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Objective Tnfrsf11 a Cre Rosa26 yfp reporter gene mice were prepared to determine the efficiency of Cre-mediated recombination using flow cytometry in different tissue-resident macrophages. Methods TheTnfrsf11 a Cre Rosa26 yfp reporter mice were generated by crossingTnfrsf11 a Cre mice withRosa26 yfp mice and identified by PCR.Brain microglia, liver macrophages, kidney macrophages, alveolar macrophages and spleen macrophages were separated from adultTnfrsf11 a Cre Rosa26 yfp reporter mouse and yellow fluorescent protein(YFP) labeling efficiency was analyzed by flow cytometry. Results YFP expression percentage was about 91.27% of brain microglia inTnfrsf11 a Cre Rosa26 yfp reporter mice, but liver, spleen, and alveolar macrophages were inefficiently targeted(63.60%,69.66%,32.76%). Conclusion Tnfrsf11 a Cre mice were qualified as conditional knockout model mice of brain microglia andTnfrsf11 a Cre Rosa26 yfp reporter mice can be used as a tool for conditional knockout of brain microgliain vivo.

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Objective To investigate the effects of p100Rband p80Rbtruncated forms of retinoblastoma protein(Rb) on HEK 293T cell cycle and apoptosis.Methods The eukaryotic expression plasmid pcDNA3. 1-p100Rb-FLAG and pcDNA3. 1-p80Rb-FLAG were constructed,plasmid was transfected into U2OS cells and immunofluorescence was performed to define the localization of p100Rband p80Rbin cells. Plasmid was also transfected into HEK 293T cells,and the total protein was extracted for Western blotting to detect the protein expression level. After overexpression of Rb,p100Rband p80Rbin HEK 293T cells,cell cycle and apoptosis were measured by flow cytometry.Results The eukaryotic expression plasmid pcDNA3. 1-p100Rb-FLAG and pcDNA3. 1-p80Rb-FLAG were constructed successfully. p100Rbwas mainly located in the nucleus while p80Rbwas mainly located in the cytoplasm,in U2OS cells. Rb,p100Rband p80Rbcould be overexpressed in HEK 293T cells. Following overexpression of Rb,p100Rbin HEK 293T cells,the percentage of G1phase of cell cycle significantly increased compared with the control group,and p100Rband p80Rbinhibited apoptosis compared with control group(P<0. 05).Conclusion Overexpression of p100rb can inhibit the apoptosis of HEK 293T cells and block the cell cycle. Overexpression of p80rb can inhibit the apoptosis of HEK 293T cells,but has no effect on cell cycle.

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Objective To explore the biocompatibility of Polydimethylsiloxane/Chlorhexidine gluconate(PDMS/CHG) composite coating on titanium surface. Methods CHG with different concentrations were covalently grafted to the surface of titanium through PDMS and divided into 5 groups: Group Ti: blank control group; Group P:PDMS coating group; Group C1:treated with PDMS and 0.2 mg/ml CHG;Group C2:treated with PDMS and 0.4 mg/ml CHG;Group C3:treated with PDMS and 0.8 mg/ml CHG;the surface morphology of titanium was observed with scanning electron microscope(SEM),and rat gingival fibroblasts(RGFs) were inoculated to the sample surface co-cultivation.Confocal laser scanning microscope(CLSM)was used to observe the cells, on the surface of titanium, morphology and the state of the cell after FITC-labeled phalloidin and AO/EB staining, besides, CCK-8 was used to detect cells proliferation. Results It could be seen by SEM that films had been formed on the surface of titanium sheets in each experimental group.Combined with elemental analysis, the films on the surfaces of C2 and C3 groups were uniform and dense; CLSM showed that the cell morphology of each experimental group was consistent with that of the titanium group, with better extension, and the results of AO/EB staining showed that there was a lot of green fluorescence in each group, and the cells were in good shape.CCK-8 results showed that there was no difference between each group(P>0.05). Conclusion Titanium surface combined with concentration of no more than 0.8 mg/ml PDMS/CHG coating has good biocompatibility.

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Objective Dendrobium polysaccharide(DOP-1-1) was prepared and its effects on the proliferation, differentiation and mineralization of pre-osteoblast MC3 T3-E1 cells were investigated. Methods A homogeneous polysaccharide(DOP-1-1) was obtained from Dendrobium officinale polysaccharide(DOP) through systematic separation and purification, and the structure of DOP-1-1 was studied by high-performance gel permeation chromatography, monosaccharide analysis, infrared spectroscopy, methylation analysis, GC-MS and NMR spectroscopy.In vitroexperiments were performed to detect the effects of DOP-1-1 on the proliferation, differentiation and mineralization of MC3 T3-E1 cells by MTT method, ALP activity determination and Alizarin Red S staining. At the same time, Western blot was used to determine the effect of DOP-1-1 on the expression of bone-related proteins(Pin1, BMP2, RUNX2) in MC3 T3-E1 cells. Results DOP-1-1 was a homogeneous polysaccharide with relative molecular weights of 3 611, which was composed of mannose, glucose and galactose. DOP-1-1 had excellent activity of promoting osteoblast proliferation in a low concentration, and the effects of 2.0 and 4.0 μmol/L of DOP-1-1 were equivalent to Positive Control 17β-estradiol(E2). Compared with the control group, E2 and DOP-1-1(4.0, 8.0 μmol/L) increased the ALP activity and mineralization rate in MC3 T3-E1 cells(P<0.01). In particular, the ALP activity and mineralization rate of DOP-1-1(8.0 μmol/L) were higher than those of the positive control E2(P<0.001). In addition, the expression of Pin1, BMP2, and RUNX2 protein in MC3 T3-E1 cells in the DOP-1-1 group was higher than that in the control group(P<0.05). Conclusion DOP-1-1 can promote the proliferation, differentiation and mineralization of MC3 T3-E1 cells in vitro, and its mechanism is related to the activation of Pin1/BMP2 signaling pathway.

