【作者】 刘敏；

【导师】 梅浙川；

【作者基本信息】 重庆医科大学 ， 内科学， 2017， 硕士

【摘要】 目的:探讨AQP9基因过表达后对人肝癌细胞SMMC-7721增殖的影响及其可能的机制。方法:将空载体慢病毒(LV-Luciferase)和靶向AQP9基因过表达慢病毒(LV-AQP9)分别转染人肝癌细胞SMMC-7721,并用嘌呤霉素筛选细胞,从而得到空载体稳定细胞株(SMMC-7721/LV-Luciferase)和AQP9过表达稳定细胞株(SMMC-7721/LV-AQP9),最后通过Western blotting检测AQP9在肝癌细胞内表达。实验分组:阴性对照组(NC组)为空载体稳定细胞;AQP9过表达组(AQP9组)为AQP9过表达稳定细胞。并且运用流式细胞术检测过表达AQP9基因对人肝癌细胞周期的影响;实时荧光定量PCR检测SMMC-7721/LV-Luciferase细胞和SMMC-7721/LV-AQP9细胞的周期相关调控因子mRNA水平表达情况;Western blotting技术检测细胞周期相关调控因子,PCNA,pser675β-catenin,β-catenin蛋白水平表达。同时采用免疫荧光染色观察PCNA和β-catenin在两组肝癌细胞中表达情况。两组数据比较采用了独立样本t检验。结果:Western blotting检测结果显示AQP9过表达组较阴性对照组AQP9的蛋白水平有显著升高,差异有统计学意义(P值<0.01)。通过流式细胞技术检测细胞周期,结果显示过表达AQP9基因后影响SMMC-7721细胞周期分布,具体地,SMMC-7721细胞G0-G1期细胞比例由52.24%±0.83%升高到65.68%±0.63%(P值<0.05);S期细胞比例由34.3%±0.65%降低至16.56%±0.85%(P值<0.05);G2-M期由13.46%±0.2%上升到17.4%±1.12%,差异有统计学意义(P值<0.05)。对细胞周期调控因子,PCNA,β-catenin分别进行Western blotting检测,其结果显示:与阴性对照组相比,过表达AQP9组人肝癌细胞细胞周期调控因子Cyclin D1,CDK2,CDK4表达下调,P27表达上调,差异均有统计学意义(P均值<0.05);同时细胞内PCNA,核内β-catenin,pser675-β-catenin表达均降低,差异同样有统计学意义(P值均<0.05)。同样,细胞免疫荧光染色及光密度分析结果显示,与阴性对照组相比,AQP9过表达组PCNA蛋白表达水平下调,差异有统计学意义(P<0.05)。AQP9过表达后影响β-catenin在细胞内的分布。结论:肝癌细胞中过表达AQP9基因后可抑制人肝癌细胞SMMC-7721细胞增殖,根据实验结果分析,其机制可能是AQP9基因下调细胞核内β-catenin的表达水平,进而抑制Cyclin D1蛋白表达水平,使得肝癌细胞阻滞在G1/S期。更多还原

【Abstract】 Objective: To investigate the effects of aquaporin 9 on the proliferation and its probable mechanism in hepatocellular carcinoma.Methods: SMMC-7721 cells were transfected by lentiviral vector with luciferase and lentiviral vector targeting the coding region of human AQP9 respectively,and then we selected them by puromycin to obtain both stable cells with luciferase and cells with lentiviral vector targeting the coding region of human AQP9.Further,the expression of AQP9 in SMMC-7721 cell line was tested by Western blot at protein level.There were two groups.The one is the negative control group(NC group),which was the SMMC-7721 cell transfected by empty carrier.In contrast,the other one is overexpressed AQP9 group that contained the cells transfected by lentiviral vector targeting the coding region of human AQP9(AQP9 group).Flow cytometry was used to evaluated the effects on the cell cycle;expression of cell cycle regulators at mRNA levels were measured by RT-PCR in two both group;Western blotting techniques were adopted to test the expression of protein levels of cell cycle regulators,PCNA,pser675-β-catenin andβ-catenin.Simultaneously,expression of PCNA and β-catenin were detected by immune fluorescence staining.Finally,we use t-test to compare these two sets of data.Results: By the detection through Western blotting,it showed that,compared to the negative control group,the protein level in the overexpressed AQP9 group was much higher,where there were statistically significant differences(p<0.01).By means of the flow cytometry detection,we could learn about the cell cycle,which indicated that the overexpressed gene of AQP9 affected the distribution of cell cycle of SMMC-7721.To be more concrete,the proportion of SMMC-7721 cells in G0-G1 and G2-M periods increased from 52.24% ± 0.83% to 65.68% ± 0.63% and from 13.46% ± 0.2% to 17.4% ± 1.12%,respectively,and the proportion in S period dropped from 34.3% ± 0.65% to 16.56% ± 0.85%,where there were statistically significant differences(p<0.05).Using Western blotting to detect cell cycle regulators,PCNA and β-catenin respectively,contrasted with the negative control group,it manifested that the cell cycle regulators,which contained Cyclin D1,CDK2,CDK4,had a low expression level after the overexpression of AQP9,while P27 had a high expression level.Moreover,the expression of PCNA,intra-nuclear β-catenin and pser675-β-catenin all reduced remarkably.There were statistically significant differences(p<0.05).Similarly,the experimental results which obtained from immunofluorescence staining and density analysis showed that the overexpressed AQP9 group had a lower PCNA level than negative control group,it has statistically significant differences(p<0.05).Also,overexpression of AQP9 affected the distribution of β-catenin in SMMC-7721 cells.Conclusion: Over-expression AQP9 gene can significantly inhibit SMMC-7721 cell proliferation.According to the experimental results,the mechanism is quite possibly that the gene of AQP9 may suppress the expression of β-catenin in the nucleus,which inhibit the expression of Cyclin D1.All these led to G1/S phase cell cycle arrest.更多还原