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Objective To construct a human G protein-coupled receptor kinase 2(GRK2) eukaryotic expression system. Methods The primers were designed,and the His-GRK2 target gene was amplified by PCR using the Pans-EGFP-GrK2( full-length) gene as the template. The His-GRK2 target gene was connected to the pcDNA3. 1-EGFP eukaryotic expression vector. The pcDNA3. 1-EGFP-His-GRK2 plasmid was transfected into HEK 293T cells. 48 h later,the expression of GRK2 protein was detected by Western blot,and the GRK2 protein was purified by nickel chelated magnetic bead method. The purification of GRK2 protein was detected by Coomassie bright blue staining and Western blot,and the activity of GRK2 protein was detected by His pull down. Results The results of double enzyme digestion and sequencing showed that pcDNA3. 1-EGFP-His-GRK2 eukaryotic expression plasmid was successfully constructed. Western blot analysis showed that the molecular weight of GRK2 protein was about 80ku,indicating that GRK2 protein was successfully expressed in HEK 293T cells( t = 6. 433,P = 0. 003). GRK2protein was purified by nickel chelated magnetic beads. His pull down experiment results showed that GRK2 was bound to prostaglandin E2 receptor 4( EP4),suggesting that GRK2 protein had biological activity( t = 13. 5,P =0. 000 2). Conclusion The pcDNA3. 1-EGFP-His-GRK2 eukaryotic expression plasmid was correctly sequenced and the GRK2 recombinant plasmid was successfully constructed. The GRK2 recombinant plasmid was successfully expressed in eukaryotic cells HEK 293T and the protein expressed was biologically active.

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Objective To investigate the distribution of toxoplasma cysts in the brain of infected mice and the effect of pathological changes on the behavior and neuropsychiatry of the mice during chronic infection withToxoplasma gondii(T.gondii). Methods Mice were infected with Prugniaud strain ofT.gondiiby oral gavage. The brain tissues of infected mice were collected on the days of 10, 30, 40, 90, 120 and 160 after infection respectively, and the hippocampal hypothalamus, prefrontal lobe, striatum and cerebellum regions were separated. The number of cysts and neuropathological changes in each infected area were observed and recorded by HE staining. The number of cysts and neuropathological changes in each infected area were observed and recorded. Results T.gondiiinfected mice showed symptoms of vertical hair and arched back, which were the most significant on the 40th day, and then gradually recovered with hemiplegia and circling in circles. At each time point, the number of toxoplasma cysts was the largest in hippocampal hypothalamus, followed by prefrontal lobe and striatum, and the least in cerebellum. The diameter of toxoplasma cysts increased with time. During chronic infection, specific pathological manifestations of toxoplasma encephalitis, such as neuronophagy, were observed in all regions of the brain tissue. The above pathological changes of toxoplasma encephalitis reached the peak on the 40th day, and gradually recovered, and increased to the stimulation peak on the 120th day, and then gradually recovered. Conclusion The behavioral and neuropsychiatric symptoms ofT.gondiiduring chronic infection were correlated with the localization and distribution of toxoplasma cysts in the brain of infected mice, and showed dynamic changes.

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Objective To investigate the expression and prognosis of protein regulator of cytokinesis 1(PRC1) in pancreatic carcinoma tissues. Moreover, to explore the effects of PRC1 on the biological functions of pancreatic carcinoma cell line SW1990 and its related mechanisms. Methods The GEPIA database was used to analyze the expression difference of PRC1 in pancreatic carcinoma tissues and normal pancreatic tissues. Overexpression and interference of PRC1 were achieved by Lipofectamine 3000 transfection plasmid or shRNA method. Then CCK-8 assay, Transwell assay and flow cytometry were used to detect the proliferation level, invasion ability and apoptosis of the SW1990 cells, respectively. The pancreatic carcinoma data were collected from the Cancer Genome Atlas(TCGA) database. The correlation between expression level of PRC1 and clinicopathological features of pancreatic carcinoma was analyzed. The STRING database was used to analyze the network of proteins interacting with PRC1. Gene set enrichment analysis(GSEA) was used to predict the possible signal pathways of PRC1 in pancreatic carcinoma. Results GEPIA database results showed that PRC1 expression in pancreatic carcinoma tissue was higher than that in normal pancreatic tissue(P<0.05). The results of CCK-8 assay, Transwell assay and flow cytometry showed that PRC1 overexpression significantly enhanced SW1990 cell proliferation, invasion and inhibited apoptosis(P<0.01). Whereas PRC1 interference significantly inhibited SW1990 cell proliferation, invasion and enhanced apoptosis(P<0.01). TCGA database data analysis identified PRC1 mRNA expression level and M stage were independent risk factors affecting the prognosis of pancreatic carcinoma(P<0.05). STRING database showed that there was an interaction between PRC1 and PLK1 and so on. GSEA research results showed that the PRC1 mRNA high expression samples were enriched into P53 signaling pathway and so on(P<0.05). Conclusion PRC1 is highly expressed in pancreatic carcinoma, and it is associated with proliferation, invasion, apoptosis and prognosis of pancreatic carcinoma. Moreover, it plays an important role in pancreatic carcinoma by regulating interacting proteins PLK1 and activating P53 signaling pathways.