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Objective To explore the potential targets, pathways and related mechanisms of artesunate(ART) in the treatment of hepatocellular carcinoma(HCC) based on network pharmacology and Bioinformatics.Methods The potential targets of ART were screened through pharmmapper database, PPI network was constructed and visual analysis was performed.The data of 424 HCC samples were downloaded from TCGA database, the data information of potential target genes were screened, and the differentially expressed target genes were analyzed.The key target genes were screened by univariate-multivariate COX regression analysis.Molecular docking software was used to conduct molecular docking between ART and key target genes.Differential expression and survival analysis of key target genes were performed using network database GEPIA2. KEGG pathway enrichment analysis was performed by R language. The effects of ART on the proliferation and migration of HCC cells were verified by MTS assay and scratch test. The expression of FABP5 in HCC cells was verified by qPCR and Western blot. Western blot was used to verify the protein changes of PI3K/AKT pathway.Results A total of 282 potential target genes were screened by network pharmacology. Finally,three key target genes were screened out. The survival analysis of FABP5 in HCC was statistically significant(P<0. 01). Molecular docking showed that ART and FABP5 had the highest binding energy. Pathway enrichment analysis showed that FABP5 was mainly enriched in PI3K/AKT signaling pathway. Cell experiments verified that ART could inhibit the expression level of FABP5,that ART could regulate the PI3K/AKT pathway,and that ART could inhibit the proliferation and migration of HCC cells.Conclusion FABP5 may regulate the PI3K/AKT pathway by inhibiting FABP5 and thus affect HCC cell proliferation and migration,and FABP5may serve as a new target gene for ART therapy of HCC.

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Objective To explore the changes of tryptophan-2,3-dioxygenase 2(TDO2) expression and its mediated kynurenine(Kyn) pathway in different inflammatory stages(initial period, peak period and remission period) of adjuvant arthritis in rats. Methods A rat model of AA was established.AA rats were sacrificed at different stages of inflammation.The pathological changes of joint synovium and liver were observed by HE staining; TDO2 expression in joint synovial tissue and liver tissue was detected by immunohistochemistry; High performance liquid chromatography was used to detect tryptophan(Trp) and Kyn levels in serum and liver. Results Compared with the normal group, AA rats showed similar clinical and pathological features to RA.The expression of TDO2 in rat joint synovium and liver tissue increased during the peak period and remission period(P<0.05).Trp levels in serum of AA rats at each inflammation stage decreased, and Kyn levels increased in the initial stage(P<0.01);Trp levels in the liver tissues of rats at the peak period decreased, Kyn levels increased, and Kyn/Trp ratio increased(P<0.01). Conclusion TDO2 may play an important role in the peak period of AA,and mediate Kyn metabolism to participate in the occurrence and development of RA.

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Objective To establish the liver conditional knockout mouse model ofNod2gene infected byKlebsiella pneumoniae(K.pneumoniae),and to explore the role and mechanism ofNod2gene in the process of liver abscess caused byK.pneumoniaeinfections. Methods Nod2flox/floxmice were obtained by self-crossing ofNod2flox/+mice, andAlb-Cre+mice were hybridized withNod2flox/+to obtainNod2flox/+;Alb-Cre+mice, then the above two genotypes mice were crossed to obtain liver conditional knockout mice ofNod2gene(Nod2flox/flox;Alb-Cre+) and negative control mice in the same litter(Nod2flox/flox).The genomic DNA of mice toe was extracted and amplified by polymerase chain reaction(PCR).The genotypes of offspring were identified by agar-gel electrophoresis and the livers of mice were extracted.Real-time fluorescence quantitative PCR(RT-qPCR) and Western blot were used to verify the knockout efficiency ofNod2gene in the liver.Both experimental group and control group mice were infected withK.pneumoniae,and the survival rate and pathological changes of livers were observed at different time points, and mRNA expression levels of Tumor necrosis factor α(TNF-α),interleukin 1β(IL-1β) and C-X-C motif chemokine ligand 1(CXCL1) in the livers of experimental group and control group were detected by RT-qPCR 24 h postK.pneumoniaeinfections. Results The expression of NOD2 mRNA in the liver ofNod2flox/flox;Alb-Cre+mice decreased, and the Western blot results showed that the expression of NOD2 protein decreased.Compared with the control group, the survival rate of mice infected withK.pneumoniaein the experimental group decreased(median survival time=60.5 h,P=0.046 9) and the liver tissue showed more serious pathological damage, furthermore the mRNA expression levels of TNF-α,IL-1β and CXCL1 in the livers of experimental group were lower than those of the control group, and the difference was statistically significant(P<0.05). Conclusion NOD2 plays a protective role in the process of liver abscess induced byK.pneumoniaeinfections.

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Objective To explore the expression of chemokine CXCL1 in proliferative infantile hemangioma(IH),and to study the effect of exogenous CXCL1 on hemangioma stem cells(HemSCs). Methods Immunohistochemistry was used to explore the expression of CXCL1 in proliferative IH specimens.Primary HemSCs were isolated from IH tissues by CD133 magnetic beads.5 groups of CXCL1 with different concentrations(0,10,20,50 and 100 ng/ml) were co-cultured with HemSCs, and the effects of exogenous CXCL1 on HemSCs were studied by cell viability and migration experiments. Results CXCL1 was expressed in the interstitial tissues of proliferative IH.The overall expression of CXCL1 in proliferative IH was low, but the expression of CXCL1 in the proliferative IH interstitial tissues was higher than that of the adjacent interstitial tissues.The CXCL1 positive area rate was(0.773±0.101)% in the tumor compared with(0.268±0.081)% in the adjacent tumor, and the difference was statistically significant(t=7.843,P<0.001).Exogenous CXCL1 promoted the proliferation of HemSCs, and there were statistical differences after adding different concentrations of CXCL1 to HemSCs for 24,48,and 72 h(F=14.610,P<0.001;F=14.430,P<0.001;F=5.388,P<0.01).But the exogenous CXCL1 did not affect the migration ability of HemSCs. Conclusion The expression of CXCL1 in proliferative IH interstitial tissues is higher than that in adjacent interstitial tissues, and exogenous CXCL1 promotes the proliferation of HemSCs.

