phospho-protein Kinase B(p-AKT) expression levels in EPC to detect the effects of ADSC and EPC co-culture and PI3K/AKT pathway on the differentiation ability of EPC into mature endothelial cells. Results CCK-8 results showed that the absorbance at 450 nm of EPC in EPC+ADSC co-culture group was higher than that in EPC group and EPC+ADSC+PI3K-inhibitor group at different time points, and the difference was statistically significant(P<0.01). The scratch test showed that the relative scratch distance of EPC+ADSC co-culture group was smaller than that of EPC group and EPC+ADSC+PI3K-inhibitor group after 24 hours, and the difference was statistically significant(P<0.01). Tube formation assay showed that the average number of tube-like structures formed in EPC+ADSC co-culture group was higher than that in EPC group and EPC+ADSC+PI3K-inhibitor group, and the difference was statistically significant(P<0.01). Western blot showed that the expression levels of VEGFA, eNOS, VE-cadherin, p-PI3K and p-AKT of EPC in EPC+ADSC co-culture group were higher than those in EPC group and EPC+ADSC+PI3K-inhibitor group. The expression level of CD133 in EPC group was lower than that in EPC+ADSC+PI3K-inhibitor group, and the difference was statistically significant(P<0.01). Conclusion Co-culture of ADSC and EPC can improve the proliferation, migration, differentiation and vasogenic activity of EPC through the regulation of PI3K/AKT pathway.

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Objective To compare the functional changes of neurotransmitters in the dopamine(DA) system of rats lesioned by 6-hydroxydopamine in unilateral medial forebrain bundle(MFB) and unilateral striatum after 4 weeks of modeling. Methods Adult male Sprague-Dawley rats(n=62) were divided randomly into four groups: single-site model(one site striatal lesion model,n=18), four-site model(four sites striatal lesion model,n=18), MFB model(MFB lesion model,n=18) and a sham operated control group(n=8). Immunohistochemical tyrosine hydroxylase(TH) staining was used to calculate the loss rate of positive DA neurons. The functional changes of dopamine transporters(DAT)and D2 receptors in rat models of four groups were detected by radioligand-receptor binding assayin vitro, behavioral test and small-animal PET. Results Immunohistochemical staining results of TH at 1-lesion, 4-lesion and MFB model showed that the TH-positive cell loss rates of the lesion side in three models were 19.1%, 84.7% and 97.1%, respectively, indicating that the model was successfully constructed. The results of DAT/D2 receptor functions in the three models showed that, compared with the sham operated control group, the DAT binding ratio of radioactivity on the lesion side of 1-lesion, 4-lesion and MFB model significantly decreased(P<0.05,P<0.05 andP<0.01). The DAT binding ratio of radioactivity on the lesion side was compared among 1, 4 lesion and MFB model. It was found that the decrease degree of MFB model was significantly higher than that of the previous two models(P<0.01), and the decrease degree of 4-lesion model was significantly higher than that of 1-lesion model(P<0.05). Compared with the sham operated control group, the D2 receptor binding ratio of radioactivity on the lesion side of 1-lesion and 4-lesion model significantly decreased(P<0.05), and there was no significant difference in the degree of decrease between the two models, but the D2 receptor binding ratio of radioactivity on the lesion side of MFB model significantly increased(P<0.05). There was no difference in the distribution of DAT/D2 receptor on the lesion side and the normal side of the sham operated control group. Methamphetamine caused ipsilateral rotations to the lesion side in all models. There were no significant differences in methamphetamine-induced rotation among 1-lesion, 4-lesion and MFB models. Bromocriptine caused ipsilateral rotations to the lesion side in 1-lesion and 4-lesion models but contralateral rotations in MFB model. There were no significant differences in bromocriptine-induced rotation between 1-lesion and 4-lesion models. The results of stepping test showed the motion initiation time of the lesion side was significantly longer than that of the normal side, the stepping length of the lesion side was significantly shorter than that of the normal side, and the adjusting steps of the lesion side was significantly less than that of the normal side(P<0.001), but there was no significant difference of the lesion side in the initiation time, stepping length, and adjusting steps among the three groups of models. Conclusion The striatal lesion and MFB lesion models showed different pathophysiological processes in terms of DA neurotransmitter functions and behavior. The MFB lesion model can mimic primary Parkinson′s disease, while the striatal lesion model is similar to some Parkinson syndromes.

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Objective The purpose of this study was to investigate the effects of sirolimus(SIR) on proliferation and apoptosis of vascular endothelial cells of venous malformation(VM) caused by mutations in TIE2-L914F and its potential molecular mechanism.Methods The expression of TEK receptor tyrosine kinase(TIE2) in human umbilical vein endothelial cell(HUVEC) was interfered to construct vascular endothelial cells of VM model(TIE2-L914F group).Subsequently part of vascular endothelial cells of VM was exposed to 1 000 ng/ml SIR for 48 h(TIE2-L914F + SIR group),and the proliferation and apoptosis of vascular endothelial cells of VM were detected by MTT and flow cytometry.The mRNA and protein expressions of CXCL1 and CXCR2 were detected by qRT-PCR and Western blot.Results Compared with the cells in TIE2-L914F group,the proliferation activity of the cells in TIE2-L914F + SIR group was inhibited,and the apoptosis rate increased(P<0.05).The expression of CXCL1increased in TIE2-L914F cells but decreased after SIR treatment(P<0.05).Compared with TIE2-L914F + pcDNA3.1 group,the cell proliferation activity increased but apoptosis rate decreased in TIE2-L914F + pcDNA-CXCL1group.Compared with TIE2-L914F + pcDNA-CXCL1 group,cell proliferation activity was inhibited but apoptosis rate increased in TIE2-L914F + pcDNA-CXCL1 + SIR Group(P<0.05).In addition,compared with TIE2-L914F group,CXCR2 expression decreased in TIE2-L914F + SIR group(P<0.05).Conclusion SIR inhibits VM cell proliferation,induces cell apoptosis of vascular endothelial cells of VM,and inhibits the expression of CXCL1/CXCR2.