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Objective To investigate the effect of peroxide reductase 1(Prdx1) on myocardial hypertrophy and fibrosis in spontaneously hypertensive rats, and to analyze its mechanism. Methods Forty five SHR rats were randomly divided into model group(SHR group), AAV9-NC group and AAV9-Prdx1 group. There were 15 WKY rats in each group, and the other 15 Wistar Kyoto rats were set as the control group. The rats in each group were administered continuously for 8 weeks, and the indexes of cardiac function were detected by echocardiography; The mean blood pressure and myocardial hypertrophy were measured; HE staining and Masson staining were used to observe the histomorphology and fibrosis of rat myocardium; The indexes of oxidative stress in rat serum were detected by ELISA; The expression level of Prdx1 mRNA in rat myocardium was detected by qRT-PCR; Western blot was used to detect the expression of Prdx1 protein and nuclear factor E2 related factor 2(Nrf2)/heme oxygenase-1(HO-1) signaling pathway related proteins in rat myocardium. Results Compared with the Control group, the expression of Prdx1 mRNA and protein, left ventricular ejection fraction(EF) and left ventricular shortening rate(FS) decreased in SHR group(P<0.05), the mean blood pressure, heart mass index(HMI) and left ventricular mass index(LVMI) of rats increased(P<0.05), and there were obvious pathological damage and collagen fiber deposition in myocardial tissue. The activities of superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px) in rat serum decreased, and the content of malondialdehyde(MDA) increased(P<0.05); The expression of Nrf2, HO-1 and quinone oxidoreductase 1(NQO1) protein decreased in myocardial tissue(P<0.05). Compared with SHR group, the expression of Prdx1 mRNA and protein, EF and FS in myocardial tissue of AAV9-Prdx1 group increased(P<0.05), the mean blood pressure, HMI and LVMI of rats decreased(P<0.05), and myocardial tissue injury and myocardial fibrosis improved; The activities of SOD and GSH-Px in rat serum increased, while the content of MDA decreased(P<0.05); The expression of Nrf2, HO-1 and NQO1 protein increased in myocardial tissue(P<0.05). Conclusion Overexpression of Prdx1 can reduce myocardial hypertrophy and fibrosis and improve cardiac function in SHR rats. Its mechanism may be related to activating Nrf2/HO-1 signaling pathway and inhibiting oxidative stress response.

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Objective To investigate the effect of glycogen synthase kinase 3β(GSK-3β) and its correlation with microtubule-associated protein 2(MAP2) during cerebral hypoxia/reoxygenation(H/R) in zebrafish. Methods The cerebral hypoxia/reoxygenation model of zebrafish was established. Healthy adult zebrafishes of the same size were divided into control group( Control),hypoxia/reoxygenation group( H/R) and hypoxia/reoxygenation +GSK-3β inhibitor group( H/R + TDZD-8) for experiment. The brain tissues of zebrafish in each group were selected to determine the mRNA expressions of hypoxia inducible factor 1αa and 1αb( Hi F-1αa and HIF-1αb) at different reoxygenation time points by qRT-PCR,and the protein expression levels of HIF-1α,GSK-3β,p-GSK-3β( Ser9) and MAP2 were detected by Western blot,TTC staining and TUNEL staining were used to detect cerebral infarction area and cell apoptosis,and immunofluorescence was used to detect the distribution and expression of MAP2 in brain. Results Compared with Control group,the mRNA levels of Hif-1αa and Hif-1αb( P<0. 01) and protein expression of Hif-1α( P<0. 01) increased in H/R group,the area of cerebral infarction( P<0. 01) and apoptotic cells( P<0. 01) increased,p-GSK-3 β( Ser 9)/GSK-3β ratio,MAP2 protein expression( P<0. 05)and immunofluorescence expression of MAP2( P<0. 01) reduced; Furthermore,TDZD-8 pretreatment could relieve the brain injury of H/R zebrafish by decreasing the infarct size and cell apoptosis,improving the ratio of pGSK-3β( Ser 9)/GSK-3β,and increasing the expression of MAP2. Conclusion Hypoxia/reoxygenation can cause brain neuron damage in zebrafish,and its mechanism may be related to inhibition of GSK-3β phosphorylation and MAP2 expression. GSK-3β specific inhibitor TDZD-8 can reverse the damage of brain neurons caused by hypoxia/reoxygenation by promoting the expression of P-GSK-3 β( Ser 9) and reducing MAP2 degradation.

