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Objective To investigate the effects of farnesyltransferase inhibitor(FTI) and geranylgeranyl transferase inhibitor(GGTI) on the proliferation of keratinocytes(KCs). Methods KCs were treated with different concentrations of FTI and GGTI. MTS analysis was used to analyze the effect of each treatment group on KCs cell proliferation. Flow cytometry was used to detect the effect of certain concentrations of FTI and GGTI on the cell cycle. Western blot was used to analyze the effects of certain concentrations of FTI and GGTI on the expressions of CyclinB1, CyclinE and P21, pAKT and pERK1/2 in each group. Results MTS showed that FTI and GGTI inhibited the proliferation of KCs in a concentration-dependent manner. The activity of KCs was the smallest in FTI(5 μmol/L) and GGTI(5 μmol/L), and the inhibition rates were 14.7% and 24.5%, respectively. The results of the flow cycle indicated that the FTI and GGTI retardation of KCs were not obvious in all phases of G1, G2 and S. Western blot results showed that FTI and GGT could down-regulate CyclinB1 and up-regulate the expression of P21 and CyclinE. At the same time, FTI and GGTI could promote the activation of P13 K-AKT signaling pathway and inhibit the activation of MAPKS-ERK1/2 signaling pathway. Conclusion FTI and GGTI have an inhibitory effect on the proliferation of primary KCs.

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Objective To evaluate the physicochemical properties of hydroxyapatite(HA)/gelatin porous scaffold and its effect on the proliferation and differentiation of rat bone mesenchymal stem cells(rBMSC). Methods HA/gelatin scaffold was fabricated by using coprecipitation method with different cycle times. And evaluated the structural/chemical characterization of the scaffolds. Cellular proliferation of rBMSC seeded on the scaffolds were examined by scanning electron microscopy(SEM), live/dead staining and CCK8, respectively. The osteogenic differentiation of rBMSC was analyzed by alkaline phosphatase(ALP) staining analysis. Results SEM showed that the surface of the composite scaffold was precipitated with rod-shaped particles and X-ray diffraction(XRD) showed that the precipitate was hydroxyapatite. Compared to 2 HA, 3 HA and gelatin groups, SEM showed that rBMSC adhered and stretched well on the 1 HA and the results of CCK8 showed that the viability and proliferation of rBMSC was enhanced in 1 HA. rBMSC seeded on 1 HA induced the ALP expression which was analyzed by ALP staining analysis, and the difference was statistically significant(P<0.05). Conclusion The fabricated 1 HA/gelatin scaffold has an excellent biocompatibility and it promotes the proliferation and differentiation of rBMSC. These results provide a promising scaffold for bone tissue engineering.

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Objective To explore the mechanism of Akt inhibitor MK-2206 in enhancing the sensitivity of pancreatic cancer cells to gemcitabine under hypoxia. Methods Hypoxic microenvironment was established using physical hypoxiain vitro. The pancreatic cancer cell lines were cultured under hypoxia conditions(oxygen concentration 1%) for 48 h, and cancer cells cultured under normoxia(oxygen concentration 21%) were used as control. Western blot was used to detect the expression changes of hypoxia inducible factor-1α(HIF-1α), p-Akt(Ser473) and Akt in pancreatic cancer cells under normoxia and hypoxia. Effect of MK-2206 on p-Akt(Ser473) of pancreatic cancer cells(MIA PaCa-2 and BxPC-3) was detected by Western blot under nomoxia and hypoxia. Effect of MK-2206 on gemcitabine sensitivity of pancreatic cancer cells(MIA PaCa-2 and BxPC-3) was investigated by CCK-8 cytotoxicity test under hypoxia. Results The results of Western blot showed that hypoxia microenvironment promoted accumulation of HIF-1α, as well as increased phosphorylation of Akt(Ser473) in pancreatic cancer cells. In addition, MK-2206 inhibited p-Akt(Ser473) in pancreatic cancer cells under normoxia and hypoxia. Cytotoxicity test results showed that the relative cell viability of co-treatment with MK-2206(1 μmol/L) and gemcitabine(10 μmol/L) group(0.549±0.001) was lower than that of co-treatment with DMSO and gemcitabine(10 μmol/L) group(0.632±0.017) in MIA PaCa-2 cells under hypoxia, and the difference was statistically significant(t=12.107,P=0.007). Furthermore, the relative cell viability of co-treatment with MK-2206(1 μmol/L) and gemcitabine(10 μmol/L) group(0.508±0.041) was lower than co-treatment with DMSO and gemcitabine(10 μmol/L) group(0.594±0.106) in BxPC-3 cells under hypoxia, and the difference was statistically significant(t=5.363,P=0.033). Conclusion MK-2206 could enhance the sensitivity of pancreatic cancer cells to gemcitabine under hypoxic microenvironment.

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Objective To investigate the roles of miR-29 c in regulating the expression of silent mating type information regulation 2 homolog-1(SIRT1) involved in atherosclerosis(AS) oxidative stress and inflammatory processes. Methods After adaptive feeding, ApoE-/-knockout mice(ApoE-/-) were fed with a high-fat diet to build the atherosclerosis model at 6 weeks of age. In addition, C57 BL/6 J mice was used as the normal control group and fed with normal feed. After 12 weeks, the expression of miR-29 c, tumor necrosis factor-α(TNF-α) mRNA, reactive oxygen species(ROS) production and nicotinamide adenine dinucleotide phosphate(NADPH) oxidase activity were detected in mouse aorta. Meanwhile, human umbilical vein endothelial cells(HUVEC) were culturedin vitro, transfected with miR-29 c mimic and antagomir respectively and stimulated with oxidized low density lipoprotein(oxLDL)(50 μg/ml). The levels of TNF-α, ROS and NADPH oxidase activity were tested in each group. Besides, the expression of SIRT1 mRNA and protein were detected in HUVEC after the stimulation of oxLDL by real time PCR and Western blot, which were also determined in miR-29 c mimic and antagomir group. Results The results showed that the expression of miR-29 c, TNF-α mRNA, ROS and NADPH oxidase activity significantly increased in AS group compared with the normal group. Under the stimulation of oxLDL, compared with the control group, the levels of TNF-α, ROS and NADPH oxidase activity in the cells decreased significantly when treated with miR-29 c antagomir. On the contrary, the levels of TNF-α, ROS and NADPH oxidase activity were significantly increased after miR-29 c mimic treatment. In addition, the expression of SIRT1 mRNA and protein were reduced in HUVEC after the stimulation of oxLDL. However, compared with the control group, there was no statistical difference in SIRT1 mRNA level in the antagomir and mimic groups, but the expression of SIRT1 protein was significant difference in the antagomir and mimic groups. The above indicated that the modulation of SIRT1 induced by miR-29 c was at the post-translational level. Conclusion miR-29 c can down-regulate the expression of SIRT1 and enhance oxidative stress and inflammatory response in the process of AS.