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Objective To investigate the molecular changes of lanosterol synthase(LSS) gene dysfunction and cholesterol levels that may affect brain function. Methods The differentially expressed genes in LSS knockout(+/-) mice and WT C57 BL/6 mice were analyzed by RNA-SEQ,and the signal pathways involved in the differentially expressed genes were analyzed by KEGG enrichment. The differential genes were detected by real-time quantitative PCR and the protein expression of differential genes was detected by Western blot. Results By RNA-SEQ analysis, LSS(+/-) heterozygous knockout resulted in the difference of 320 genes in the mouse brain, including 145 up-regulated genes and 175 down-regulated genes.KEGG enrichment analysis showed that these differentially expressed genes involved in neural-activated receptor and ligand pathways, hippocampal signaling pathways and glutamate synaptic pathways.Real-time quantitative PCR showed that CCKBR and Htr5 b were up-regulated, respectively. Western blot results showed that CCKBR gene was highly expressed in the total protein of all brain tissues of LSS knockout(+/-) mice. Conclusion LSS heterozygotic knockout may affect brain function and animal behavior.

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Objective The purpose of this study was to investigate the effect of different exposure sequence and interval on the post-antibiotic effect(PAE) and 24-hour time-kill produced by linezolid and fosfomycin in combination againstStaphylococcus aureus(S.aureus). Methods PAEs were evaluated by exposingS.aureusto linezolid and fosfomycin individually and simultaneously for 1 h.Further assess of PAEs with the organism exposed first to linezolid for 1 h, followed by a second 1 h fosfomycin exposure at the beginning(Tb),middle(Tm)and end(Te) of the PAE phase induced by linezolid.The next study was similar to the above, but with the sequence of the two antibiotics reversed, i.e.fosfomycin then linezolid.The 24-hour time-kill studies were performed with three exposure regimens: simultaneous exposure of the two antibiotics; first exposure to linezolid for 1 h and then combined with fosfomycin, and first exposure to fosfomycin for 1 h and then combined with linezolid to draw the sterilized curve. Results Compared with the PAE of individual antibiotics [ linezolid(1.2±0.1) h, fosfomycin(1.6±0.1) h],the PAE produced by simultaneous exposure of the combination(2.9±0.1) h presented additive interaction.However, the PAE of fosfomycin decreased or even disappeared(-0.3 h for Tb, 0 h for Tm and 0.9 h for Te) during the PAE phase of linezolid, and with a stronger antagonism at the beginning.Differently, an additive interaction was observed again when the test organism was exposed to fosfomycin first.In the 24-hour time-kill assays, the antibacterial effect of the first exposure to fosfomycin regimens was significantly stronger(difference of 1 log10)than that of the simultaneous exposure and the first exposure to linezolid, while the latter two were similar. Conclusion The pharmacodynamic behaviour of linezolid combined with fosfomycin againstS.aureuswas influenced by exposure regimen.The first exposure to fosfomycin regimens rather than simultaneous exposure may exert the optimal antibacterial activity.Therefore, the exposure regimen is one of the important factors to be considered for the clinical application of this combination.

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Objective To investigate the function and mechanism of golgi protein 73(GP73) neutralizing antibody in Methionine-choline deficient(MCD) diet-induced non-alcoholic fatty liver disease(NAFLD). Methods Total cholesterol(TC),aspartate aminotransferase(AST),triglyceride(TG) and serum alanine aminotransferase(ALT) levels were detected after MCD diet and GP73 neutralizing antibody intervention. Lipid deposition in liver tissue was observed by oil red O staining; qRT-PCR was used to detect the gene expression ofACC1,HMGR,TIMP1andTGF-βin liver tissue; Western blotting was used to detect α-SMA and SREBP1 protein expression levels in liver tissues. Results GP73 neutralizing antibody could reduce the accumulation of serum ALT and AST(P<0.001) induced by MCD diet, and increase the levels of serum TG and TC(P<0.001);The gene expression levels ofHGMR(P<0.001),TGF-β(P<0.05),ACC1(P<0.01) and the protein expression levels of α-SMA(P<0.05) and SREBP1(P<0.05) in liver tissue significantly decreased, and the lipid deposition was also improved. Conclusion GP73 neutralizing antibody inhibits the formation of MCD-induced NAFLD by slowing down the progression of liver fibrosis and reducing the deposition of liver lipids.

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Objective To prepare the gelatin methacryloyl(GelMA)/hydroxyapatite(HA) composite hydrogel scaffold by additive manufacturing(AM) technology, and to explore its feasibility as a scaffold for repairing in bone tissue engineering via characterizing microstructure and testing biocompatibility. Methods The GelMA solution and hydroxyapatite particles were uniformly mixed to prepare a bio-ink firstly.Then, AM technology was used to fabricate the GelMA/HA composite hydrogel scaffold.The microstructure and components of the composite scaffold were characterized and analyzed by scanning electron microscope(SEM) and transmission electron microscope(TEM).The Young′s modulus of pure GelMA scaffold and the composite scaffold was measured by a mechanical tester.Cell counting kit-8(CCK-8) and Live/dead cell staining were used to evaluate the cytocompatibility of scaffolds.Alkaline phosphatase(ALP) staining was used to explore the osteogenic properties of scaffolds. Results The GelMA/HA hydrogel scaffold showed a porous and lattice-like structure and exhibited stiffer than pure GelMA scaffold.The live-dead cell staining experiment exhibited that BMSCs grew well and displayed a spread morphology on the composite scaffold after 7 days of culture.Cell proliferation experiments showed that the proliferation rates of BMSCs in pure GelMA hydrogel scaffold group and the GelMA/HA composite hydrogel scaffold group cultured for 3 and 7 d were higher than those of the blank group(P<0.05).The ALP staining experiment revealed that the positive ALP stained area of the GelMA/HA composite hydrogel scaffold significantly increased(P<0.05) compared with the blank group and pure GelMA hydrogel scaffold group. Conclusion The GelMA/HA composite hydrogel scaffold processes good biocompatibility and promotes the osteogenic differentiation of BMSCs, which has a potential to be used as a filling and repair scaffold for bone tissue engineering.