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Objective The CRISPR/Cas9 technology was applied to construct PDE4D homozygous knockout mice to provide a basis for in-depth investigation of PDE4D gene function and mechanism of action. Methods A vector was constructed for PDE4D gene exon 4, 5 microinjected into fertilized eggs of C57BL/6J mice, and PDE4D-/-mice were obtained after maternal breeding and offspring mating, and the mice genotypes were determined by PCR product sequencing and genotype identification techniques. Changes in morphology and function of the major organs of the mice were detected using an ultrasound imaging system and H&E staining, and the expression of PDE4D protein in the mice was verified by Western blot assay. Results The PDE4D-/-mouse genotype was stably inherited, the mice were small, and there were no obvious morphological and histological changes in the major organsin vivo. The PDE4D expression was reduced or largely absent in the major tissues of PDE4D heterozygous or pure knockout mice, and the knockout effect was better. Conclusion PDE4D-/-mice were successfully established using CRISPR/Cas9 technology, and no significant physiological abnormalities were found, which could be used for disease pathogenesis and drug research using PDE4D as the target.

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Objective To investigate the expression pattern, underlying function and clinical significance of Guanylate-binding protein 1(GBP1) in pulmonary tuberculosis(pTB). Methods Immunohistochemical staining was applied to detect the expression of GBP1 in pTB specimensand control samples. Combined with Gene Expression Omnibus(GEO) datasets, including GSE83456 and GSE34608, receiver operating characteristic(ROC) curve was depicted to assess the diagnostic value of GBP1 in pTB. Then, the correlation between GBP1 and related regulatory factors was analyzed by protein-protein interaction network(PPI); Finally, the potential molecular mechanism of GBP1 in pTB was probed by Gene Set Enrichment Analysis(GSEA). Results Compared with the control group, GBP1 was significantly overexpressed in human pTB samples, including lung tissue and blood.The positive rate of GBP1 protein in pTB was 73.9%. ROC curve analysis revealed that GBP1 might have important value in early diagnosis of pTB. GSEA analysis suggested that the hyper-expression of GBP1 was closely related to the host inflammatory response, IFN-γ/α signaling pathway and TNF-α/IL-6 signal transduction. Conclusion GBP1 is highly expressed in pTB tissues and is involved in the process of inflammatory response and host anti-tuberculosis infection; GBP1 may be used as an early diagnostic marker or therapeutic target for pTB.

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Objective To construct the full-length prokaryotic expression plasmid of the wild type of androgen receptor(AR) and the truncated body of four functional domains, and to identify the fusion protein by Western blot and electrophoretic mobility shift assay(EMSA). Methods Based on the pGEX-4T-1 vector, the recombinant plasmids were constructed to express the full-length and functional domains of AR. IPTG was used to induce the expression of the recombinant proteins,which were isolated and purified by glutathione sepharose 4B beads under the optimized condition. The specific protein expression in the bacterial lysate and the purified protein isolated with glutathione sepharose 4B beads was identified by Western blot with AR antibody and GST labeled antibody. The purified protein was incubated with a fluorescent probe of the virus,and the complex was detected by electrophoresis in a non-denaturing gel. Results The prokaryotic recombinant plasmids of full length and three functional domain truncated AR were successfully constructed. The recombinant clones were identified by using bacterial culture as a template,and further verified by double enzyme digestion. It showed that there were identical bands in the same sizes as the inserted fragments. The nucleotide and the amino acid sequences were aligned to the reference sequence in NCBI GenBank. The GST fusion protein,GST-AR-NTD + DBD( 96 ku) and GST-AR-NTD( 86 ku)were successfully induced and verified. The purified protein could be directly combined with the viral genome DNA. Conclusion The prokaryotic expression conditions of truncated AR plasmid from the same gene sequence are different. The purified AR protein can be used to understand the direct interaction mechanism between functional domains of AR and other molecules.

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Objective To explore method for isolating and culturing human epidermal stem cells(EPSCs)in vitroand isolating and purifying epidermal stem cell exsomes(EPSCs-Exo) by optimizing the technical process. Methods Firstly, the improved separating enzyme was used to isolate the EPSCs derived from human skin tissue. Then, an improved serum-free culture medium and 10 specific factors were combined to construct optimized 2D culture medium which could stimulate the growth of EPSCs, promote the secretion of EPSCs-Exo, maintain the stemness and proliferation of EPSCs, and delay the differentiation and maturation of EPSCs. Further, the conditions of differential centrifugation was optimized, and then the human EPSCs-Exo was successfully extracted with high efficiency and high purity. Results The human skin tissue was confirmed with the expressions of markers for epidermal cells. EPSCs were verified with high expression levels of integrin-α6, integrin-β1, P63 and CK19 by immunofluorescence staining and Western blot. The nanoparticle tracking analysis results showed the particles separated for EPSCs supernatant was saucepan with the detected diameter between 30-150 nm. The Western blot results showed the positive expression of membrane markers Tsg101, CD9 and CD63 and the negative expression of intracellular markers Calnexin and GAPDH. Conclusion The results show that the human-derived EPSCs have been successfully isolated and culturedin vitro, and the EPSCs-Exo have been successfully isolated and identified.