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Objective To investigate the inhibitory effect of microRNA(miR)-100 on breast cells and breast cancer cell proliferation. Methods Immunohistochemistry and q-PCR were used to detect expressional difference of miR-100 among clinic breast carcinoma specimen cells, normal breast epithelial MCF-10 A cells and MCF-7 cells in breast cancer. miR-100 knock-down MCF-10 A cell line and miR-100 over-expression MCF-7 cell line were established by stable transfection of lentiviral vector of miR-100 inhibitor or lentiviral vector of miR-100 mimics. The cell activity and colony formation ability of the influence of miR-100 were compared between normal breast epithelial cells and breast cancer cells. Results MiR-100 expression in normal mammary tissues and cell lines evidently surpassed those in breast cancer tissues and cells. MiR-100 stable knock-down MCF-10 A cell line and miR-100 stable over-expression MCF-7 cell line were established and tested. It showed that the proliferation of breast cells was up-regulated after inhibiting its expression. On the contrary,the up-regulation of its expression could inhibit the malignant behavior of breast cancer cells,which was manifested by the decrease of proliferation level. Conclusion The expression of miR-100 is negatively correlated with the malignant level of breast cells,and its mechanism may be related to negatively regulated cells proliferation capacity directly,suggesting that miR-100 may be a potential effective target for prevention and treatment of breast cancer.

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Objective To explore the effects ofSOX4 on the regulation of Wnt, TGF-β and other intracellular and extracellular factors on the proliferation, migration and invasion of lung cancer cells and the possible molecular mechanisms. Methods The pEX-2-SOX4 overexpression plasmid, GFP empty plasmid, shRNA interference plasmid and shNC control plasmid were constructed. The cells were transfected by liposome method, and cell biology experiments were used to verify the effects of overexpression and silencing ofSOX4 gene on cell proliferation, migration and invasion ability and the changes of EMT-related genes in transcription and translation. The transcription level of key factors of EMT-related pathways in the target cells was verified by PCR to speculate on the possible mechanism ofSOX4-mediated EMT on cell proliferation, migration and invasion. Results Cell function experiments confirmed thatSOX4 can promote the proliferation, migration and invasion of lung cancer cells, accompanied by the increase in the expression of EMT positive regulators N-Cad, FN, Vim, Snail, ADAM28 and β-catenin. EMT inhibition was down-regulated by E-Cad expression, but the over expression ofSOX4 in 16 HBE and SK-MES-1 cells mainly showed the up-regulation of EMT inhibitors Smad7, TJP1(ZO-1), TJP2(ZO-2) and WIF1. Whereas in the silent group, the level of factor expression described above was opposite to that of the pEX-2-SOX4 group. Most of these factors directly or indirectly participate in the regulation of lung cancer EMT through Wnt and TGF-β. Conclusion SOX4 can mediate the occurrence of EMT in tumor cells through the signaling pathway represented by Wnt and TGF-β, and promote the proliferation, migration and invasion of lung cancer.

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Objective To investigate the effect of SHP-2 shRNA combined with matrine on proliferation of Hela by contrsucting the lentiviral RNA interference vector of SHP-2 gene with interference technology. Methods The lentiviral vector of shRNA sequence targeting SHP-2 was constructed and transfected into human cervical cancer Hela cells to silence SHP-2 gene. The expression of SHP-2 mRNA and protein in transfected Hela cells was detected by Real-time PCR and Western blot respectively,and the effect of SHP-2 gene silencing associated with matrine on cell proliferation was detected by CCK8. Results The interference sequences of three groups of SHP-2 genes( SHP-2 shRNA1,SHP-2 shRNA2 and SHP-2 shRNA3) and the control sequence( NS) were designed and successfully constructed,and RT-PCR showed that the one with best interference effect was the SHP-2 shRNA2 sequence. After screening stable cells,fluorescence microscopy showed that the infection efficiency was high. Western blot demonstrated that the expression of SHP-2 in interference group was lower than that in control group. CCK-8 assay showed that the proliferation of Hela cells in the combined group was slower than that in the control group with time longer( P<0. 05). Conclusion A cervical cancer cell model with stable SHP-2 down-regulation was successfully established,and SHP-2 gene silencing combined with matrine can affect cervical cancer cells proliferation.

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Objective To investigate the changes of vascular endothelial growth factor(VEGF) and epithelial kinase A2(EphA2) in human umbilical vein endothelial cells(HUVECs) apoptosis induced by brucine. Methods HUVECs cultured in conventional culture,which were divided into blank control group, treatment group with low brucine(10 μmol/L), medium(20 μmol/L) and high(40 μmol/L). HUVECs cell activity was detected by CCK-8 assay, migration of HUVECs was detected by scratch assay, and apoptosis of HUVECs cells was detected by immunofluorescence of Hoechst 33258 and flow cytometry with Annexin V/PI double staining. The expression of VEGF and EphA2 in HUVECs cells was detected by Western blot. Results Brucine could inhibit HUVECs proliferation in a concentration-dependent manner. Scratch test showed that brucine could inhibit HUVECs migration. Immunofluorescence and flow cytometry showed that brucine promoted HUVECs apoptosis in a concentration-dependent manner, and Western blot results showed that brucine down-regulated protein expressions of VEGF and EphA2 in HUVECs in a concentration-dependent manner. Conclusion Brucine can inhibit the proliferation and migration of HUVECs, and induce apoptosis, which may be related to the down-regulated expression of VEGF and EphA2.