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Objective To investigate the effect of acid-sensing ion channel 1(ASIC1) gene knockout on articular cartilage injury in adjuvant arthritis(AA) of mice. Methods Wild-type mice and ASIC1 knockout mice were divided into normal group and model group. Arthritis score was performed by observing the inflammation of paws, and the right paw swelling was measured. HE staining, immunohistochemistry, TUNEL and ELISA were used to detect the injury of articular cartilage and arthritic inflammation. Results The arthritis score and paw swelling of AA mice with ASIC1 knockout was lower than that of the AA wild-type mice. AA mice with ASIC1 knockout showed less destruction and increased expression of collagen-Ⅱin articular cartilage. Moreover, the lower apoptosis rate was observed by TUNEL assay in AA mice with ASIC1 knockout by comparing with AA wild-type mice. Furthermore, ELISA assay showed that the levels of IL-1β、TNF-α in the serum decreased in AA mice with ASIC1 knockout. Conclusion Knockout of ASIC1 may have protective effect on articular cartilage injury.

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Objective To investigate the relevance between vitamin D receptor(VDR) gene promoter methylation level and idiopathic hypocitraturia of the Bai nationality in Yunnan province. Methods Fifteen patients with idiopathic hypocitrouria of the Bai nationality in Yunnan province with double dominant expression(FF type) of single nucleotide polymorphism shot(SNP shot) rs2228570(FokⅠ) genotype were selected as the experimental group. Fifteen people of the Bai nationality in Yunnan province with normal content of urinary citric acid were the control group. First, blood samples were taken from both groups. Next, the blood samples were treated with sulfites, RNA products of each sample were obtained by PCR amplification andin vitrotranscription of T7 DNA polymerase. Then the corresponding RNA fragments were digested by base-specific enzymes. Finally the degree of methylation at each test site was obtained through the EpiTYPER procedure. Results In the statistical results of DNA methylation level, the methylation level ofVDRⅠ fragment experimental group was 4.136%(1.655%, 5.152%) which was higher than 1.261%(0.827%, 1.930%) in the control group, and the difference was statistically significant(P<0.001). Among the 33 CpG sites onVDRⅠ fragment, there were significant differences in DNA methylation levels of CPG-5(F=8.831,P=0.008) and CpG-8(F=16.155,P=0.001) between the experimental group and the control group. Conclusion The increased methylation level ofVDRgene promoter is related with idiopathic hypocitric of the Bai nationality of Yunnan province. And compared with the normal Bai nationality people, the DNA methylation level ofVDRgene promoter significantly increased in the Bai nationality patients with FF type ofVDRgene SNP shotFokⅠ.

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Objective To explore the effect of C1q/tumor necrosis factor-related protein 9(CTRP9) on the expression of genes and proteins related to lipid metabolism of brown adipose tissue(BAT) in mice after cold stimulation. Methods C57BL/6J male mice were injected with adenovirus Ad-GFP(control group) or Ad-CTRP9(experience group) into the scapular region and kept for 7 days. After cold stimulation at 4 ℃ for 10 hours, the expression levels of BAT marker genes and proteins were detected by real time PCR and Western blot. Results Overexpression of CTRP9 induced by cold stimulation significantly increased the mRNA level of iodothyronine deiodinase 2(Dio2) in BAT(P<0.01). Additionally, there was no significant difference in the expression of BAT marker genes(UCP-1,PGC-1α,PRDM16andARβ3), and liposynthesis and lipolysis related genes(PPARγ,HSLandATGL). Uncoupling protein 1(UCP-1) protein expression was upregualted in Ad-CTRP9 compared to the Ad-GFP control group, while the expression of lipolysis related protein adipose triglyceride lipase(ATGL) decreased significantly(P<0.05). Conclusion In cold environment, overexpression of CTRP9 promotes the accumulation of UCP-1 protein in BAT, upregulates the expression of thyroid hormone signal related geneDio2, and inhibits triglyceride hydrolysis to maintain a constant body temperature.

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Objective To solve the problems of low throughput of current cell migration research methods, which was difficult to establish a stable concentration gradient and observe cell migration behavior in real time, a six-channel array microfluidic chip was designed in this paper.Methods In this paper, a six-channel array microfluidic chip is designed.Firstly, multiphysics modeling and numerical simulation were performed using the finite element analysis software COMSOL Multiphysics 5.5 to analyze the flow behavior of the main pipeline of cell migration.Then, the throughput advantage of the device was verified by testing the chemotaxis response of healthy human neutrophils to different types of chemokine gradients in this microfluidic chip.Subsequently, by analyzing the migration rate of neutrophils in 6 patients with type II diabetes mellitus and 3 healthy people, the clinical applicability of the annular six-channel array microfluidic chip was further verified.Finally,the Pearson coefficient was used to analyze the correlation between neutrophil chemotaxis function and some physiological indicators in patients with diabetes.Results The concentration gradient data inside the pipeline simulated by the simulation software was compatible with the real-time fluorescence test data of the pipeline.The average migration rate of healthy human neutrophils was(0.21±0.01) μm/s in 100 nmol/L interleukin-8(IL-8) environment and(0.22±0.01) μm/s in 100nmol/L N-Formyl-Met-Leu-Phe(f MLP) environment.In the comparison of neutrophil migration experiments between healthy people and diabetic patients,the chemotaxis rate of neutrophils in healthy people was(0.19±0.01)μm/s,and the neutrophil chemotaxis rate in diabetic patients was(0.15±0.02) μm/s.Correlation analysis showed that neutrophil migration rate in patients with type II diabetes mellitus was inversely correlated with glycated hemoglobin.Conclusion The high-throughput microfluidic chip proposed in this paper allowed rapid and selective detection of cell migration characteristics at the single-cell level,and it could be used as a new tool for cell migration research.

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Objective To explore and optimize the primary culture method of neonatal mouse hippocampal neuronsin vitro. To construct a G-protein-coupled receptor kinase 2(GRK2) knockout HT22 cell line. Methods Hippocampal tissue of C57BL6/J mice on day 1-2 was taken, digested with trypsin and pipetted to form a cell suspension, and supplement was added to Neurobasal-A medium to maintain cell growth. CRSIPR/Cas9 gene editing technique was used to construct HT22-GRK2-/-cell line, and the knockout efficiency ofGRK2was detected by immunofluorescence staining and Western blot. Results Primary hippocampal neurons of newborn mice were put into six-well plates with 3×107/well using a serum-free culture method, which could get a high purity and good activity; HT22-GRK2-/-cell line was constructed successfully. Conclusion The primary culture method of mouse hippocampal neurons was successfully established and optimized, and HT22-GRK2-/-cell line was successfully constructed by CRSIPR/Cas9 gene editing technique.