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Objective To explore the method of intraperitoneal injection of allogenic fecal filtrate to establish the rat model of moderate and severe sepsis. Methods The preparation method of allogeneic fecal filtrate was determined. Allogeneic fecal filtrate of different concentrations(0.5, 1, 2 g/kg) was injected intraperitoneally to observe the general situation, survival time and severe degree of sepsis of rats. After determining the optimal concentration, the success rate of the model, serum inflammatory factors, serum concentration of D-lactic acid(D-LA) and serum intestinal fatty acid binding protein(I-FABP), lung function changes, lung, liver and kidney tissue injury were further observed. Results After intraperitoneal injection of allogenic fecal filtrate for 24 h, the rats of 1 g/kg group presented fever, tachypnea and hypotension, the survival rate was 83.3% at 24 h and 16.7% at 48 h, 2 g/kg group rats all died within 24 h, the dose of 1 g/kg was determined for subsequent experiments. Injected fecal filtrate for 24 h, the success rate of the sepsis model was 77.8%. The levels of interleukin-6(IL-6), tumor necrosis factor-α(TNF-α), D-LA and I-FABP in serum significantly increased. There were severe edema and bleeding in lung tissue, Pulmonary function appeared respiratory dysfunction, included functional residual capacity(FRC), quasi static compliance(Cdyn), forced expiratory volume for the first 100 milliseconds(FEV100), peak expiratory flow(PEF) decreased, airway resistance(RI), inspiratory capacity(IC) increased. Liver and kidney tissues also showed varying degrees of edema and inflammatory cell infiltration, the levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST), blood urea nitrogen(BUN) and creatinine(Cr) in serum significantly increased. Conclusion Intraperitoneal injection of allogenic fecal filtrate(1 g/kg) can produce a relative typical septic model in rats.

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Objective To prepare an adhesive hemostatic hydrogel and examine its hemostatic performance and biocompatibility. Methods The precursor components were homogenously dissolved and photo-crosslinked in order to form dual-network hydrogel. The electron microscopy morphology was then analyzed; mechanical properties were tested;in vitrohemostatic performances were investigated by whole blood clotting test and simulation trial of cutaneous bleeding. Further, the hemocompatibility was evaluated. Results A characteristic porous network structure was presented through microscopy observation of the sample. The tensile strength of hydrogel reached 46 kPa, and strong adhesion was achieved between the hydrogel andex vivobiological tissues. Hydrogel had significant effect on wound closure and clotting time could be shortened to 1 minute. Conclusion The hydrogel was capable of accelerating coagulation due to its ability to accumulate platelets and red cells after blood contact. The dual-network hydrogel with good hemocompatibility enabled excellent hemostatic performance by the synergistic effects of the chemical activation mechanism and physical hemostatic effect.

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Objective To investigate the expression of Kin of IRRE-like protein 1(KIRREL) in gastric cancer tissues and further explore its effect on angiogenesis. Methods Western blot and IHC were used to detect the expression of KIRREL in gastric cancer tissues and adjacent tissues. Human gastric cancer cell lines SNU-5 and AGS were selected to construct KIRREL-interfering and overexpressing stable cell lines by lentiviral infection of gastric cancer cells. The expression of hypoxia-inducible factor 1α(HIF-1α) and vascular endothelial growth factor(VEGF) in the control, null, interfering and overexpression groups were measured by Western blot, and the effect of KIRREL on the tube-forming ability of human umbilical vein endothelial cells(HUVECs) was observed by angiogenesis assay. Results The expression of KIRREL in gastric cancer tissues was higher than that in adjacent tissues(P<0.05). After silencing KIRREL, the protein expression of HIF-1α and VEGF was significantly reduced, and the tube length and nodes of HUVECs were also significantly reduced; the overexpression of KIRREL was the opposite(P<0.05). Conclusion KIRREL is overexpressed in gastric cancer tissues and may promote angiogenesis in gastric cancer.

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Objective To explore the regulatory mechanism of ginsenoside Rb1 on focal cerebral ischemia-reperfusion injury(CIRI). Methods A total of 60 C57/BL mice were randomly divided into 6 groups(n=10): sham-operated group, CIRI model group, ginsenoside Rb1 low-, medium-, and high-dose group and nimodipine(positive control) group. The surgical method was used to construct the focal CIRI mouse model. The neurological function scores and behavioral tests were performed, and Nissl staining was utilized to detect the number of nissl bodies in the hippocampus. The effect of ginsenoside Rb1 on the molecule expression of the Wnt signaling pathway in the hippocampus was detected by qPCR, Western blot and immunohistochemistry assays. The regulatory mechanism of ginsenoside Rb1 was investigated through molecular docking and co-precipitation assays. Results Compared with the CIRI model group, the addition of ginsenoside Rb1 reduced the neurological function scores of mice(P<0.05), shortened the time passing the balance beam(P<0.05), but increased the time entering the correct arm(P<0.05) and the swinging time and climbing time of mice(P<0.05), indicating that ginsenoside Rb1 could effectively resume the function of the nervous system in mice and improve the behavioral ability of model mice. After ginsenoside Rb1 treatment, axis inhibition protein 2(Axin2) and glycogen synthase kinase-3β(GSK-3β) in the hippocampus decreased, whereas the expression of Wnt3a, Wnt1 and β-catenin increased. Conclusion The ginsenoside Rb1 can improve neurological function of the CIRI mouse model and increase the number of Nissl bodies in the hippocampus, which is correlated with the activation of the Wnt signaling pathway, and it may be neuroprotective against focal CIRI during stroke treatment.