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Objective To explore the immunologic signature genes(ISGs) associated with cisplatin resistance in gastric cancer based on gene set enrichment analysis(GSEA). Methods GSE94714 dataset from GEO database was used, and the differentially expressed genes(DEGs) were analyzed with GEO2 R. The effects of conditional gene screening on gene number were observed. GSEA included all DEGs from gastric cancer cells in drug resistant and non-resistant groups. The DEGs were compared with the molecular signatures database(MSigDB), and the obtained ISGs were screened for intersection, and the effects of ISGs on the prognosis of gastric cancer were analyzed by Kaplan Meier Plotter method. Results A total of 34 183 DEGs included 12 452 up-regulated and 17 381 down-regulated genes. The increased fold change(FC) added the number of excluded genes. Six entries with the top normalized enrichment score(NES) were identified by GSEA(P<0.01). The intersection ISGs included mitochondrial ribosomal protein L 12(MRPL12), proline-rich protein 13(PRR13), coactosin like F-actin binding protein 1(COTL1), poly(RC) binding protein 1(PCBP1), iduronate 2-sulfatase(IDS), LIM domain containing 2(LIMD2), purine rich element binding protein A(PURA), small optic lobes homolog(SOLH), CCR4-NOT transcription complex subunit 3(CNOT3), transforming growth factor beta 1(TGFβ1), diacylglycerol kinase zeta(DGKZ), and docking protein 2(DOK2). Twelve ISGs were associated with the overall survival time of gastric cancer, all of which were statistically significant(P<0.05). Conclusion The GSEA method can effectively extract the ISGs of cisplatin resistance in gastric cancer. Newly discovered ISGs, as potential targets, can enhance the study of chemoresistant mechanisms in gastric cancer.

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Objective To investigate the changes of histone 3 lysine 9(H3 K9), methylated transferase(SUV39 H1) and glial fibrin acidic protein(GFAP), an active marker of astrocytes, in focal cerebral ischemia reperfusion injury. Methods The left middle cerebral artery occlusion model(MCAO model) was established by Koizumi method in the C57 BL/6 mouse model. The expressions of SUV39 H1, H3 K9 and GFAP in the cerebral ischemia reperfusion mice cerebral ischemia 2 h reperfusion 24 h group(the experimental group), the sham operation group and the blank control group were observed by behavioral analysis, slice staining, qRT-PCR, correlation analysis and Western blot. Results 2,3,5-triphenyltetrazolium chloride(TTC) staining showed that the cerebral infarct size of the experimental group was significantly higher than those of the sham operation group and the blank control group. The experimental group had obvious cerebral edema. qRT-PCR and Western blot showed that the expressions of SUV39 H1, H3 K9 and GFAP in focal cerebral ischemia-reperfusion group were significantly higher than those in the sham operation group and blank control group(P<0.05). Conclusion Focal cerebral ischemia reperfusion injury can stimulate the expression of SUV39 H1 and H3 K9. Meanwhile, the expression of GFAP also increases significantly. These results suggest that astrocyte activation may play an important role in increasing SUV39 H1 and H3 K9 expression in brain tissue of mice with focal ischemia reperfusion injury.

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Objective To study the effect of TiO2nanotubes(TNT) with differe-nt diameters on the loading of minocycline and explore its sustained release e-ffect. Methods TNT with different diameters were fabricated on the surface of titanium by anodizing at 10, 20 and 30 V, and minocycline hydrochloride(MH) was loaded on the surface to construct an antibacterial coating. The samples were characterized by electron microscopy, contact angle measurement,X-ray diffraction, Fourier transform infrared spectroscopy and UV-Visible spectrophotometer, to compare the sustained release effects. Results Anodization at 10, 20 and 30 V voltage produced 30, 70 and 120 nm nanotube coating. The 70 nm TNT had the highest drug loading and the best sustained release effect, followed by 120 nm TNT and 30 nm TNT. Conclusion The 70 nm TNT has the highest drug loading and the best sustained release effect, which is expected to improve sustained antibacterial properties of the titanium implant surface.

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Objective To explore the localization and expression of nanos1 gene in Schistosoma japonicum at different developmental stages.Methods The nanos1 gene of Schistosoma japonicum was searched in NCBI, amplified by PCR and analyzed by BLAST software. DNAMAN 6.0 and ClustalX 2.0.9 were used for multiple sequence alignment, and MAGA 5.05 was used to construct phylogenetic tree for phylogenetic analysis. Digoxin(DIG) labeled RNA probes were transcribed in vitro and used for whole mount in situ hybridization to determine the localization of nanos1 gene in Schistosoma japonicum at 3 developmental stages: 18, 24 and 42 days.Results The nanos1 gene of Schistosoma japonicum was amplified by PCR. Bioinformatics analysis showed that the highest homology was found between its coding protein and nanos2 protein of Schistosoma mansoni. RNA probes were successfully transcribed in vitro for whole mount in situ hybridization of Schistosoma japonicum at different developmental stages.Positive signals were detected in the ovaries and vitellarium of 24-day-old and 42-day-old females,and in the ovaries of 18-day-old females. No significant positive signal was observed in males at all developmental stages. Conclusion Nanos1 gene is highly expressed in the ovaries and vitellarium of 24-day-old and 42-day-old females,and in the ovaries of 18-day-old females,but no significant positive signal is observed in males at any developmental stages. The expression pattern of nanos1 gene in Schistosoma japonicum is observed successfully,which provided an important clue for studying the function of nanos1 gene.