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Objective To explore the difference in H4 clustered histone 6(H4C6) methylation level and circulating cell-free DNA(cfDNA) concentration between 94 normal group and 122 tumor groups(65 patients with lung cancer, 22 patients with gastric cancer, 23 patients with colorectal cancer, and 12 patients with liver cancer), and the age of total 216 subjects were between 18 and 85 years old.To construct a cancer risk prediction model based on H4C6 methylation level and cfDNA concentration and evaluate the predictive performance of the model.Methods cfDNA was extracted from blood samples using magnetic beads.Qubit 4.0 fluorescence quantitative meter was used to detect the concentration of cfDNA.Real-time quantitative PCR(RT-qPCR) technology was used to detect the methylation level of H4C6 in cfDNA.Logistic regression algorithm was used to construct a cancer risk prediction model of H4C6 methylation level combined with cfDNA concentration.The accuracy of the model was assessed using receiver operating characteristic(ROC) curve and calibration curve.The clinical benefit of the model was assessed using decision curve analysis(DCA).Results The model was constructed by combining H4C6 methylation level and cfDNA concentration to distinguish lung cancer,liver cancer,colorectal cancer,gastric cancer,pancancer from healthy control group had the area under curve(AUC) of 0.769,0.988,0.934,0.922,0.830,respectively.The mean absolute error of the calibration curve was less than 0.05; the net benefit of the DCA curve was greater than 0.Conclusion The cancer risk prediction model based on H4C6 methylation level and cfDNA concentration has good predictive performance,which helps to provide reasonable and effective suggestions for preclinical decision-making,and ultimately may provide patients with targeted and personalized cancer detection and diagnosis program.

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Objective To investigate the effects of retinoid-related orphan receptor γ(RORγ) gene on proliferation and metastasis of human colon cancer cells. Methods RORγknockdown cell lines were constructed and the knockdown efficiency was detected by RT-qPCR and Western blot assays; MTT, colony formation, Transwell and wound healing assays were used to detect cell proliferation and metastasis; the expression of epithelial-mesenchymal transition(EMT) related proteins was detected by Western blot. The relationship betweenRORγgene expression and immune cell infiltration in tumor microenvironment was analyzed using TIMER 2.0 database. Results The knockdown ofRORγenhanced the viability(F=157.40,P<0.01), clonogenesis(F=61.35,P<0.01), migration(F=13.00,P<0.01), invasion(F=21.26,P<0.01) and wound healing ability(F=877.2,P<0.01) of colon cancer cells, inhibited the expression of E-Cadherin, and promoted the expression of vimentin and N-Cadherin. TIMER 2.0 database analysis showed thatRORγexpression in colon adenocarcinoma(COAD) tissues was associated with multiple immune cell infiltrates. Conclusion Downregulation ofRORγexpression promoted the proliferation and metastasis of colon cancer cells.

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Objective To establish a laboratory model of heroin addiction in C57BL/6 mice based on associative learning mechanisms. Methods The black box and white box were selected as the memory training environment, and three behavioral training paradigms were studied:(1) Pavlovian conditional position preference(CPP) training paradigm, mice were placed in the white box for memory reinforcement training for 30 min after intraperitoneal injection of 0.1 ml of corresponding concentrations(5.0, 10.0, 20.0 mg/kg) of heroin at 9:00 a.m., and 24 h later 0.1 ml of 0.9% NaCl solution was injected intraperitoneally into the black box for training, and after the training, the mice were tested for their memory preference for the black and white boxes(movement time of different boxes).(2) A naloxone conditional position aversion(CPA) training paradigm was conductedbased on the results of the CPP training paradigm.(3) Behavioral sensitization training paradigm, heroin addiction rating scale was established based on the statistical results of 3 behavioral experiments and the lethality of experimental animal disease mice after drug administration. Three different doses of heroin(5.0, 10.0, 20.0 mg/kg) were selected to induce heroin addiction, and the most appropriate heroin concentration was selected by the results on the rating scale. Results In the CPP training paradigm, CPP was observed in all heroin groups(P<0.05,P<0.001,P<0.05).In the CPA training paradigm, the CPA induction rate was highest in the 10.0 mg/kg heroin group compared to the control group(P<0.01).In the behavioral sensitization training paradigm, all heroin groups caused behavioral sensitization changes(P<0.001);but the 5.0 and 10.0 mg/kg heroin groups did not cause animal mortality. Overall, the 10.0 mg/kg heroin group had the highest dose score on the rating scale. It could be used as a concentration to establish a stable experimental animal model of heroin addiction. Conclusion The study was effective in establishing a heroin addiction model in mice, and it was suitable for modeling drug concentration screening, with high animal survival rate and simple and practical. The combined learning mechanism can effectively shorten the model establishment period.