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Objective To explore the effects and possible mechanism of 2-hydroxy-3-methylanthraquinone(HMA) on the growth inhibition of colon cancer cells(HCT116, HT-29). Methods The target of HMA was searched and analyzed by TCMSP database. The colon cancer HCT116 and HT-29 cells were culturedin vitro, and then they were intervened with different concentrations of HMA. The effects of HMA on the viability, growth, survival ability and apoptosis of colon cancer cells were detected by CCK-8, trypan blue count, colony formation and Annexin V/PI double staining respectively. The effects of HMA on potential targets and apoptosis-regulating proteins were detected by Western blot. Results TCMSP search analysis showed that there was HMA in Hedyotis diffusa, Scutellaria barbata and Morinda officinalis, which contained 32 potential targets, including apoptosis-related gene Caspase-3. Different concentrations of HMA could inhibit cells viability, growth and number of formed colonies, and induce apoptosis. The results of Western blot showed that HMA could down-regulate anti-apoptosis gene Bcl-2, up-regulate pro-apoptosis gene Bax, and promote the activation and cleavage of apoptosis-related proteins(cleaved Caspase-9, cleaved Caspase-3), thus promoting apoptosis. Conclusion HMA can inhibit growth and induce apoptosis of colon cancer cells, which may be related to regulating expressions of Bcl-2, Bax, cleaved Caspase-9, cleaved Caspase-3 and other apoptosis proteins.

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Objective To investigate whether the cyclic adenosine monophosphate(cAMP)/exchange proteins directly activated by cAMP(Epac)/ras-related protein 1(Rap1) signalling pathway is involved in the intervening mechanisms of interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), brain-derived neurotrophic factor(BDNF) and Jujuboside A(JuA) secretion by NG2 cells. Methods NG2 cells were cultured in vitro and the experiment was divided into control group,pertussis toxin( PTX) group,ESI-09 group,JuA group and positive drug group. The effect of different concentrations of JuA on the survival rate of NG2 cells was detected by CCK-8 method,and the expression of IL-1β,TNF-α,BDNF,cAMP,Epac,Rap1 mRNA and protein in each group was detected by RT-PCR and Western blot. Results Compared with the control group,the PTX group decreased the expression of IL-1β and TNF-α mRNA and protein( P<0. 01) and increased the expression of cAMP and BDNF mRNA and protein( P<0. 01); the ESI-09 group increased the expression of IL-1β and TNF-α mRNA and protein( P<0. 05) and decreased the expression of BDNF,Epac and Rap1 mRNA and protein expression( P<0. 01); the JuA group and positive drug group increased IL-1β,TNF-α,BDNF,cAMP,Epac,Rap1 mRNA and protein expression( P<0. 01). Conclusion The cAMP/Epac/Rap1 signaling pathway is involved in the secretion of IL-1β,TNF-α,and BDNF by NG2 cells. JuA may act on cAMP/Epac/Rap1 signaling pathway to affect the secretion of BDNF by NG2 cells.

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Objective To study the effect of Er, Cr: YSGG laser on the mineralization of dentin collagen fiber, accelerate the mineralization process and shorten the mineralization time. Methods Human health third molarswere selected to remove the teeth of the tooth enamel, degenerate the teeth, leave the dentin collagen fiber web, place the degenerative collagen fiber in the configured re-mineral fluid. Using Er, Cr: YSGG, the teeth before and after the test were used to analyze the surface morphology using SEM scanning electron microscopy, and the EDS X-ray energy spectrum was analyzed by the distribution of calcium phosphorus. Results Er, Cr: YSGG laser irradiation laser irradiated tooth in the teeth in the teeth, new minerals were generated, which covered the inner wall of the teeth, the calcium phosphorus element was enriched in the inner wall of the teeth. Conclusion YSGG laser can help dentin collagen fibers mineralized in mineralization fluid, form new minerals.This method can shorten the mineralization time of dentin collagen fibers in mineralization fluid, which is the re-ore.