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Objective To investigate the effect and mechanism of cordycepin(CP) on autophagy in oral squamous cell carcinoma SCC9 cell line. Methods The SCC9 blank group, CP(5 μg/ml) group, CP(10 μg/ml) group and CP(20 μg/ml) group were set in the experiment. Cell proliferation was detected by 5-ethynyl-2′-deoxyuridine(EdU) staining. Apoptosis was detected by Hoechst staining. Western blot was used to detect the expressions of Ki67, PCNA, Caspase-3, Caspase-9, Beclin 1, p62, LC3 A/B, PI3 K, p-PI3 K, AKT, p-AKT, mTOR and p-mTOR. The autophagy marker molecule LC3 Ⅱ was detected by immunofluorescence. After adding PI3 K inhibitor LY294002, cell proliferation was detected by EdU staining, apoptosis was detected by Hoechst staining, and autophagy marker molecule LC3 Ⅱ was detected by immunofluorescence. Results Compared with the SCC9 blank group, the cells in each CP treatment group showed significant inhibition of proliferation, accompanied by apoptosis, down-regulation of proliferation-related molecules, up-regulation of apoptosis-related molecules, and increased concomitant with increasing concentration of CP(P<0.01); The expression of autophagy-promoting related molecules(Beclin 1 and LC3 Ⅱ) was up-regulated, the expression of autophagy-inhibiting molecules(p62) was down-regulated, and the activity of the upstream signal molecules(PI3 K/AKT/mTOR) decreased, and the difference increased with the increase of CP concentration(P<0.01). Compared with CP treatment, LY294002 combined with CP treatment inhibited cell proliferation and promoted apoptosis and autophagy. Conclusion CP can increase the level of autophagy in oral squamous cell carcinoma, inhibit cell proliferation and promote apoptosis. The main reason may be related to the down-regulation of PI3 K/AKT/mTOR pathway.

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Objective To screen synovitis-related core differential genes and interacting microRNAs(miRNAs) in rheumatoid arthritis(RA) using bioinformatics. Methods The gene chip GSE55235 was downloaded from the GEO database, and the differentially expressed genes were screened by R software 3.5.0, and functional enrichment analysis was performed by David online database. The protein interaction network was established by using String 10.5,Cytoscape v3.6.1 and MCODE to screen out the core genes during RA development. miRNAs that interact with core genes were predicted by CyTargetLinker. Results 605 differentially expressed genes were screened, of which 314 were up-regulated and 291 were down-regulated.Their functions were mainly concentrated in the process of immune reaction and biosynthesis and binding of macromolecules. The protein interaction network contained a total of 552 nodes and 5 163 interaction edges. The first four cluster modules were listed and 10 core genes were screened: protein tyrosine phosphatase receptor type C(PTPRC), vascular endothelial growth factor(VEGF), fibronectin 1(FN1), integrin Subunit Alpha M(ITGAM), epidermal growth factor(EGFR), CD86, matrix metalloproteinase-9(MMP-9), integrin subunit Beta 2(ITGB2), TYRO protein tyrosine kinase binding protein(TYROBP) and MYC. It was predicted that 36 miRNAs could interact with three core genes by CyTargetLinker. Conclusion The selected core genes and interacting miRNAs may be potential targets for RA treatment.

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Objective To investigate the expression of sorting nexin 4(SNX4) in mouse testis and its possible role in early endosomal substance transport.Methods Real-time quantitative polymerase chain reaction(Real-timePCR) was used to detect the difference in the expression of SNX 23 family members in mouse testis. Real-time PCR was used to detect the differences in the expression of mRNA in liver, spleen, kidney, brain and testisof mice. Immunohistochemistry and immunofluorescence staining localized the localization of SNX4 and Rab5 in testis and TM4 cells. Results The relative expression of SNX 4 in the testis of mice was about( 15. 49 ± 4. 92) times that in the liver( P<0. 01). In the testis,the relative expressions of SNX1 and SNX4 in SNX family were higher.Immunohistochemical staining showed that SNX4 was localized in Sertoli cells of testis. Immunofluorescence staining showed that SNX4 and Rab family member of rat sarcoma( Ras) protein superfamily( Rab5),a marker of early endosome,were co-localized in Sertoli cell lines in testis of normal mice( TM4 cells). Conclusion SNX4 is highly expressed in Sertoli cells of mouse testis and co-localized with Rab5. The distribution of SNX4 protein suggests that it may be involved in the endosomaltransportation and sorting of Sertoli cells.

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Objective To investigate the effect and mechanism of RasGAP binding protein 2(G3 BP2) on senescence of colon cancer cells. Methods The G3 BP2-specific siRNA(siG3 BP2#1 and siG3 BP2#2) and G3 BP2 overexpressing plasmids were used to construct G3 BP2 knockdown and overexpression colon cancer cell models, and the expression of G3 BP2 was detected by Western blot. Cell proliferation was detected by CCK8 assay. The proportion of senescent cells was detected by β-galactosidase staining, the protein levels of p53 and p21 were determined by Western blot. p53 siRNA or p53 overexpressing plasmid was used to conduct repression experiments. The cell proliferation ability was detected by the above methods. Results The siG3 BP2#1和siG3 BP2#2 could down-regulate the expression of G3 BP2 while G3 BP2 overexpressing plasmid could up-regulate the expression of G3 BP2. G3 BP2 knockdown significantly decreased the proliferation of colon cancer cells and increased the proportion of senescent cells from(32.00±5.10)% to(57.33±3.68)% and(56.00±6.68)%(P<0.05). After transfected with G3 BP2 overexpressing plasmid, the proliferation of colon cancer cells increased, and the proportion of senescent cells decreased from(35.33±2.87)% to(25.00±2.94)%(P<0.05). Compared with the control group, the levels of p53 and p21 protein in G3 BP2 knockdown cells were significantly increasedwhile the expression of p53 and p21 protein in G3 BP2 overexpressed cells was significantly decreased(P<0.05); low expression of p53 reversed inhibition of tumor proliferation and promoted cellular senescence induced by down-regulation of G3 BP2, while overexpression of p53 reversed the effect of G3 BP2 up-regulation on promoting tumor proliferation and inhibiting cellular senescence. Conclusion G3 BP2 promotes cell senescence by inhibiting p53/p21 signaling pathway in colon cancer, which may provide the potential target for tumor therapy.