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Objective To elucidate the effect of phosphorylation modification at the threonine 592(Thr592) site on the inhibition of gastric cancer proliferation by sterile alpha motifs and HD structural domain-containing protein 1(SAMHD1) and the potential mechanism of action. Methods Post-translational modifications(PTMs) of SAMHD1 protein in gastric cancer tissues and cell lines in the database were analyzed, and immunohistochemical staining was performed to detect SAMHD1 Thr592 phosphorylation in paired tissues of gastric cancer patients. In gastric cancer cells, SAMHD1 Thr592 variants were constructed and transiently transfected, and cell proliferation was detected using the cell counting kit 8(CCK-8) method. The phosphorylation of the cyclin-dependent kinases(CDK)2 protein threonine 160(Thr160) site was inhibited by the addition of different concentrations of the CDK6 inhibitor, Palbociclib, which reduced the level of SAMHD1 protein Thr592 phosphorylation. Three online databases were used to analyze the SAMHD1 reciprocal proteins and take the intersection to derive the Nik-related kinase(NRK) protein. Immunoprecipitation(Co-IP), mass spectrometry and Western blot were used to verify the interactions between SAMHD1 and NRK proteins and detect the effect of NRK on the phosphorylation of the SAMHD1 Thr592 site. Results Compared with PTMs such as ubiquitination, the highest level of phosphorylation modification of SAMHD1 was observed in tumors, and the difference was statistically significant(P<0.01). Immunohistochemical experiments showed that phosphorylated SAMHD1(Thr592) was expressed higher in gastric adenocarcinoma than that in normal mucosal tissue adjacent to the cancer, and the difference was statistically significant(P<0.01). Western blot assay showed that SAMHD1 protein expression was elevated in MKN-45 cells in the overexpression wild type and mutant groups, and phosphorylated SAMHD1 levels were also elevated in the wild type, T592E and HD/AA groups. CCK-8 assay showed that both SAMHD1 wild type and T592A could inhibit gastric cancer cell proliferation, while T592E and HD/AA had no effect on gastric cancer proliferation. On the basis of overexpression of SAMHD1, CCK-8 suggested that cell proliferation was inhibited after adding different concentrations of Palbociclib treatment, and Western blot assay suggested that the phosphorylation level was also reduced. NRK protein was obtained by Co-IP and mass spectrometry identification to screen the SAMHD1 reciprocal protein profile and database intersection, and NRK was found to interact with SAMHD1 protein and promote phosphorylation at SAMHD1 Thr592 site by Co-IP and Western blot assay. Conclusion Phosphorylation of the Thr592 site contributes to the loss of SAMHD1′s ability to inhibit gastric cancer cell proliferation, which is reversed by Palbociclib. NRK interacts with SAMHD1 protein, promoting phosphorylation of the SAMHD1 Thr592 site.

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Objective To study the expression of long non-coding RNA LINC01152 in glioma and its influence on the malignant biological behavior of glioma cells. Methods LINC01152 expression in glioma was analyzed by LncSpA V2.0 and GEPIA database. qRT-PCR was applied to detect the expression of LINC01152 mRNA in 10 samples of human normal brain tissues, 40 samples of glioma tissues and 5 glioma cell lines. GO and KEGG enrichment analysis of LINC01152 co-expressed genes were performed using the DAVID database to predict the related functions. The AnoLnc2, TargetScan, LinkedOmics and miRDB databases were used to predict the LINC01152 related miRNAs and target genes to construct a ceRNA regulatory network. LINC01152 expression was knocked down in glioma cell lines by small interfering RNA(siRNA) transfection. The CCK-8 test, scratch healing experiments, Transwell, flow cytometry and Western blot experiments were used to measure the influence of LINC01152 on the proliferation, migration, invasion and apoptosis of glioma cells. Results Database analysis showed that compared with other tumor types, LINC01152 was highly expressed in glioblastoma(GBM) and low grade glioma(LGG), and was higher than normal brain tissue. qRT-PCR showed that the expression of LINC01152 mRNA in glioma tissues was significantly higher than that in normal brain tissues(P<0.01). The expression of LINC01152 was correlated with Ki-67(P<0.05), but not with clinical parameters such as gender, age, tumor size, P53 protein, glial fibrillary acidic protein(GFAP), O-6-methylguanine-DNAmethyltransferase(MGMT) and WHO grade of glioma patients. Functional enrichment analysis of co-expressed genes indicated that the LINC01152 was mainly involved in biological processes such as cell adhesion and synaptic signaling. LINC01152-miRNA-mRNA regulatory network was constructed according to predicted target genes. After down-regulation of LINC01152 expression, the proliferation, migration and invasion abilities of A172 and U87 cells decreased(P<0.01), while the apoptosis of glioma cells significantly increased(P<0.001). Conclusion LINC01152 is highly expressed in glioma, which can promote the malignant biological behavior of glioma cells by enhancing proliferation, migration as well as invasion and inhibition of apoptosis.

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Objective To study the effect of miR-28-5p on cisplatin(DDP)-resistant A549 lung adenocarcinoma cell line(A549/DDP), and to explore whether its mechanism is related to ferroptosis suppressor protein 1(FSP1)-mediated ferroptosis. Methods The A549 lung adenocarcinoma cell line and A549/DDP cells were selected as the research objects. RT-qPCR method was used to detect the expression level of miR-28-5p in the cells. Cell proliferation was measured by CCK-8 method and colony formation assay. The target gene of miR-28-5p was identified and verified by luciferase reporter gene and Western blot analysis. A549/DDP was transfected with miR-28-5p simulant or FSP1 overexpression plasmid to evaluate cell proliferation, mitochondrial morphology was evaluated by transmission electron microscope, and the levels of reactive oxygen species(ROS), glutathione(GSH) and malondialdehyde(MDA) were measured by kit. A cell line-based xenograft model was established to evaluate the effect of miR-28-5p on tumor growthin vivo. Results Compared with A549 cells, the expression level of miR-28-5p in A549/DDP cells was significantly reduced(P<0.001). Compared with A549 cells, the cell viability(F=49.542,P<0.001) and colony forming ability(t=4.412,P<0.01) of A549/DDP cells increased significantly. Compared with miR-NC group, the cell viability(t=4.612,P<0.01) and colony number(t=4.503,P<0.01) of A549/DDP cells in miR-28-5p group significantly decreased. The luciferase activity decreased in the cells transfected with the miR-28-5p mimic, being significantly more so in the presence of the pGL3-FSP1 3′UTR-WT vector. In addition, the expression of FSP1 in cells overexpressing miR-28-5p was significantly suppressed. Compared with the Vector+miR-28-5p group, the FSP1+miR-28-5p group significantly increased cell viability and colony formation, cell mitochondrial length and GSH(P<0.01), and significantly increased cell apoptosis, ROS production and MDA formation decrease(P<0.05).In vivoexperiments showed that compared with the miR-NC+DDP group, the size and weight of tumors formed in the miR-28-5p+DDP group were significantly reduced(P<0.05), and the expression of Ki-67 and FSP1 protein was significantly reduced(P<0.05). Conclusion The mechanism of miR-28-5p reversing cisplatin resistance in lung adenocarcinoma cells may be related to the inhibition of FSP1-mediated ferroptosis.