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Objective To study the effect of bufalin on the proliferation and apoptosis of human colorectal cancer cell line HCT116, and to explore the role of endoplasmic reticulum stress(ERS) in this process. Methods The effect of bufalin on the proliferation of HCT116 cells was determined by CCK-8 assay. After HCT116 cells were treated with different concentrations of bufalin for 48 hours, cell apoptosis was detected by Annexin V/PI assay, and the expression of apoptosis-related proteins Bax and Bcl-2 was detected by Western blot. At the same time, the expression of ERS-related proteins glucose regulated protein 78(GRP78), phosphorylated protein kinase R like endoplasmic reticulum kinase(p-PERK), eukaryotic translation initiation factor 2α(eIF2α), phosphorylated eukaryotic translation initiation factor 2α(p-eIF2α) and C/EBP homologous protein(CHOP) was detected by Western blot. HCT116 cells were divided into control group, bufalin group and combination group(bufalin+4-phenylbutyric acid), and the expression of apoptosis-related proteins Bax and Bcl-2 was observed by Western blot. Results CCK-8 assay showed that bufalin could inhibit the proliferation of HCT116 cells. Apoptosis assay showed that bufalin could induce apoptosis of HCT116 cells. The results of Western blot showed that bufalin could up-regulate the expression of pro-apoptotic protein Bax and down-regulate the expression of anti-apoptotic protein Bcl-2. It could also induce ERS and activate PERK/eIF2α/CHOP pathway. When bufalin combined with 4-phenylbutyric acid, the apoptosis-promoting effect of bufalin was inhibited. Conclusion Bufalin can effectively inhibit the proliferative activity and induce apoptosis of HCT116, which is achieved to some extent by activating ERS.

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Objective To explore the clinical dosimetry advantages of automated plan of IMRT for postoperative esophageal cancer and the dose prediction accuracy of the constructed 3D U-Res-Net model.Methods A total of110 postoperative esophageal cancer (middle and upper) cases treated by IMRT were considered in the study,of which 90 cases were randomly selected for training of deep learning prediction model.The deep learning prediction model and Auto-Plan module (Philips pinnacle316.2) were used to predict the three-dimension dose distribution and redesigned the remaining 20 cases respectively,and the results obtained were compared with manual plan.Results The average DSC value between the deep learning prediction plan and the manual plan was greater than0.92 in isodose surface,and the average Hausdorff distance HD95of the isodose surface was 0.58-0.62 cm;The V20,V30,Dmeanof total lung were slightly lower than those of manual plan (P<0.05) for the prediction model,meanwhile,the D2,D50,Dmean,HI of the target area and V30of total lungs were better than those of manual plan(P<0.05) for Auto-Plan;Three-dimensional dose distribution of the three groups and the corresponding DVH curve showed that the three-dimensional dose distribution of the three groups had a little differences,and the DVH curves of the target area and organs at risk had a good agreement.Conclusion Auto-Plan can realize the design of automated plan for postoperative esophageal cancer,while the deep learning prediction model can realize the accurate prediction of the 3D dose distribution.

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Objective To develop an endoscopic automatic detection system in early gastric cancer(EGC) based on a region-based convolutional neural network(Mask R-CNN). Methods A total of 3 579 and 892 white light images(WLI) of EGC were obtained from the First Affiliated Hospital of Anhui Medical University for training and testing, respectively. Then, 10 WLI videos were obtained prospectively to test dynamic performance of the RCNN system. In addition, 400 WLI images were randomly selected for comparison with the Mask R-CNN system and endoscopists. Diagnostic ability was assessed by accuracy, sensitivity, specificity, positive predictive value(PPV), and negative predictive value(NPV). Results The accuracy, sensitivity and specificity of the Mask R-CNN system in diagnosing EGC in WLI images were 90.25%, 91.06% and 89.01%, respectively, and there was no significant statistical difference with the results of pathological diagnosis. Among WLI real-time videos, the diagnostic accuracy was 90.27%. The speed of test videos was up to 35 frames/s in real time. In the controlled experiment, the sensitivity of Maks R-CNN system was higher than that of the experts(93.00%vs80.20%,χ2=7.059,P<0.001), and the specificity was higher than that of the juniors(82.67%vs71.87%, χ2=9.955,P<0.001), and the overall accuracy rate was higher than that of the seniors(85.25%vs78.00%, χ2=7.009,P<0.001). Conclusion The Mask R-CNN system has excellent performance for detection of EGC under WLI, which has great potential for practical clinical application.

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Objective To investigate the 90 day prognosis of acute ischemic stroke patients treated with mechanical thrombectomy in the real world and analyze the related factors of prognosis. Methods A total of 1 033 acute ischemic stroke patients treated with mechanical thrombectomy in tertiary hospitals of Anhui province were retrospectively investigated. The 90 day modified Rankin Scale(mRS) score was evaluated by follow-up telephone, 0～2 points were good prognosis and 3～6 points were poor prognosis. Results A total of 770 patients were followed up, with an average age of(67.7±11.8) years. The good prognosis rate was 32.1% and the mortality rate was 31.8%. With the increase of age, the good prognosis rate showed a downward trend while the mortality rate showed an upward trend. The best cut-off value for age prediction of 90-day mortality was 69 years old. There was no gender difference in the good prognosis rate between women(70.6±10.6)years and men(65.6±12.2)years, but females experienced a higher mortality than males(37.1%vs27.9%) before adjusting for age. Comparison was made among provincial, municipal and county hospitals, only the good prognosis rate of provincial hospitals was significantly higher than that of county hospitals(37.2%vs15.6%). In the grouping of annual number of endovascular treatment cases, the good prognosis rate of only ≥40 cases group was significantly higher. There was no significant difference in mortality among the groups. Conclusion At present, the good prognosis is low and the mortality is high for the whole acute ischemic stroke patients treated with mechanical thrombectomy. Age was positively correlated with poor prognosis and mortality. The higher mortality rate in women than in men is due to the higher average age of onset in women. The good prognosis rate of provincial hospitals and hospitals with annual number of endovascular treatment ≥40 cases is higher than that of other hospitals.