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Objective To study the effect of sirtuin-2(SIRT2) on proliferation and migration of human breast cancer MDA-MB-231 cells. Methods Real-time fluorescence quantitative PCR(qRT-PCR) was applied to detect SIRT2 mRNA expression in breast cancer MDA-MB-231 cells. MDA-MB-231 cells were transfected with small interference RNA(siRNA), targeting SIRT2 in the experimental group, and also with negative control sequence in the control group. The proliferation ability of MDA-MB-231 cells was detected by MTT assay and cell colony formation assay, and the migration abilityof MDA-MB-231 cells was assessed through wound healing assay. Results The expression of SIRT2 mRNA in MDA-MB-231 cells transfected with SIRT2-siRNA was lower than that in the negative control group(P<0.01). After transfection with SIRT2-siRNA, cell proliferation, clonality and migration of MDA-MB-231 cells decreased(P<0.05). Conclusion The proliferation and migration of breast cancer MDA-MB-231 cells were inhibited after SIRT2 knockdown, which suggested that SIRT2 might function as an oncogene in triple negative breast cancer cells.

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Objective To investigate the effects of liraglutide on ERp46 protein, adiponectin and oxidative stress in liver tissues of rats with non-alcoholic fatty liver disease(NAFLD), and to explore the possible mechanism of liraglutide in improving fatty liver. Methods Thirty two male rats were randomly divided into high-fat diet group(HFD,n=20) and normal diet group(ND,n=12). After NAFLD models were successfully established by high-fat diet for 12 weeks, HFD were randomly divided into liraglutide group and placebo group, and hypodermic injection of liraglutide 0.6 mg/(kg·d) and isopyknic saline respectively. Rats were all killed at the end of 28 weeks. The levels of weight, liver mass, biochemical index and inflammatory factors were measured, and liver tissues were stained with hematoxylin-eosin. Real-time PCR and Western blot were used to detect the expression of ERp46 mRNA and protein in liver tissue. Results Compared with placebo group, the expression levels of ERp46 and adiponectin in liver tissue of liraglutide group were increased, and the body weight, liver index, alanine aminotransferase, fasting insulin, insulin resistance index, cholesterol, triglyceride, low densith lipoprotein, and the liver levels of triglyceride, cholesterol, free fatty acids, tumor necrosis factor α and malonaldehyde decreased,the hepatic homogenate superoxide dismutase(SOD) increased and the degree of hepatic steatosis decreased(P<0.05). Conclusion The mechanism of liraglutide in improving non-alcoholic fatty liver may be related to the up-regulation of ERp46 protein expression and adiponectin level in liver tissue, and inhibition of endoplasmic reticulum stress and alleviation of oxidative stress in liver tissue.

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Objective To investigate the effect of interleukin-1β(IL-1β) on lung squamous cell proliferation and its mechanism. Methods CBA kit was used to detect the content of IL-1β in plasma of patients with lung squamous cell carcinoma(n=20) and healthy controls(n=20). The effect of IL-1β on lung squamous cell proliferation was evaluated by plate cloning formation experiment. The effect of IL-1β on the expression of miR-223-3 p in SK-MES-1 cells was detected by qRT-PCR. The potential downstream target genes of miR-223-3 p were verified by TargetScan software, double lucigenase reporting experiment and Western blot. CCK-8 assay was used to detect the effect of down-regulated p27 kip1 on cell proliferation. Confocal microscope was used to observe the distribution of p27 kip1 in SK-MES-1. Western blot was used to analyze the effect of IL-1β on the expression of p27 kip1 in SK-MES-1 cells. Results The median IL-1β content in plasma of patients with lung squamous cell carcinoma was 11.96(7.55, 29.20)ng/L, which was higher than the healthy control group by 2.52(1.64, 3.48)ng/L, and the difference was statistically significant(Z=-5.06,P<0.001). IL-1β can promote the cloning formation of SK-MES-1 cells in lung squamous cell carcinoma and significantly reduce the expression of miR-223-3 p in cells. miR-223-3 p directly binded to the 3′UTR of CDKN1 B(p27 kip1) and inhibited its expression. After p27 kip1 was down-regulated, SK-MES-1 cell proliferation ability was significantly reduced. p27 kip1 was mainly distributed in the cytoplasm of SK-MES-1 cells, and IL-1β can promote the expression of p27 kip1. Conclusion IL-1β promotes cell proliferation by regulating the expression of miR-223-3 p and p27 kip1 in lung squamous cell carcinoma cells.

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Objective To study the effect of human oxidized low density lipoprotein(OxLDL) on rat vascular endothelium-dependent relaxation and its mechanism. Methods Acetylcholine(ACh)-induced endothelium-dependent relaxations of rat coronary artery(CA) and mesenteric artery(MA) were examined by using examination of vascular tone; nitric oxide(NO) and H2S concentrations in supernatant of cultured human coronary artery endothelial cells(HCAECs) were measured by commercial kit; store-operated calcium entry(SOCE) in HCAECs were measured by using fluorescent dye Fura-2 Am. Results 100 mg/ml OxLDL pretreatment markedly decreased ACh-induced endothelium-dependent relaxation of rat CA and MA; pretreatment of OxLDL 100 mg/ml for 24 h significantly inhibited the production of H2S rather than NO in the HCAECs; OxLDL significantly inhibited the release of Ca2+from the store and subsequent SOCE in HCAECs. Conclusion OxLDL could damage rat vascular endothelium-mediated vasodilation, and its mechanism may be related to the inhibitions of H2S production and SOCE in endothelial cells.