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Objective To evaluate the effects of low temperature plasma modification on the surface morphology and bonding durability of zirconia ceramics.Methods 180 pieces of zirconia ceramic(Y-TZP) were randomly divided into 5 groups(n= 36). Group A:blank group; Group B:sandblast group; Group C:nitrogen( N2) plasma group; Group D:oxygen(O2) plasma group; Group E:argon(Ar) plasma group. The surface hydrophilicity and microstructure of zirconia ceramics were observed,and the changes of surface elements and functional groups were detected. Each group of bonded specimens was randomly divided into two subgroups(n= 15). After receiving 0 and10 000 cycles of heat and cold,the shear strength was measured. The measured results were statistically analyzed and the fracture mode was analyzed.Results The surface contact angles of groups B,C,D and E were significantly lower than those of group A,with statistically significant differences(P<0. 001). Compared with group A,the surface morphology of group C,D and E did not significantly change,while that of group B significantly changed. XPS showed that the surface carbon decreased,oxygen increased and carbon/oxygen ratio decreased after plasma treatment. SBS results showed that the immediate shear bonding strength of group A was lower than that of the other four groups(P<0. 001),and there was no significant difference in the immediate shear bonding strength of groups B,C,D and E(P>0. 05);After artificial aging,the shear bonding strength of group A was significantly lower than that of the other four groups,and the difference of shear bonding strength between groups A and B before and after artificial aging was statistically significant(P<0. 001).Conclusion The zirconia ceramics achieves better bonding strength and durability with resin cement through three kinds of low temperature plasmas,without changing the surface morphology.

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Objective To investigate the effects of chronic intermittent hypoxia(CIH) on depressive and anxiety-like behaviors in CIH mice model and the possible mechanisms. Methods Fourteen 8-week-old male C57 BL/6 mice were randomly divided into control group(Ctrl group) and CIH group. The depressive-like behavior was evaluated by the tail suspension test; while the basic motor function and anxiety-like behavior were evaluated by the open field test. Western blot was performed to quantitatively detect the expression of mTOR, p-mTOR, Atg7, p62, NLRP3 and NLRP1 in the hippocampus and prefrontal cortex of mice. Results Compared with the Ctrl group, CIH group mice showed reduced moving distance(t=6.377,P<0.001), moving speed(t=6.362,P<0.001), line crossing(t=6.131,P<0.001) and central time(t=9.295,P<0.001) in open field test. Besides, the immobility time(t=4.684,P<0.001) in tail suspension test was increased in CIH mice. In addition, Western blot results showed an increased NLRP3(t=15.320,P<0.001), p-mTOR(t=5.076,P<0.05) and a decreased Atg7(t=3.511,P<0.05) protein levels in hippocampus, leaving unchanged in prefrontal cortex. Conclusion CIH can lead to depressive and anxiety-like behaviors in mice through activating mTOR to inhibit autophagy and increasing NLRP3 inflammasome protein level.

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Objective To explore the effect of long-chain non-coding RNA(lncRNA) SPINT1-AS1 on the growth and invasion of ovarian cancer cells and the molecular mechanism. Methods The expression of lncRNA SPINT1-AS1 in ovarian cancer tissues and normal tissues was analyzed by GEPIA database.Fluorescence real-time quantitative polymerase chain reaction(qRT-PCR) method was used to determine the expression of lncRNA SPINT1-AS1 in ovarian cancer cell lines SKOV-3,A2780,OC3,HO-8910 and immortalized ovarian epithelial cell line IOSE80.The SKOV-3 cells were divided into si-SPINT1-AS1 group(transfected with lncRNA SPINT1-AS1 siRNA) and si-NC group(transfected with siRNA control).The effect of down-regulated lncRNA SPINT1-AS1 on the proliferation and invasion of SKOV-3 cells was determined by MTT method and Transwell chamber experiment.Western blot was used to detect the expression of proliferative phenotypic proteins(PCNA,CyclinD1) and metastatic phenotypic proteins(MMP2,MMP9).The dual luciferase reporter system was used to identify the targeting relationship between lncRNA SPINT1-AS1 and miR-211-5 p.The effect of down-regulated lncRNA SPINT1-AS1 on the expression of miR-211-5 p was detected by qRT-PCR. Results Compared with normal tissues, the expression level of lncRNA SPINT1-AS1 in ovarian cancer tissues was higher(P<0.01).Compared with IOSE80 cells, the expression level of lncRNA SPINT1-AS1 in ovarian cancer cell lines SKOV-3,A2780,OC3,HO-8910 increased(P<0.05),and the expression level of lncRNA SPINT1-AS1 in SKOV-3 cells was the highest(P<0.01).Compared with the si-NC group, the absorbance of SKOV-3 cells in the si-SPINT1-AS1 group was reduced(P<0.05),the number of invasive cells was reduced(P<0.05),and the expression of proliferation phenotype proteins PCNA and CyclinD1 were reduced(P<0.01),the expression of metastatic phenotypic proteins MMP2 and MMP9 was reduced(P<0.01).lncRNA SPINT1-AS1 could directly bind to miR-211-5 p(P<0.01).Compared with the si-NC group, the expression level of miR-211-5 p in SKOV-3 cells of the si-SPINT1-AS1 group increased(P<0.01). Conclusion The expression of lncRNA SPINT1-AS1 in ovarian cancer tissues and cell lines increased.Down-regulation of lncRNA SPINT1-AS1 can inhibit the proliferation and invasion of ovarian cancer SKOV-3 cells by targeting up-regulation of miR-211-5 p.