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Objective To investigate the difference of clinical characteristics of recurrent spontaneous abortion(RSA) in patients with losses after spontaneous gestation and afterin vitrofertilization and embryo transfer(IVF-ET). Methods 237 patients diagnosed with RSA were divided into spontaneous gestation group(n=185) and IVF group(n=52) according to their previous modes of fertilization. The clinical characteristics of the two groups were analyzed. Results Compared with the spontaneous gestation group, the age of the first pregnancy and the age at the time of RSA in the IVF group were statistically greater than those in the natural pregnancy group(P<0.05). The number of biochemical pregnancies in IVF group was statistically higher than that in spontaneous gestation group, and the number of spontaneous abortions in IVF group was statistically lower than that in spontaneous gestation group(P<0.001). The proportion of patients with irregular menstruation in IVF group was statistically higher than that in spontaneous gestation group(P<0.05). The serum activated partial prothrombin time(APTT), R, K, high density lipoprotein cholesterol(HDL-C), thyrotropin(TSH) and homocysteine(HCY) in IVF group were statistically lower than those in spontaneous gestation group, and the Angel, Ma, low density lipoprotein cholesterol(LDL-C) and body mass index(BMI) in IVF group were statistically higher than those in spontaneous gestation group(P<0.05). In the comparison of etiological composition between the two groups, the proportion of anatomical factors in IVF group was statistically higher than that in spontaneous gestation group(P<0.05). Conclusion The abortion risk factors in RSA patients after IVF-ET is more serious than that in RSA patients after natural pregnancy. It is suggested to further improve and implement the pre-pregnancy examination and education of infertile patients during IVF-ET treatment, so as to reduce the risk of RSA and obtain a better pregnancy outcome.

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Objective To measure and analyze the position and shape of implant-related anatomical structure such as mental foramen, mental canal and the submandibular fossa in the adult mandibular posterior tooth area based on Cone Beam Computed Tomography(CBCT)images, and to detect the relationship between anatomical structures and the anatomice buccalingual angulation at implant site in order to help determine the implant angulation at the implant treatment-planning phase. Methods Random samples of 181 CBCT images were selected and analyzed, including 109 patients with dentition defect: 59 males and 50 females, and 72 patients with non dentition defect: 36 males and 36 females. The anatomic marks and alveolar bone inclination of mandibular posterior tooth area were measured respectively, and the results were statistically analyzed. Results The inclination angles of alveolar bonewere(5.08±3.86) on the left and(3.07±5.20) on the right of the mental foramen area,(8.57±5.03) and(9.54±5.19) on the first mandibular molar,(15.48±5.23) and(14.21±6.67) on the second mandibular molar, and the inclination angles of the left and right mental canal were(41.81±10.07) and(44.77±8.96) respectively. The distance from the upper edge of the mental foramen to the top of the alveolar ridge was(12.35±2.38) mm, and the distance between the mental canal at 1.5 mm inside the mental foramen and the top of the alveolar ridge was(14.14±2.46) mm. The depth of the submandibular gland fossa in the first and second molars were(0.80±0.56) mm and(1.00±0.50) mm, the distance from the most concave point to the alveolar crest top were(14.15±5.95) mm and(14.52±3.55) mm, the distance from the most convex point of the crown to the alveolar crest top were(10.71±4.72) mm and(9.25±2.81) mm, and the distance from the upper edge of the mandibular canal to the alveolar crest top were(15.52±3.48) mm and(13.83±2.76) mm. Conclusion The buccallingual inclination of mandibular alveolar bone is gradually inclined from buccal side to lingual side from premolar area to molar area, and the lingual inclination in the second molar area is the most obvious; The implant tip can stop at the upper edge of the mental foramen to obtain better initial stability; The existence of submandibular fossa limits the depth and direction of implant implantation to a certain extent. It is necessary to accurately measure and analyze this part with CBCT before operation to avoid complications such as mandibular lingual perforation and improve the success rate of implant repair.