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Objective To investigate the expression changes of insulin-like growth factor binding protein-3(IGFBP-3) in rat myocardial fibrosis and cardiac fibroblasts(CFs) activation and proliferation.Methods Forty SD rats were randomly divided into model group and control group. The model group rats were subcutaneously injected with isoproterenol to establish a model of myocardial tissue fibrosis. Transforming growth factor β1(TGF-β1) was used to stimulate the CFs of neonatal rats to make them proliferate and activate as a model group. The control group was CFs that were cultured normally without any stimulation. The rat myocardial tissue fibrosis model was used to detect pathological changes using HE and Masson staining, CCK-8 was used to determine the degree of CFs proliferation and activation, and qRT-PCR and Western blot were used to detect IGFBP-3 and α-smooth muscle actin(α-SMA) and type I collagen procollagen A1(COL1 A1) mRNA and protein expression.Results Compared with the control group,myocardial tissue collagen fibers proliferated and myocardial cells were disordered in the animal model group. CFs stimulated by TGF-β1 in the cell model group had significant proliferation and activation. Compared with the control group,the protein and mRNA expressions of IGFBP-3,α-SMA and COL1 A1 were significantly increased in the animal and cell model groups. Conclusion The expression of IGFBP-3 in fibrotic myocardium and CFs stimulated by TGF-β1 is significantly increased,suggesting that IGFBP-3 may promote or inhibit myocardial fibrotic lesions,which may be helpful to explore the pathological mechanism of myocardial fibrosis.

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Objective To investigate whether advanced glycation end products(AGEs) have an effect on the release of endothelial microparticle(EMP). Methods Cultured human umbilical vein endothelial cells(HUVECs) were incubated with AGEs(100, 200, 400 μg/ml) for 12 or 24 h. Pretreatment with anti-RAGE antibody or reactive oxygen species(ROS) scavenger, HUVECs were incubated with 400 μg/ml AGEs for 24 h. EMP, RAGE and ROS were assessed. Results AGEs induced EMP releasing in a dose-dependent manner, not a time-dependent manner. Meanwhile, AGEs increased the levels of RAGE and ROS. Pretreatment with anti-RAGE antibody or antioxidant treatment could reverse the release of AGEs inducing EMP. Conclusion AGEs-RAGE interaction induced EMP generation, which was mediated by oxidative stress.

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Objective To investigate the factors related to the depression of couples preparing for assisted reproduction, and to evaluate whether depression is related to the live birth rate. Methods Based on China National Birth Cohort, the center for epidemiological survey(CES-D) was used to evaluate depression among the couples undergoingin vitrofertilization and embryo transfer(IVF-ET) or intracytoplasmic sperm injection(ICSI), and live birth rate were calculated. Results A total of 424 undergoing assisted reproductive couples were enrolled in this study. There were 33.0% of the female patients with depression, and 25.9% of the male cases with depression. Education level was the influencing factor of depression in female patients. Female patients with high education level were more likely to have depression than those with low education level, and the difference was statistically significant(P<0.05). Male patients who do not drink alcohol were more likely to be depression(P<0.05). The result of logistic regression analysis showed that the depression of couples undergoing assisted reproduction was not related to live birth rate. Conclusion Higher education is a risk factor for depression of female patients. Male patients who do not drink are more prone to depression. This study does not suggest that depression of assisted reproductive couples had any influence on the live birth rate. Follow-up studies should focus on alleviating the problem of infertility treatment.

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Objective To investigate the correlation between metabolic parameters of18F-fluorodeoxyglucose(FDG) PET/CT and clinical features and tumor markers of colorectal cancer(CRC). Methods The clinical data and preoperative PET/CT data of 76 patients with CRC were retrospectively analyzed. Maximum standardized uptake value(SUVmax), mean standardized uptake value(SUVmean) and metabolic tumor value(MTV) of primary lesion were measured, and total lesion glycolysis(TLG) was calculated. Relationship between metabolic parameters and clinicopathological features and tumor markers was analyzed. Results SUVmax, SUVmean, MTV and TLG of 76 primary lesions were 13.15(9.10, 16.40), 7.50(5.20, 9.70), 16.30(9.32, 24.10) cm3and 114.75(61.45, 201.12) g, respectively. MTV and TLG of primary lesion were positively associated with the length of lesion along the intestine(ρ=0.773, 0.704,P<0.05), while SUVmaxand SUVmeanwere not significantly associated with length of lesion along the intestine(P>0.05); SUVmax, SUVmean, MTV and TLG were all positively associated with the degree of differentiation(ρ=0.258, 0.235, 0.238, 0.295,P<0.05); SUVmaxand SUVmeanwere positively associated with Kras gene mutation(ρ=0.321, 0.317,P<0.05), but MTV and TLG were not significantly associated with Kras gene mutations(P>0.05); SUVmax, SUVmean, MTV and TLG were all positively associated with T stage(ρ=0.274, 0.256, 0.444, 0.541,P<0.05). Only MTV was positively correlated with N stage(ρ=0.290,P<0.05), SUVmax, SUVmeanand TLG had no significant correlation with N stage(P>0.05); MTV and TLG were positively associated with vascular endothelial growth factor(VEGF)(ρ=0.250, 0.288,P<0.05), SUVmaxand SUVmeanwere not significantly associated with VEGF(P>0.05). None of the parameters had correlation with gender, age, M stage, carcinoembryonic antigen(CEA) and carbohydrate antigen199(CA199)(P>0.05). Conclusion Metabolic parameters of CRC primary lesion have certain correlation with some clinical features and serum tumor markers.