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Objective To analyze the expression and clinical prognostic significance of nuclear Dbf2-related kinase 1(NDR1) in hepatocellular carcinoma, and to investigate the biological function and regulatory mechanism ofNDR1in hepatocellular carcinoma cells. Methods ENCORI database was used to analyze the correlation ofNDR1and c-Myc. Cycloheximide(CHX) experiment analyzed the relationship betweenNDR1and c-Myc protein stability. The expression levels ofNDR1in liver cancer tissues and normal tissues and its relationship with the survival rate of liver cancer patients were analyzed using the ENCORI database. MYC-NDR1eukaryotic expression vector was constructed, transfected with hepatocellular carcinoma HepG2 cells, and its expression was verified by protein immuno blotting(Western blot); cell proliferation and migration ability were detected by CCK-8 assay, cell clone formation assay and scratch assay, respectively. The correlation betweenNDR1and c-Myc expression was analyzed using the ENCORI database, and the relationship betweenNDR1and c-Myc protein was investigated using a protein synthesis inhibitor CHX dosing assay. Results The results of the ENCORI database showed that the expression ofNDR1in liver cancer tissues was higher than that in normal tissues and the overall survival rate of patients with highNDR1expression was lower than that of patients with lowNDR1expression, and the difference was statistically significant(P<0.001). The results of the CCK-8 assay showed that the MYC-NDR1group grew faster than the empty vector group(P<0.001). The clone formation assay showed that the number of clones in the MYC-NDR1group was higher than that in the empty vector group(P<0.001). The cell scratch assay showed that the mean migration distance in the MYC-NDR1group was greater than that in the empty vector group(P<0.001). ENCORI database analysis showed thatNDR1correlated with c-Myc expression(R=0.184,P<0.001); CHX dosing assay showed that the reduction of c-Myc protein in the MYC-NDR1group was lower than that in the empty vector group during the same time. Conclusion NDR1is highly expressed in hepatocellular carcinoma tissues, closely correlated with poor prognosis of hepatocellular carcinoma patients, and positively correlated with the expression ofc-Mycgene. The study successfully constructes MYC-NDR1eukaryotic expression vector, and the expression product MYC-NDR1can increase the stability of c-Myc protein and promote the proliferation and migration of human hepatocellular carcinoma cells.

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Objective To establish a rat model of preeclampsia(PE) in early pregnancy and to observe the changes in phenotype, pregnancy outcome and cognitive ability of offspring. Methods The pregnant rats were randomly divided into model group and control group. Ultra-low dose lipopolysaccharide(LPS)(0.5 μg/kg) and an equal volume of normal saline were injected into the tail vein of pregnant rats on the fifth day of pregnancy. The levels of blood pressure, 12-hour urinary protein, peripheral blood coagulation factors and placental cytokines in the two groups were measured. Furthermore,placental pathology,pregnancy outcomes,and cognitive abilities of offspring were observed. Results Blood pressure and urinary protein levels of model group were significantly higher than those of control group levels. Compared with the control group,the levels of platelet and antithrombin Ⅲ(AT Ⅲ)in the peripheral blood of pregnant rats in the model group were lower than those in the control group,while D-dimer was higher than that in the control group,the weight of the fetus and placenta in the model group decreased(P<0.001),the expression levels of interleukin(IL)-6,tumor necrosis factor α(TNF-α) and interferon gamma(INF-γ) in peripheral blood increased,while the expression level of transforming growth factor β1(TGF-β1) decreased(P<0.001). The water maze test showed that the latency of the offspring of the model group to the platform was longer than that of the control group(P<0.05),while the frequency of crossing the platform quadrant and the time of staying in the platform quadrant of the model group were lower than those of the control group(P<0.05). HE and PAS staining showed that there were infiltration of inflammatory cells in the basal layer of placenta,obvious decrease of blood vessels in labyrinthine area,slight edema of renal interstitium and degeneration of local renal tubular epithelial cells in the model group,while there were no above pathological changes in placenta and kidney in the control group. Conclusion A single injection of LPS in early pregnancy can successfully induce PErelated symptoms and adverse pregnancy outcomes such as fetal growth restriction and lead to the decline of cognitive ability of offspring.

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Objective To investigate the therapeutic effect and mechanism of isoliquiritigenin(ISL) on hepatic fibrosis(HF). Methods SD rats were randomly divided into normal group, model group, colchicine tablet group(positive control, 0.1 mg/kg), and high-dose and low-dose ISL groups(40, 10 mg/kg).Except for the normal group, the other groups were given intraperitoneal injection of 50% carbon tetrachloride(CCl4) olive oil solution(1.5 ml/kg) to establish liver fibrosis models, twice a week for 8 weeks. After modeling, rats in each group were given the corresponding drugs by gavage at a volume of 10 ml/kg per day for 4 weeks.After the last administration, the levels of alanine aminotransferase(ALT) and aspartate aminotransferase(AST) in the serum of rats were determined; liver index was calculated; HE and Masson staining were used to observe the pathological changes of liver tissue; Real-time quantitative PCR was used to detect the mRNA expression levels of hypoxia-inducible factor-1α(HIF-1α) and vascular endothelial growth factor(VEGF); the expressions of HIF-1α and VEGF protein in liver tissue were detected by immunohistochemistry and Western blot. Results Compared with the normal group, the serum ALT and AST contents of the rats in the model group increased(P<0.01), the liver index increased(P<0.01), the rat fibrous tissue hyperplasia, the collagen volume fraction increased(P<0.01), and the liver the expression levels ofHIF-1αandVEGFmRNA in the tissue increased(P<0.01). Immunohistochemistry and Western blot showed that the expression levels of HIF-1α and VEGF protein increased in the model group(P<0.01). Compared with the model group, the ISL high and low dose groups could reduce the levels of ALT and AST in serum(P<0.01), the liver index(P<0.01), the proliferation of fibrotic tissue and the collagen volume fraction(P<0.05), down-regulate the expression levels ofHIF-1αandVEGFgenes(P<0.01). Immunohistochemical detection showed that the high and low dose groups of ISL could reduce the protein expression levels of HIF-1α(P<0.01,P<0.05),the expression level of VEGF protein decreased(P<0.01), Western blot detection showed that the high-dose group of ISL could reduce the protein expression levels of HIF-1α and VEGF(P<0.05). Conclusion ISL has a significant therapeutic effect on CCl4-induced HF model rats, and its mechanism may be related to the regulation of HIF-1α and VEGF protein expression levels.