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Objective To study the effect of hydroquinone on autophagy of human L-02 hepatocytes.Methods L-02 cells were treated with different concentrations of hydroquinone(0,10,20,40 and 80 μmol/L) for 24 hours, the formation of autophagosomes was observed by transmission electron microscope;autophagosome formation was traced by mCheery-GFP-LC3B fusion protein. Western blot experiment was used to detect the expression of autophagy marker proteins LC3,Beclin-1,P62;immunofluorescence experiment was used to detect the subcellular localization and expression of autophagy proteins LC3,Beclin-1,P62.Results Under the transmission electron microscope,it was observed that the autophagosomes and lysosomes of L-02 cells increased after the action of hydroquinone. Under the fluorescence microscope,the number of GFP-LC3B punctate yellow spots increased with the increase of hydroquinone dose,while the red flake spots decreased. Western blot results showed that hydroquinone increased the ratio of LC3Ⅱ/LC3Ⅰ protein in L-02 cells,and the difference between treatment groups and the control group was statistically significant(P<0. 05). Hydroquinone had no significant effect on Beclin-1 and P62 protein expression in L-02 cells(P>0. 05). The results of immunofluorescence experiments showed that LC3 and Beclin-1 were both expressed in the nucleus and cytoplasm of L-02 cells,hydroquinone had no effect on the subcellular localization of LC3 and Beclin-1 in L-02 cells,while 40 and 80 μmol/L hydroquinone could induce the translocation of P62 in L-02 cells from the cytoplasm to the nucleus.Conclusion Hydroquinone induces the increase of autophagosomes in L-02 cells,which may be related to the obstacles to autophagosome clearance.

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Objective To compare the effect of BoneCeramic and Bio-OSS on osteogenic after PAOO and bone marrow stromal osteogenic differentiation in rats. Methods 36 8-week-old SD rats were enrolled and divided into 3 groups: corticotomy group(CO group);corticotomy+Bio-Oss implantation group(BO group);corticotomy+BoneCeramic implantation group(BC group).Immediately, a standard orthodontic spring(50 N/kg) was activated to pull the right upper first molars toward midrange movement.At 28,60 days after activation, 6 rats were randomly selected from each group and sacrificed.The mesial movement distance, palatal alveolar bone height loss(ABHL) and bone volume fraction(BVF) of the first molars were measured by micro-CT.The effects of the two materials on the osteogenic differentiation of rat bone marrow mesenchymal stem cells(BMSCs) were investigatedin vitro. Results There was no difference in tooth movement between the three groups; The ABHL value of the control group was higher than that of the experimental group at 28 and 60 days, and the BVF value was lower than that of the two groups.Meanwhile, the ABHL value of the BO group was higher than that of the BC group at 60 days, and the BVF value was lower than that of the BC group.CCK-8 showed that the two materials did not inhibit the growth of BMSCs.SEM showed that BMSCs were more distributed on the surface of BoneCeramic material.Quantitative ALP detection showed that BMSCs exhibited higher ALP activity on the surface of BoneCaremic material, and mRNA expressions of RUNX-2,OPN and OCN were higher. Conclusion BoneCeramic is more beneficial to osteogenesis after PAOO and osteogenic differentiation of BMSCs in SD rats.

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Objective To investigate the effect of hypoxia on human leukocyte antigen-G(HLA-G) and endothelial PAS domain protein-1(EPAS1) in placenta of pregnant rats and its mechanism in pathogenesis of preeclampsia. Methods Experimental grouping and modeling: SD pregnant rats were randomly divided into normoxia group and acute hypoxia group.Both groups were subdivided into different groups of 48 h, 72 h and 1 week; the third group is chronic hypoxia group lasting for 2 weeks or 3 weeks.The preeclampsia model was established by intraperitoneal injection of L-NAME solution.Tail artery blood pressure of pregnant rats was measured with a noninvasive sphygmomanometer, serum alanine aminotransferase(ALT),serum album(ALB),blood urea nitrogen(BUN),creatinine(Cr) and urinary protein in pregnant rats was detected by the automatic biochemical analyzer, HLA-G protein and EPAS1 protein expressions were detected with the immunohistochemical method in the placenta of the pregnant rats. Results (1) The mean systolic blood pressure, mean diastolic blood pressure and urinary protein on the 21 st day of pregnancy in each pre-eclampsia group were significantly higher than those in normal pregnant rats under the same conditions(P<0.05),but hypoxia had no effect on blood pressure and urinary protein;(2) There were differences in ALT and BUN among normal pregnant rats and preeclampsia rats under hypoxic environment, and the difference was statistically significant(P<0.05).In hypoxia environment, the ALT and BUN values of normal pregnant rats and pre-eclampsia rats were shown significant differences, namely, the ALT and BUN values of normal pregnancy and preeclampsia group began to rise after 72 hours, hypoxia induced them to the maximum value in 1 week, and then decreased in 3 weeks, but it was still higher than that of normoxia group(P<0.05).(3) The positive rate of HLA-G protein expression in preeclampsia group was lower than that in normal pregnancy group(P<0.05);Hypoxia also affected the positive rate of HLA-G protein expression in pregnant rats.The positive rate of HLA-G protein decreased first and then increased.(4) The positive rate of EPAS1 protein expression in preeclampsia group was significantly higher than that in normal pregnancy group(P<0.05);Hypoxia also affected the positive rate of EPAS1 protein expression in pregnant rats.The positive rate of EPAS1 protein increased first and then decreased. Conclusion The overall expression of HLA-G protein in placenta of pregnant rats decreased first and then increased under hypoxic environment.Acute hypoxia may aggravate maternal immune aggression to placenta, but after the body adapts to hypoxia, the immune relationship between mother and placenta returns to normal.The overall expression of EPAS1 protein increased first and then decreased.The response of EPAS1 to the high selective pressure of hypoxia may lead to the acclimation to high altitude hypoxia.