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Objective To investigate the role and significance of La-related protein 1(LARP1) in gallbladder cancer progression. Methods We selected 76 cases of gallbladder cancer tissue specimens underwent radical cholecystectomy, 27 cases of gallbladder adenoma tissue specimens underwent cholecystectomy, and 21 cases of gallbladder tissue specimens underwent hepatectomy and cholecystectomy for hepatic hemangioma. The expression level of LARP1 in paraffin samples was detected by immunohistochemistry, and the difference of LARP1 expression in tissue samples of three groups was statistically analyzed, as well as the relationship between LARP1 andclinicopathological data and prognosis of patients with gallbladder cancer. Results ① The average optical density of LARP1 expression in three groups of non-disease gallbladder, gallbladder adenoma and gallbladder cancer were 0.166(0.157,0.192), 0.219(0.185,0.281) and 0.294(0.217,0.368), respectively. There was significant difference in average optical density of LARP1 expression among three groups(χ2=48.076,P≤0.001). There were statistically significant differences between the gallbladder cancer group and the gallbladder adenoma group or the gallbladder non-disease group(P<0.001), and there was statistically significant difference between the gallbladder adenoma group and the gallbladder non-disease group(Z=-3.118,P=0.002). ② The expression level of LARP1 in advanced(TNM Ⅱ-ⅢB) gallbladder cancer tissues was significantly higher than that in early(TNM 0-Ⅰ) gallbladder cancer tissues(Z=-2.361,P=0.018), and the expression level of LARP1 in gallbladder cancer tissues was correlated with the histological grade of the tumor(Z=-2.849,P=0.004). ③ The complete follow-up data were obtained for 52 patients with gallbladder cancer. Further Kaplan-Meier analysis showed that the postoperative survival period of the LARP1 high-expression subgroup in the gallbladder cancer group was significantly shorter than that of the LARP1 low-expression subgroup(Log Rank=7.132,P=0.008). The survival time of progress(TNM stage Ⅱ-ⅢB) gallbladder cancer subgroup was obviously shorter than the early(TNM stage 0-Ⅰ) gallbladder subgroup(Log Rank=18.972,P<0.001). Furthermore, univariate and multivariate analyses showed that LARP1 expression level was an independent prognostic factor for patients with gallbladder cancer [HR=2.552, 95%CI(1.235,5.271),P=0.011]. Conclusion The expression of LARP1 in non-disease gallbladder, gallbladder adenoma and gallbladder cancer shows a progressive increase. The expression level of LARP1 is positively correlated with gallbladder cancer TNM stage and tissue grade. The expression level of LARP1 is correlated with the prognosis of gallbladder cancer patients.

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Objective To investigate the expression, clinical significance and correlation with cell cycle related proteins c-myc and CDC25 A of TRIM59 in non-small cell lung cancer(NSCLC). Methods 40 cases with NSCLC tissues(NSCLC group) and 40 cases with normal lung tissues(normal group) were selected and the expression levels of TRIM59 mRNA in two groups were detected by reverse-transcription polymerase chain reaction(RT-PCR). The expressions of TRIM59, c-myc,and CDC25 A in these two groups were detected by immunohistochemistry. Meanwhile, clinical data of the patients were collected to analyze the correlation between TRIM59, c-myc,and CDC25 A expression and the clinical pathologic features. The correlation among the three proteins were analyzed by spearman test. The survival curve was drawed to analyze the correlation between TRIM59 and the prognosis of NSCLC patients, and Cox regression model was established to analyze the prognostic factors of NSCLC patients. Results RT-PCR showed that the expression level of TRIM59 mRNA(0.87±0.14) in the NSCLC group was significantly higher than that in the normal group(0.16±0.05)(t=2.901,P=0.002). Immunohistochemistry showed that the expression levels of TRIM59, c-myc and CDC25 A in the NSCLC group(72.50%, 67.50%, 60.00%) were significantly higher than those in the normal group(15.00%, 10.00%, 17.50%)(allP=0.000). The expression levels of TRIM59, c-myc and CDC25 A in the NSCLC group were all correlated with the degree of lesion differentiation, TNM stage, vascular invasion, lymph node metastasis and liver metastasis(allP<0.05), but not related to age and gender(allP>0.05). Spearmen correlation analysis showed that TRIM59 was significantly correlated with c-myc and CDC25 A in the NSCLC group(r=0.451,P=0.000;r=0.421,P=0.002). Survival analysis showed that the survival time of NSCLC patients with TRIM59 positive expression was significantly shorter than that of NSCLC patients with TRIM59 negative expression(P<0.05). Cox regression model confirmed that TRIM59 expression was an independent prognostic factor of NSCLC patients. Conclusion The high expression of TRIM59 plays an important role in the occurrence and development of NSCLC, which is considered to be related to the up-regulation of cell cyclin-related proteins c-myc and CDC25 A.TRIM59 can be used as a potential biological indicator for the early diagnosis and prognosis monitoring of NSCLC.

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Objective To investigate the expression of cell division cycle protein 27 gene(cdc27) on peripheral blood mononuclear cell(PBMC) from systemic lupus erythematosus(SLE) patients and its significance. Methods The expression of cdc27 on PBMC from 32 SLE patients and 21 healthy controls(HC) was examined by reverse transcription-polymerase chain reaction(RT-PCR). The expression of cdc27 on PBMC was compared between SLE patients and HC. Correlations of cdc27 on PBMC with several clinical indexes were analyzed. We also analyzed regular treatment influence on expression of cdc27. Moreover, the receiver operating characteristic(ROC) was used to analyze the diagnostic value of cdc27. Results The expression of cdc27 on PBMC in SLE patients was significantly lower than that in HC(U=126.0,P<0.001). The expression of cdc27 on PBMC in SLE patients was negatively correlated with CRP(r s =-0.462,P=0.009),ESR(r s =-0.358,P=0.048),monocytes numbers(r s =-0.386,P=0.029). The expression of cdc27 on PBMC in SLE patients was significantly increased after effective treatment(P=0.005). The area under ROC curve(AUC), sensibility and specificity of cdc27 on PBMC between SLE patients and HC were 0.813(95%CI:0.681～0.944;P<0.001), 81.25%, 80.95%. Conclusion The expression of cdc27 on PBMC is decreased in SLE patients and correlated with inflammation and treatment. This study suggests that the cdc27 on PBMC may be a potential diagnosis biomarker of SLE patients.