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Objective To investigate the role of nucleotidebinding oligomerization domain-like receptor protein 3(NLRP3) inflammasome in hyperalgesia induced by temporomandibular joint osteoarthritis(TMJOA) in rats.Methods Twenty 6-week-old male SD rats were randomly divided into NS group and MIA group. The rat model of TMJOA was established by injection monosodium iodoacetate(MIA) into the upper cavity of temporomandibular joint(TMJ). The changes of pain threshold in the TMJ region of rats after MIA injection were detected by Von Frey. The changes of condyle structure were observed by Hematoxylin-eosin(HE) and Safranin O-fast green stains. Histopathological changes of trigeminal ganglion(TG) were observed by HE stains. The expression and distribution of TG NLRP3 protein were detected by immunohistochemistry. Western blot was used to detect the protein levels of NLRP3 and interleukin(IL)-1β in TG. The mRNA levels of NLRP3,apoptosis-associated speck-like protein(ASC),cysteinyl aspartate specific proteinase(Caspase-1),IL-1β and IL-18 in TG were detected by quantitative real-time polymerase chain reaction(qRT-PCR). Results Compared to saline group,the pain threshold of experimental TMJ osteoarthritis rats decreased(P<0.05). TMJ and TG showed obvious pathological changes. The protein and mRNA levels of NLRP3 expressed in the tissues of rats in the TMJOA group increased(P<0.05).Conclusion NLRP3 inflammasome may be involved in the regulation of hyperalgesia in TMJOA rats.

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Objective To study the relationship between different doses of subchronic sodium fluoride exposure and lung histopathological changes in rats, and to explore the effect and related mechanism of sodium fluoride induced lung tissue injury in rats. Methods Thirty-two Wistar male rats were randomly divided into control, low, medium and high dose groups, each group included 8 rats, intragastric administration of sodium fluoride toxin solution 0, 12, 24, and 48 mg/(kg·d). The experiment lasted for 16 weeks. The incidence of dental fluorosis and weight in each group of rats was recorded, the lung coefficient was calculated, the pathological changes of lung tissue was observed by HE staining, enzyme-linked immunosorbent assay(ELISA) was used to detect the content of superoxide dismutase(SOD) and malondialdehyde(MDA) levels in rats serum, apoptosis level of lung cells was assessed by TUNEL staining, and the Caspase-3 protein expression levels in lung tissues were determined by immunohistochemistry. Results At the end of exposure, the upper and lower incisors of rats in different dose groups showed different degrees of dental fluorosis. The increase in body weight of the rats in the high-dose group was lower than that in the other three groups(P<0.05). The coefficient of lung organs and the content of MDA in serum of the rats in the high-dose group were higher than those in the control group(P<0.05). The rats in the low, medium and high dose groups had different degrees of pulmonary interstitial inflammatory infiltration and alveolar morphological changes.Compared with the control group, the lung cell apoptosis rate and Caspase-3 protein expression of the rats in the low, middle and high groups significantly increased, and the differences were statistically significant(P<0.05). Conclusion Subchronic exposure to sodium fluoride can cause the lung tissue damage in rats, and the mechanism may be related to the apoptosis induced by oxidative stress.

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Objective To investigate the effect of mesencephalic astrocyte-derived neurotrophic factor(MANF) on the adaptive expression of bile acid transporter in human hepatoellular carcinomas(HepG2) induced by rifampicin(RFP). Methods The control group cell line(Y07) and the knockdown group cell line(Y25) were constructed by lentiviral stable transfection technology. The Y07 and Y25 cells were treated with RFP of 200 μmol/L for 48 h, and qRT-PCR and Western blot were used to detect the protein and gene expression levels of MANF, bile salt export pump(BSEP), multidrug resistance-related proteins 2/3/4(MRP2, MRP3, MRP4), multidrug resistance protein 1(MDR1), organic solute transporter a/β(OSTα/β), organic anion transporter(OATP2B1). The protein and gene expression levels of proliferating cell nuclear antigen(PCNA), proliferating cell marker Ki67 were used to evaluate the proliferation of cells in each group changes in levels. Changes in the protein and gene expression levels of C/EBP homologous protein(CHOP) and cysteinyl aspartate specific proteinase-3(Caspase-3) were used to evaluate the apoptosis of cells in each group.The relative contents of alanine aminotransferase(ALT),aspartate aminotransferase(AST), alkaline phosphatase(ALP), total bilirubin(TBIL),indirect bilirubin(IBIL)and total bile acid(TBA)in the supernatant of cell culture medium of each group were detected by kits. Results RFP could induce the protein and gene expression of MANF, BSEP, MRP2, MRP3, MRP4, MDR1, OSTα, OSTβ, OATP2B1 in HepG2 cells(P<0.05), while the protein and gene expression levels of BSEP, MRP2, MRP3, MRP4, MDR1, OSTα、OSTβ、OATP2B1 decreased after MANF knockdown(P<0.05). Moreover, under the action of RFP, the protein expression of PCNA and Ki67 in the knockdown group was still higher. The protein and gene levels of CHOP and Caspase-3 significantly increased after MANF knockdown(P<0.05). The levels of the hepatic cell injury markers in the cell supernatant increased significantly(P<0.05). Conclusion RFP can induce the expression of bile acid transporter such as BSEP, MRP2, MRP3, MRP4, MDR1, OSTα, OSTβ and OATP2B1 to increase in HepG2 cells(P<0.05), but the expression of bile acid transporter of HepG2 after MANF knockdown will significantly decrease under the induction of rifampicin(P<0.05),and cell indury is aggravated, indicating that MANF plays a protective role in RFP-induced adaptive responses by regulating the bile acid transporter.