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Objective To explore sex differences in 3D T1texture features in the progression of Alzheimer's disease(AD) and to predict the diagnosis of AD patients of different sex. Methods Seventy-seven AD patients(34 males and 42 females), 74 amnestic mild cognitive impairment(aMCI) patients(35 males and 39 females) and 75 healthy controls(HC)(35 males and 40 females) were recruited and high-resolution 3-dimensional T1 structural images were collected. Brain regions closely related to AD brain damage were selected as regions of interest(ROIs), texture feature extraction and feature screening were performed. Analyses were performed by sex, and the support vector machine(SVM) was used for classification and prediction. Results In the AD vs HC, AD vs aMCI and aMCI VS HC groups by different sex, we obtained some brain regions with relatively high recognition index in different subgroups, and found that there were significant differences between female patients and male patients with high recognition index, and the recognition index of female patients(area under the curve, accuracy, sensitivity and specificity were generally higher than that of male. Conclusion There are significant sex differences in texture features in AD process, and the classification and prediction ability of texture features in female patients is better, suggesting the importance of sex differences in AD research. This study provides some reliable biomarkers for early sex-specific identification of AD, which may be helpful for the early diagnosis and treatment of AD in the future.

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Objective To investigate the value of clinical data and qualitative and quantitative computed tomography(CT) in predicting the pathological grade of thymoma. Methods The clinical data and CT images of 81 patients with confirmed pathological diagnoses of thymoma were retrospectively analyzed. The patients were divided into low-risk and high-risk groups according to the pathological results, and the clinical features and qualitative and quantitative CT findings were compared between the groups. The efficacy of different parameters in predicting the pathological thymoma grade was evaluated by logistic univariate and multivariate regression analyses and receiver operating characteristic(ROC) curves. Results Sixty-two tumors were classified as low-risk thymoma and 19 as high-risk thymoma. The efficacies of both the maximum CT values on enhancement and maximum degree of enhancement for risk prediction were statistically significant(P<0.001). Logistic regression analysis showed that both the maximum CT values on enhancement(OR=0.88, 95% CI: 0.83-0.94) and maximum degree of enhancement(OR=0.92, 95% CI:0. 88-0. 97) were independent predictors of thymoma risk( P<0. 001). The area under the ROC curve( AUC) of the maximum CT values on enhancement was 0. 884,with a sensitivity of 68. 4% and a specificity of 96. 8%; the AUC of the maximum degree of enhancement was 0. 833,with a sensitivity of 89. 5% and a specificity of 72. 6%.Conclusion Quantitative parameters based on enhanced CT are helpful for the preoperative prediction of thymoma risk stratification. The predictive efficacy of the maximum CT values on enhancement is superior to that of the maximum degree of enhancement.

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Objective To explore the correlation between lipoprotein-associated phospholipase A2(Lp-PLA2) level and albuminuria in patients with type 2 diabetes mellitus(T2DM). Methods 200 T2DM patients were chosen to collect general data and relevant laboratory indicators. According to the urinary albumin/creatinine ratio(UACR), they were divided into normal group(UACR<30 mg/g,n=66), microalbuminuria group(30 mg/g≤UACR<300 mg/g,n=64) and macroalbuminuria group(UACR≥300 mg/g,n=70). On the basis of Lp-PLA2 tertile, they were divided into low Lp-PLA2 group(Lp-PLA2<104 ng/ml,n=66), medium Lp-PLA2 group(104 ng/ml≤Lp-PLA2<161 ng/ml,n=67) and high Lp-PLA2 group(Lp-PLA2≥161 ng/ml,n=67). Group differences were compared by analysis of variance and nonparametric test. Associations between Lp-PLA2 and other indicators were performed by Pearson correlation and Spearman correlation. Related factors of albuminuria in T2DM patients were explored by multivariate Logistic regression analysis. In addition, receiver operating characteristic(ROC) curve analysis was applied to evaluate the predictive value of Lp-PLA2 for albuminuria in T2DM patients. Results Lp-PLA2 was significantly higher in the macroalbuminuria group than that in both the normal group and the microalbuminuria group, and the differences were statistically significant(P<0.05). Compared with the normal group, Lp-PLA2 in the microalbuminuria group increased(P<0.05). With the increase in Lp-PLA2 tertile, there was gradual increase in UACR, and the difference was statistically significant(P<0.05). Correlation analysis showed that Lp-PLA2 was positively correlated with duration of DM,systolic blood pressure(SBP),glycosylated hemoglobin(HbA1c),fasting blood glucose(FBG),blood urea nitrogen(BUN), serum creatinine(Scr), UACR and negatively correlated with estimated glomerular filtration rate(eGFR)(P<0.05). Multivariate Logistic regression analysis showed that Lp-PLA2 [OR=1.046,95%CI(1.031,1.060)] was an independent risk factor for albuminuria(P<0.05). The AUC of Lp-PLA2 for predicting albuminuria was 0.902 [95%CI(0.862,0.942)]. The cut-off value of Lp-PLA2was 148 ng/ml, the sensitivity was 65.7% and specificity was 98.5%. Conclusion Lp-PLA2 is closely related to the albuminuria in T2DM patients, which provides a new method for the diagnosis and treatment of diabetic kidney disease(DKD).