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Objective To investigate the effects of SETDB1 on the proliferation and apoptosis of human colon cancer cells. Methods The senescence of colon cancer cells was induced by doxorubicin, and the protein expression of SETDB1 in the senescent cells was detected by Western blot.Colon cancer cell models with low expression of SETDB1 and overexpression of SETDB1 were constructed by using shRNA(shSETDB1#1 and shSETDB1#2) of SETDB1 and overexpression plasmid, respectively.Western blot was used to detect the expression of SETDB1,the CCK8 assay was performed to detect cell proliferation while a Senescence β-Galactosidase Staining Kit was used to detect senescent cells, and Western blot was used to detect the changes of p53 and p21.The reverse experiment was carried out with p53 overexpression plasmid. Results Cell senescence evoked by doxorubicin could down-regulate the protein expression of SETDB1.After low expression of SETDB1,the proliferation ability of colonic cancer cells decreased, and the proportion of senescent cells increased from(22.00±4.35)% to(54.00±5.56)% and(53.33±4.93)%(P<0.001).After overexpression of STEDB1,the proliferation ability of colon cancer cells increased, and the ratio of senescent cells decreased from(43.33±6.11)% to(21.33±3.51)%(P<0.01).Through GSE56496 enrichment and analysis, it was found that SETDB1 was related to p53 signal pathway, silencing SETDB1 could increase the levels of p53 and p21 protein, while overexpression of SETDB1 could decrease the level of p53 and p21 protein, and overexpression of p53 reversed the decrease of cell senescence caused by overexpression of SETDB1. Conclusion SETDB1 inhibits the senescence of colon cancer cells by inhibiting p53/p21 signal pathway, which provides a potential target for individualized tumor therapy.

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Objective To investigate the changes and recovery of gut microbiota in rats after broad-spectrum antibiotic exposure and its effect on cognitive behaviors. Methods Rats were randomly divided into antibiotic(AT) group and control group.AT group was treated with antibiotic cocktail containing ampicillin, ancomycin, metronidazole and imipenem for 3 days, and control group was gavaged with the same amount of normal saline.On the 4 th day(24 h after the last antibiotic treatment) and 2 months, feces were collected for 16 S rRNA gene sequencing to detect the changes of gut microbiota; At 2 months, the cognitive behaviors were detected by operant-based delayed matching to position(DMTP) task and Y maze. Results On day 4,the gut microbiota in AT group decreased, including a decrease in microbiota diversity(P<0.000 1),a reduction ofBacteroidetes(P<0.000 1) andFirmicutes(P=0.000 1),and an increase ofProteobacteria(P<0.000 1).After discontinuation of antibiotics, the microbiota began to recover, which had no difference from control group at 2 months.And there was no significant difference in cognitive behaviors between AT group and control group. Conclusion The broad-spectrum antibiotics can decrease gut microbiota in rats.However, this decrease is temporary.The microbiota returns to normal at 2 months after discontinuation of antibiotics.And it does not affect the cognitive behaviors of rats.

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Objective 16 S rDNA sequencing technology was used to analyze the changes of intestinal flora in rats with anti-tuberculosis drug-induced liver injury(ADLI) induced by rifampicin(RFP). Methods Twenty-four male SD rats were randomly divided into control group(D0 group),RFP gavage group for 10 days(R10 group),and RFP gavage group for 28 days(R28 group).The fecal specimens of 4 rats in each group were selected by random number table method for 16 S rDNA sequencing. Results Compared with the D0 group, the α diversity and β diversity of the R10 and R28 groups both changed(allP<0.05),the abundance ofBacteroidetesincreased at the phylum level, the abundance ofFirmicutesdecreased(allP<0.05),and the abundance ofUnclassified＿f＿Prevotellaceae,Blautia,Prevotellaceae＿NK3B31＿group,Erysipelotrichaceae＿uCG-003,Fournierella,increased at the genuslevel,Lactobacillus,Romboutsia,Ruminococcaceae＿uCG-014 decreased in abundance(allP<0.05). Conclusion In the process of RFP-induced ADLI in rats, the number of intestinal flora decreased, the structure and composition changed, the beneficial bacteria decreased, and the pathogenic bacteria increased, but the diversity of the flora remained unchanged.

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Objective To observe the pathological changes of brain tissue in neonatal rats after hypoxic-ischemic brain damage(HIBD), the distribution and expression changes of endoplasmic reticulum stress(ERS) related factors includingactivated transcription factor 6(ATF6), inositol demand factor 1(IRE1) and double-stranded RNA-activated protein kinase-likeendoplasmic reticulum kinase(PERK) in brain tissue. Methods Seven-day-old newborn rats were divided into sham operation group(Sham group) and hypoxic-ischemic brain damage group(HIBD group). The modified rice method was used to establish the HIBD model. Hematoxylin-eosin(HE) staining was performed on the brain tissue at the 72 h point of modeling to observe the pathological changes of the brain, Nissl staining was used to observe the neuron damage in the cortex and hippocampus, and immunohistochemistry was used to detect the expression of ATF6, IRE1 and PERK in the cerebral cortex and hippocampus of rats in each group. The models were sacrificed at 6 h, 24 h, and 72 h respectively, Western blot was used to detect the protein expression of ATF6, IRE1, and PERK at each time point at 6 h, 24 h, and 72 h. Results 72 hours after HIBD, HE staining showed that no nerve cell injury was observed in the Sham group, while nerve cell injury was observed in the brain tissue of the HIBD group, and the main injury sites were the cerebral cortex and hippocampus. Nissl staining showed that the proportion of damaged neurons in the cortex and hippocampus of the HIBD group was higher than that in the Sham group, and the difference was statistically significant(P<0.001). Immunohistochemical staining showed that compared with the Sham group, the number of ATF6, IRE1, and PERK positive cells(brown-yellow) in the cerebral cortex and hippocampus of the HIBD group increased(P<0.001). In the HIBD group, the expression of ATF6 in the cytoplasm and nucleus of nerve cells increased, and the expression of IRE1 in the nucleus and PERK in the cytoplasm increased. The relative protein expression levels of ATF6, IRE1, and PERK at each time point in the HIBD group were higher than those in the Sham group, and the differences were statistically significant(allP<0.05). Conclusion The brain damage of neonatal rats after hypoxic-ischemic brain injury is mainly in the cerebral cortex and hippocampus, and the expression of ER stress factors in cerebral cortex and hippocampus is increased, suggesting that ER stress related factors are involved in its pathological injury.