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Objective To investigate the effects of estrogen deficiency on depression-like behavior and mitochondrial function in hippocampus, and to study the underlying mechanisms. Methods The female adult C57BL/6 mice were randomly assigned to Sham group, Ovx group and Ovx+E2group. On the 7th day after operation, Ovx+E2group was subcutaneously injected with 17β-estradiol(E2) for 4 weeks. Then another 48 female adult C57BL/6 mice were randomly divided into Sham group, Sham+MnTBAP group, Ovx group and Ovx+MnTBAP group. After operation for one week, MnTBAP was injected into the hippocampus of mice in Sham+MnTBAP group and Ovx+MnTBAP group. The depression-like behaviors of the mice were monitored by sucrose preference test and forced swimming test. Then the mice were decapitated, and the hippocampus samples were collected. Mitochondria of hippocampus were isolated using mitochondria fractionation kit. The concentrations of adenosine triphosphate(ATP), reactive oxygen species(ROS) and mitochondrial membrane potential were determined, respectively. Results Compared with Sham mice, Ovx showed a decrease in the percentage of sucrose consumption, an increase in immobility time and the alterations of hippocampal mitochondrial function, which were reversed by estrogen treatment. Intrahippocampal injection of MnTBAP significantly increased the percentage of sucrose consumption and decreased immobility time in Ovx mice. Conclusion Hippocampal mitochondrial dysfunction contributes to estrogen deficiency-induced depression-like behaviors.

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Objective To investigate the protective effect of Paeoniflorin-6′-o-benzene sulfonate(CP-25) on methotrexate(MTX)-induced liver injury in rats.Methods 40 SD rats were randomly divided into normal group, model group, CP-25 treatment group, CP-25 prevention group and resveratrol prevention group.A rat model of liver injury was established by single intraperitoneal injection of MTX(20 mg/kg), the levels of serum alanine aminotransferase(ALT), aspartate aminotransferase(AST), superoxide dismutase(SOD), glutathione(GSH) and malondialdehyde(MDA) were determined by ELISA, the levels of interleukin-1β(IL-1β), interleukin-6(IL-6), tumor necrosis factor-α(TNF-α), B-lymphocyte tumor-2(Bcl-2), Bcl-2-associated X(Bax),cytochrome C(Cyt-C), cleaved cysteinyl aspartate specific proteinase8(cleaved-cas8), cleaved cysteinyl aspartate specific pro-teinase9(cleaved-cas9) and reduced nicotinamide adenine dinucleotide phosphate oxidase 4(NOX4) containing cysteine were assessed by Western blot.Results Compared with the normal group,the levels of ALT and AST in MTX-induced liver injury model rats increased(F=70.23,P<0.01; F=11.09,P<0.05),and the liver histopathology showed extensive inflammatory cell infiltration,hepatic lobular structure disorder and hepatocyte swelling.Compared with MTX model group,the levels of ALT and AST in CP-25 prevention group and treatment group were lower(F=62.32,86.86,P<0.01; F=16.35,7.14,P<0.01),and the pathological score of liver tissue was lower(F=18.33,P<0.01; F=15.47,P<0.05).Further studies showed that compared with MTX model group,both CP-25 prevention group and treatment group could significantly down-regulate the expression of pro-apoptotic proteins Bax(F=21.37,P<0.01; F=19.35,P<0.05),Cyt-C(F(CP-25 prevention group)= 7.21,P<0.01),cleavedcas8(F(CP-25 prevention group)= 12.32,P<0.05) and cleaved-cas9(F=8.41,P<0.01; F=6.91,P<0.05),and upregulate the expression of anti-apoptotic protein Bcl-2(F=13.11,P<0.01; F=9.93,P<0.05).At the same time,compared with MTX model group,CP-25 prevention group and treatment group decreased the levels of IL-1β,IL-6 and TNF-α(F(CP-25 prevention group)= 160.7,46.55,28.89,P<0.01); F(CP-25 treatment group)= 127.4,53.70,26.46,P<0.01), down-regulated the levels of MDA and NOX4(F(CP-25 prevention group)= 31.71,38.45, P<0.01;F(CP-25 treatment group)=27.89,31.27,P<0.01),and up-regulated the level of GSH(F=39.65,29.04,P<0.01).Conclusion CP-25 has a good therapeutic and protective effect on hepatotoxicity induced by MTX,and this effect may be related to its inhibition of oxidative stress,inflammatory response,and reduction of apoptosis in liver tissue.

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Objective To investigate the value of combined stool syndecan-2(SDC2) and tissue factor pathway inhibitor 2(TFPI2) gene methylation testing in the early screening of colorectal cancer. Methods 106 patients with colorectal cancer(colorectal cancer group), 75 patients with advanced adenoma(advanced adenoma group) and 35 patients with non-advanced adenoma(non-advanced adenoma group) were selected as study subjects, and 153 patients with other gastrointestinal disorders and 182 patients with negative colonoscopy results during the same period were selected as the control group. The quantitative methylation-specific PCR(qMSP) method was used to detectSDC2andTFPI2gene methylation in the stool specimens of all subjects. The sensitivity and specificity of the combinedSDC2andTFPI2gene methylation assay for the detection of colorectal cancer and adenoma were evaluated using colonoscopy and pathology results as the gold standard. Results Among 106 patients with colorectal cancer, the sensitivity of combined methylation test was 93.4%; among 75 patients with advanced adenoma, the sensitivity of combined methylation test was 62.7%; among 35 patients with non-advanced adenoma, the sensitivity of combined methylation test was 34.3%; the specificity of the combinedSDC2andTFPI2gene methylation test for colorectal cancer and adenoma screening was 94.6%. Conclusion The combinedSDC2andTFPI2gene methylation test has high sensitivity for colorectal cancer and its early lesions, and it also maintains high specificity.