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Objective To explore the effect of body mass index(BMI) on sarcopenia in patients with rheumatoid arthritis(RA). Methods 468 RA patients and 223 age and gender-matched healthy controls were selected. The BMI, skeletal muscle mass, skeletal muscle mass of the extremities, body fat percentage and protein content were measured using a bioelectrical impedance Inbody720 body composition analyzer.BMI was calculated according to weight(kg)/height(m)2and four groups were divided according to BMI,weight loss(BMI<18.5),normal(18.5≤BMI<24),overweight(24≤BMI<28)and obesity(BMI≥28). Appendicular skeletal muscle index(ASMI) was calculated according to limb skeletal muscle mass(kg)/height(m)2as a criterion for sarcopenia. Results ① The percentage of weight loss in the RA group was higher than that in the control group(12.6% vs 4.5%, χ2=11.086, P=0.001). ② The incidence of sarcopenia in the RA group was 51.1%, which was higher than that in the control group(24.2%)(χ2=44.593, P<0.000 1).③ The BMI, skeletal muscle mass, limb skeletal muscle mass and protein content of the RA group were lower than those of the control group, while the percentage of body fat was higher than that of the control group(P<0.05). ④ There was a difference in the incidence of sarcopenia between different BMI groups in the RA group(χ2=82.437, P<0.000 1). The incidence of sarcopenia in the BMI weight loss group and the normal group was higher than that in the overweight or obesity group( P<0. 000 1). The incidence of sarcopenia was the highest in the BMI weight loss group,and there was no significant difference in the incidence of sarcopenia between the overweight and obesity groups( P>0. 05).(5) The age of RA patients with sarcopenia was higher than that of RA patients without sarcopenia,and the BMI of RA patients with sarcopenia was lower than that of RA patients without sarcopenia( P<0. 05).(6) There was a statistically significant difference in the BMI between the RA with sarcopenia and RA without sarcopenia( χ2= 82. 437,P<0. 000 1). The BMI weight loss and normal percentage in the RA with sarcopenia were higher than those in RA without sarcopenia; and the BMI overweight and obesity percentage in the RA with sarcopenia were less than those in RA without sarcopenia( P<0. 01).(7) Multivariate logistic regression analysis showed that referring to the RA patients in the normal BMI group,the risk of sarcopenia in the weight loss group was increased,while the risk of sarcopenia in the overweight or obesity group was reduced; in addition,protein content was a protective factor for sarcopenia in RA patients,age and body fat percentage were risk factors for sarcopenia in patients with RA. Conclusion The incidence of sarcopenia is higher in patients with RA. BMI is a protective factor for the incidence of sarcopenia in patients with RA.

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Objective To discuss prognostic factors of exdranodal NK/T cell lymphoma from clinical indicators, immunohistochemistry and other perspectives. Methods The clinical follow-up data of 85 cases of exdranodal ENKTL patients diagnosed by pathology were collected. Clinical indicators(Ann arbor staging, performance status score, international prognostic index score, symptoms of group B, bone marrow infiltration or not, complete remission or not after the first treatment or not), peripheral blood indicators(blood routine, biochemical and immune), immunohistochemistry, and genetic testing indicators were used. Kaplan-Meier method was used for univariate analysis. Cox proportional risk model was used for multivariate analysis, and χ2test was used to analyze the two-factor correlation, so as to explore the influence of these indicators on the prognosis of exdranodal NK/T cell lymphoma. Results Among the patients in the whole group, 50 patients survived for 1 year; 27 patients survived for 3 years, and 14 patients survived for 5 years. The overall survival rates at 1 year, 3 years, and 5 years were 58.8%, 31.8%, and 16.5%. The presence or absence of symptoms in group B, Ann arbor staging, IPI score, PS score, presence or absence of bone marrow invasion, complete remission after the first treatment, hemoglobin count, platelet count, proalbumin, ferritin, aspartate transaminase, lactate dehydrogenase, and β2 microglobulin were all single factors affecting the poor prognosis of extrapolecular NK/T cell lymphoma.Complete remission after the first treatmentand PS score, platelet count were independent prognostic factors for extrapolecular NK/T cell lymphoma. Conclusion Hypoproproteinemia, high ferritin, high glutamic oxalacetic transaminase(AS) and bone marrow invasion were all associated with poor prognosis of extracellular NK/T cell lymphoma. Among these factors, the efficacy of the first treatment(with or without CR) and PS score, platelet count could be used as independent factor indicators affecting the prognosis of extracellular NK/T cell lymphoma.

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The modified tissue adherent method was used to extract and culture rat pulmonary arterial smooth muscle cells(PASMCs), and the effects of hypoxia on the proliferation of PASMCs were explored. The lungs of SD rats were separated from chest under aseptic condition. Pulmonary artery was isolated and pulmonary artery tissue was planted with the adherent method of tissue explants. The cellular morphology was observed by inverted phase contrast microscope. The purity of the cells was identified by immunofluorescence assay using α-smooth muscle actin(α-SMA). The primary in vitro cultured PASMCs were exposed to normoxic and hypoxia condition respectively, then CCK-8 assay was used to detect the proliferation of PASMCs. The primary rat PASMCs were isolated and cultured successfully, the cell growth and passage were stable. Immunofluorescence assay showed that the positive rate of α-SMA was beyond 90%. The cells grew stably, culture and purification could perform in the same time. Immunology results showed that the positive rate of α-SMA was beyond 90%. CCK-8 assay demonstrated that the proliferation of PASMCs expose to hypoxia was lower than that of normoxia. The primary culture model of rat PASMCs was built successfullyin vitro. PASMCs with appropriate inner diameter and separated from the male rats will be selected as the study objectin vitro.

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To analyze the risk factors of multi-drug-resistantAcinetobacter baumannii(MDR-Ab) for intracranial infection resistance, and to provide suggestions for clinical prevention and treatment. Case control and retrospective analysis were used to study the reported cases ofAcinetobacter baumannii(Ab) infection. Patients were divided into MDR-Ab and non-MDR-Ab groups based on drug resistance. Univariate and Logistic regression analysis were used to analyze the risk factors of intracranial drug resistance in MDR-Ab with multiple drug resistance. Univariate analysis showed thathospitalization time, ICU stay, pulmonary infection, cerebrospinal fluid(CSF) leakage, CSF drainage obstruction, antibiotics use before infection Ab and hormone use after surgery were all related risk factors of MDR-Ab intracranial infection resistance(P<0.05). Logistic multivariate analysis showed thathospitalization time, ICU stay, inpatency of CSF drainage and antibiotics use before infection with Ab were all independent risk factors for MDR-Ab intracranial infection resistance(P<0.05). For patients with long hospital stay, ICU stay, unobstructed CSF drainage,and antibiotics use before Ab infection, drug resistance was easy to occur. Therefore, in the process of clinical treatment, the hospital stay should be shortened as far as possible and the patients should be moved out of ICU in time. The drainage channel should be maintained unobstructed and antibiotics should be reasonably applied.