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Objective To investigate the incidence and possible risk factors of small intestinal bacterial overgrowth(SIBO) in patients with chronic kidney disease(CKD). Methods 92 patients with CKD were selected as subjects.SIBO was assessed by lactulose hydrogen methane breath test(LHMBT),fat tissue index(FTI) was determined using multifrequency bioelectrical impedance analysis, and gastrointestinal symptoms were evaluated by gastrointestinal symptom rating scale(GSRS).According to the results of LHMBT,the patients were divided into SIBO group and non-SIBO group.The demographic data, laboratory and clinical indicators, gastrointestinal symptoms and other differences between the two groups were compared.Binomial logistic stepwise regression was used to analyze the possible risk factors of SIBO in CKD patients. Results The incidence of SIBO in these 92 patients was 47.8%.Compared with the non-SIBO group, the GSRS score of the SIBO group was higher, and the difference was statistically significant [(23.27±3.97)vs(21.13±4.39),t=2.451,P=0.016].Multivariate stepwise logistic regression analysis showed that low serum potassium(OR=0.396,95%CI0.176-0.893,P=0.025) and high FTI(OR=1.182,95%CI1.037-1.348,P=0.013) were independent risk factors for SIBO in patients with CKD. Conclusion The incidence of SIBO is high in CKD patients.Symptoms of dyspepsia are more prominent in SIBO positive patients.Low serum potassium and high FTI are independent risk factors for SIBO in patients with CKD.

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Objective To evaluate risk factors for NEC in in term infant. Methods Risk factors associated with NEC were investigated using a retrospective case-control design.43 patients with Bell's stage NEC≥Ⅱ were identified.Each case was paired with two GA-and weight-matched controls.Data were collected from medical records, including oligohydramnios, ICP,neonatal asphyxia, MAS,neonatal septicemia, CHD,neonatal shock, gestational diabetes, PROM,fetal distress, hypertensive disorders in pregnancy, and univariate/logistic regression analysis was employed. Results A total of 43 cases and 86 controls were enrolled.The demographic characteristics of the two groups were not statistically significant.The onset time of 43 cases was 2～17 d after birth and the average time was 6.3 d.The single factor analysis indicated that the two group had statistical significance in oligohydramnios, MAS,CHD,neonatal septicemia, neonatal asphyxia, gestational diabetes, neonatal shock, PROM,fetal distress and hypertensive disorders in pregnancy.Logistic regression analysis suggested that CHD(OR=4.27,P=0.029),neonatal septicemia(OR=2.77,P=0.007),Oligohydramnios(OR=2.42,P=0.010),PROM(OR=2.11,P=0.013),neonatal asphyxia(OR=2.06,P=0.031),MAS(OR=1.89,P=0.017),hypertensive disorders in pregnancy(OR=1.86,P=0.015),gestational diabetes(OR=1.75,P=0.032),neonatal shock(OR=1.65,P=0.024)and fetal distress(OR=1.35,P=0.021) were significantly associated with NEC. Conclusion Oligohydramnios, MAS,CHD,neonatal asphyxia, neonatal septicemia, neonatal shock, PROM,fetal distress, hypertensive disorders in pregnancy and gestational diabetes may be associated with an increased risk of NEC in term infant.

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Objective To investigate the correlation between co-signaling molecule B7-H3 and epidermal growth factor receptor tyrosine kinase inhibitor(EGFR-TKI) target therapy in acquired drug resistance of lung adenocarcinoma. Methods Immunohistochemical method was used to detect B7-H3 expression in 56 lung adenocarcinoma patients, and the cohort was divided into low and high B7-H3 expression groups.The proportion and occurrence time of acquired drug resistance after EGFR-TKI targeted therapy were compared between the two groups.Gefitinib-resistant EGFR mutant lung adenocarcinoma cell lines H3255 and HCC827 were induced and cultured in vitro.The wild-type cell lines were used as control, and the expression level of B7-H3 on resistant cell lines B7-H3 was analyzed by flow cytometry. Results In 56 lung adenocarcinoma patients, 31 cases and 25 cases had low and high B7-H3 expression, respectively.By the end of this study, the cases of acquired drug resistance in patients with low and high B7-H3 expression were 12 cases(38.7%) and 14 cases(56.0%)(P=0.19,χ2=1.663),and the median drug resistance time was 13.2 months and 5.1 months(P=0.004,t=3.193),respectively.Compared with wild-type H3255 and HCC827 cell lines, the expression level of drug-resistant strain B7-H3 further increased. Conclusion B7-H3 expression level is correlated with acquired drug resistance of EGFR-TKI targeted therapy in lung adenocarcinoma.