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Objective To analyze the pregnancy outcome of preimplantation genetic testing for aneuploidy(PGT-A) in cycles with different indications.Methods The clinical information of 549 couples who underwent PGT-A were retrospectively analyzed.The cycles were divided into 6 groups according to the indication for PGT-A,namely: recurrent pregnancy loss group(n=304),repeated implantation failure group(n=57),advanced age group(≥38years old,n=80),history of adverse pregnancy group(chorionic trisomy or adverse pregnancy,n=24),male factor infertility group(n=67),and abnormal sex chromosome number group(n=17).The basic information,the number of retrieved oocytes,embryo biopsy result and pregnancy outcome were compared among different indication groups.Results The average age and days of gonadotropin(Gn) used among the six groups were statistically different(P<0.001).The average number of retrieved oocytes,the rate of good-quality embryos,mosaic embryos,abnormal embryos and normal embryos,the average ovarian sensitivity index(OSI) among the six groups were statistically different(P=0.03,P<0.001,P=0.03,P<0.001,P<0.001,P<0.001).Advanced age group had the highest rate of abnormal embryos,the least average number of retrieved oocytes,the lowest OSI and the lowest rate of normal embryos.There were statistical differences in clinical pregnancy rate,ongoing pregnancy rate and cumulative pregnancy rate per oocyte retrieved cycle(P<0.001) among the six groups,but there were no statistical differences in clinical pregnancy rate,ongoing pregnancy rate per transfer cycle and cumulative pregnancy rate among the five groups except for the male factor infertility group.Conclusion PGT-A can detect euploid embryo to transfer thereby improving pregnancy efficiency.The advanced age women have normal embryo to transfer and can obtain a better pregnancy rate,which may shorten their time of “take-baby-home”.At the same time,PGT-A can significantly improve the pregnancy outcome of those with male factor infertility.

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Objective To evaluate the accuracy and feasibility of coronary artery calcium score(CACS) on virtual non-contrast scan(VNC) images obtained from coronary artery CT angiography(CCTA) scan with dual-layer spectral detector CT(SDCT). Methods The data of 197 patients who underwent CCTA scan in hospital were analyzed retrospectively, and 88 patients with CACS>0 were further analyzed. Linear regression analysis of CACS and coronary artery calcium volume(CACV) of true non-contrast(TNC) images and VNC images(CACS-TNC, CACS-VNC, CACV-TNC, CACV-VNC) was performed to obtain linear regression equation and correction coefficients λ1AVGand λ2AVG. CACS-VNC and CACV-VNC were corrected by the corresponding regression equation and recorded as CCACS-VNC and CCACV-VNC, respectively. Spearman correlation coefficient was used for correlation analysis and Bland-Altman plot was used for consistency test. Mann-WhitneyUtest was used to compare the difference between the two groups. Results For the total coronary artery, there was a strong correlation between CACS-TNC and CACS-VNC(rs=0.952,P<0.001, λ1AVG=2.19), CACV-TNC and CACV-VNC(rs=0.954,P<0.001, λ2AVG=1.93). The results of Mann-WhitneyUtest showed that there was no significant difference between CACS-TNC and CCACS-VNC or between CACV-TNC and CCACV-VNC, and the Bland-Altman plot showed good consistency between CACS-TNC and CCACS-VNC, CACV-TNC and CCACV-VNC. Conclusion VNC images based on SDCT can accurately measure CACS and be used for cardiovascular risk classification, which is expected to replace TNC scan and reduce the radiation dose of patients.

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Objective To investigate the clinical value of coefficient of variation(RDW-CV) and standard deviation(RDW-SD) of erythrocyte distribution width in the diagnosis of colorectal cancer(CRC) metastasis. Methods 91 CRC inpatients were selected as the research subjects. According to whether the tumor was metastatic, they were divided into two groups: 61 cases in the non-metastatic group and 30 cases in the metastasis group. The laboratory indicators of the two groups of patients included: neutrophils count, lymphocyte count, platelet count, carcinoembryonic antigen, plasma prothrombin time, activated partial thromboplastin time, fibrinogen, thrombin time, RDW-CV and RDW-SD and other indicators. Thettest was used to compare the means of the two groups, and the pearson correlation was used to analyze the relationship between RDW-CV and RDW-SD with neutrophil count, lymphocyte count, neutrophil count-lymphocyte count ratio(NLR) and carcinoembryonic antigen. Correlation coefficient, receiver operating characteristic curve(ROC) to assess the area under the curve(AUC) of RDW-CV, RDW-SD, carcinoembryonic antigen and their combined diagnosis in assessing CRC metastasis. Results Compared with non-metastatic patients, the RDW-CV and RDW-SD values of metastatic patients were higher(bothP<0.05). Both RDW-CV and RDW-SD levels were positively correlated with carcinoembryonic antigen content. Compared with the AUC of carcinoembryonic antigen in diagnosis of CRC metastasis, the AUC of RDW-CV or RDW-SD combined with carcinoembryonic antigen was higher. Conclusion RDW-CV and RDW-SD have potential clinical application value in differential diagnosis of CRC metastasis.

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Objective To explore the effects of different implant sites combined with pterygomaxillary implantation on the biomechanics of implant and bone tissue, and to provide a basis for clinical selection of implant design in accordance with biomechanical principles. Methods The cone-beam CT scan data of an edentulous maxillary with inadequate posterior bone were selected to complete the establishment of the three-dimensional entity model of the maxilla. The prosthesis and implant-abutment integrated three-dimensional entity models were established using the prosthesis and implant data, and five groups of three-dimensional finite element models of different implant sites were designed. A unilateral vertical load of 200 N and an oblique load of 100 N were applied to the bilateral posterior dental area, respectively. ANSYS finite element analysis software was used to calculate the stress distribution on the surface of the implant and surrounding bone tissue. SPSS 26.0 software package was used to statistically analyze the data. Results (1) All the five models showed that the maximum stress was concentrated in the neck of the implants and cortical bone.(2) The maximum stress value of the implant under oblique loading was greater than that under vertical loading(P<0.05), but there was no significant difference in the maximum stress value around bone tissue in the 5 groups of models.(3) There was no significant difference in the maximum stress of the implant and bone between the different implant sites combined with pterygomaxillary implants. Conclusion In the fixed restoration of maxillary edentulous implants, pterygomaxillary implants implanted symmetrically on both sides and changing the position point of the anterior implant do not affected the stress distribution of the whole design. In clinical practice, suitable sites can be selected according to the residual bone mass of patients, and combined with pterygomaxillary implantation for implant design.