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Objective To explore the influential factors on tumor diameters in rectal cancer. Methods Cross sectional study data were collected from patients undergoing radical resection of rectal cancer.Patients were grouped by median value of tumor diameter, and the clinical pathological characteristics were compared between the two groups.The associations between tumor diameter and continuous variables were analyzed using locally weighted scatterplot smoothing(LOWESS) method.The associating factors of tumor diameter were further analyzed using generalized additive model(GAM). Results Patients with rectal cancer were divided into two groups with tumor diameter either<4 centimeters or ≥4 centimeters.Proportions of T,TNM stages differed significantly between different groups.Median carcinoembryonic antigen(CEA) in≥4 centimeters group were 4.90 ng/ml(inter-quartile range [IQR] 2.39,9.53 ng/ml),much higher than that in<4 centimeters group(2.93 ng/ml [IQR:1.78,4.90 ng/ml]),P<0.01.LOWESS analysis showed that there were non-linear relationships between tumor diameter and the following variables: age, the distance between tumor and anal verge, CEA,neutrophils, lymphocytes, platelets, neutrophil-to-lymphocyte ratio, and platelet-to-lymphocyte ratio.GAM showed that increased blood platelets(P<0.05) and later T stage(P<0.01) were significantly associated with increased tumor diameters, and increased CEA levels were also associated with larger tumors(P<0.01),but there were saturation effects. Conclusion Platelets, T stage and CEA are associated with tumor diameter in rectal cancer, indicating that the above factors should be considered when studying associated factors of tumor diameter in rectal cancer.

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Objective To detect the differences in the expression of Yes-associated protein(YAP) in non-cardia gastric cancer and normal gastric tissue and analyze the associations between single nucleotide polymorphisms(SNP) in the YAP gene andits protein expression,and to preliminarily discuss the correlationbetween YAP and the development of non-cardia gastric cancer,so as to provide new ideas for the prevention and treatment of non-cardia gastric cancer.Methods 126 casesofnon-cardia gastric cancer tissues and 104 cases of paracancerous normal tissues were collected and set as case group and control group,respectively. Immunohistochemical SP assay was used to detect the expression of YAP in non-cardia gastric cancer tissues and adjacent normal tissues. Taqman method was used for genotyping. The unconditional logistic regression method was used to calculate the odds ratio(OR) and its 95%confidence interval(CI) to detect the associations between YAP SNPs and the expression of YAP protein under Codominant,Dominant,Overdominant,Recessive,and Log-additive genetic models.Results The expression of YAP protein in non-cardia gastric cancer tissues was significantly higher than that in normal gastric tissues(P<0. 001).The expression level of YAP protein was negatively correlated with the degree of tumor differentiation(P<0. 05).There was no correlationbetween the expression level of YAP protein and gender,age,lymph node metastasis and tumor size respectively(P>0. 05). No associations were found between SNP rs11225163 and rs1820453 and the expression of YAP protein under Codominant,Dominant,Overdominant,Recessive and Log-additive genetic models.Haplotype was constructed by Haploview 4. 2 software,and the association between haplotype and YAP protein expression was analyzed.Conclusion YAP may be involved in the development of non-cardia gastric cancer.

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Objective To investigate the predictive value of serum homocysteine(HCY) level on the risk of miscarriage in polycystic ovary syndrome(PCOS) patients undergoing frozen-thawed embryo transfer(FET). Methods 536 PCOS patients and 550 non-PCOS patients with fallopian tube factors(control group) retrospectively were analyzed.The basic characteristics and pregnancy outcomes were compared between the two groups.Also, the basic characteristics between the miscarriage group and the live birth group in PCOS patients were compared.The predictive value of Hcy on the miscarriage of PCOS patients after FET treatment was analyzed. Results Compared with the control group, body mass index(BMI),testosterone(T),Hcy, fasting insulin(FINS),homeostasis model assessment of insulin resistance(HOMA-IR),total cholesterol(TC),number of retrieved oocytes and miscarriage rate increased in PCOS group, while high density lipoprotein-cholesterol(HDL-C),high-quality embryo rate and live birth rate decreased, and the differences were statistically significant(P<0.05).Among PCOS patients, Hcy, FINS and HOMA-IR increased in the miscarriage group compared with live birth group, and the differences were statistically significant(P<0.05).Hcy, FINS and HOMA-IR were independent risk factors for miscarriage in PCOS patients, and the predictive value of Hcy for miscarriage in PCOS patients was greater than FINS and HOMA-IR,and the area under ROC curve(AUC) was 0.829(95%CI=0.755-0.902,P<0.001),the cut-off value was 15.975 μmol/L with a 79.0% sensitivity and a 87.4% specificity, which indicated high predictive value for miscarriage in patients with PCOS. Conclusion Serum Hcy level significantly increased in PCOS patients, which has an important predictive value for miscarriage after FET treatment.

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Objective To explore the biomechanical characteristics of short implants with different diameters in mandibular molars with severe jaw absorption, and to provide theoretical basis for the application of short implants in mandibular molar area. Methods CBCT data of patient were imported into Mimics and 3-Matic, and the finite element model of mandibular second molar repaired with short implant with diameter of 5.5 mm or 6.5 mm and length of 7 mm was established. The data were imported into three-dimensional finite element analysis software Marc Mentat, and high or low density bone was set up. An axial or buccal load of 150 N was applied to simulate clinical situation. A total of 8 models were established. Cortical bone stress, cancellous bone strain and implant displacement were calculated and analyzed. Results The cortical bone stress was concentrated in the neck of implant, and the cancellous bone strain was mainly distributed in the apical area of implant. The maximum values of cortical bone stress, cancellous bone strain and implant displacement were higher in buccal loading model than those in axial loading model, and they were higher in low density bone model than those in high density bone model under the same conditions. When the 5.5 mm diameter implant was loaded in buccal direction and the surrounding bone density was low, the maximum strain of cancellous bone was greater than the upper limit of physiological absorption of bone tissue 3 000 μstrain. Conclusion The application of short implants in mandibular molars with insufficient vertical bone mass is a feasible restoration scheme. But for patients with low bone mineral density, large diameter short implants can be appropriately selected, and the occlusal area and the cusp inclination can be appropriately reduced, so as to reduce the lateral force and obtain the stability and success of the implant in the short and long term.