慢病毒载体介导hTERT对肝细胞生物学的影响

发布时间：2018-12-18 05:18

【摘要】：目的：研究慢病毒介导人端粒酶逆转录酶(h-TERT)基因对人张氏肝细胞(chang liver)生物学特性的影响。方法：将表达h-TERT基因的重组慢病毒感染人肝细胞,通过杀稻瘟筛选获得阳性克隆。采用实时荧光RT-PCR方法检测转染前后chang liver肝细胞的h-TERT-mRNA基因表达水平和运用TRAP-PCR法扩增端粒重复序列以及ELISA检测转染前后chang liver肝细胞的端粒酶活性的变化。通过倒置相差显微镜动态观察转染前后肝细胞形态变化及生长增殖情况。通过MTT法观察转染前后细胞增长变化,运用流式细胞术(FCM)检测转染前后细胞周期。通过全自动生化分析仪测定转染前后细胞培养上清中ALB含量以及用RT-PCR方法检测转染前后细胞培养上清中ALB mRNA的表达。采用过碘酸希夫(periodic acid schiff, PAS)实验进行转染前后细胞糖原染色。结果：转染后的chang liver肝细胞存在h-TERT基因过表达和端粒酶活性上调。形态学观察发现h-TERT-chang liver均呈椭圆形、纺锤形或多角形,上皮样细胞贴壁生长,与转染前细胞相比无明显差异。细胞生长曲线发现h-TERT-chang liver较chang liver生长速度明显提高,进一步检测细胞周期观察到其G1期细胞数下降,G2/M期和S期的细胞数升高,P I升高,提示h-TERT-chang liver较chang liver增殖能力较强。培养上清生化指标结果表明转染后的肝细胞仍然保持了转染前肝细胞的合成分泌ALB功能,转染前后细胞PAS染色结果表明,转染后的肝细胞仍然保持转染前肝细胞的糖原合成功能。结论：利用h-TERT基因构建的h-TERT-chang liver肝细胞在体外培养条件下基本维持肝细胞原有形态,存活期显著延长,增殖能力较强；并且具有表达和分泌ALB及合成糖原的功能；为生物人工肝在临床的广泛应用提供了廉价易得且安全可靠的肝细胞源,为肝细胞移植和体外药物筛选建立了新的实验基础和生物学模型。
[Abstract]:Aim: to study the effect of human telomerase reverse transcriptase (h-TERT) gene mediated by lentivirus on the biological characteristics of human (chang liver) in Zhang's hepatocytes. Methods: the recombinant lentivirus expressing h-TERT gene was infected with human hepatocytes and positive clones were obtained by killing rice blast. The expression level of h-TERT-mRNA gene in chang liver hepatocytes before and after transfection was detected by real-time fluorescence RT-PCR, telomere repeat sequence was amplified by TRAP-PCR method, and telomerase activity of chang liver hepatocytes before and after transfection was detected by ELISA. The morphological changes and proliferation of hepatocytes before and after transfection were observed by inverted phase contrast microscope. Cell growth before and after transfection was observed by MTT, and cell cycle was detected by flow cytometry (FCM) before and after transfection. The content of ALB in supernatant of cell culture before and after transfection and the expression of ALB mRNA in supernatant of cell culture before and after transfection were detected by automatic biochemical analyzer and RT-PCR method. Cell glycogen staining was performed before and after transfection by (periodic acid schiff, PAS) assay. Results: overexpression of h-TERT gene and up-regulation of telomerase activity were observed in chang liver hepatocytes after transfection. Morphological observation showed that h-TERT-chang liver were oval-shaped, fusiform or polygonal, epithelioid cells were adherent to the wall, and there was no significant difference between the cells before transfection and those before transfection. The cell growth curve showed that the growth rate of h-TERT-chang liver was significantly higher than that of chang liver. Further cell cycle analysis showed that the number of cells in G1 phase decreased, and the number of cells in G 2 / M phase and S phase increased in G 2 / M phase and S phase. The results suggest that h-TERT-chang liver has stronger proliferative ability than chang liver. The biochemical index of culture supernatant showed that the hepatocytes still maintained the function of synthesis and secretion of ALB before and after transfection, and the PAS staining showed that before and after transfection. After transfection, the hepatocytes maintained the function of glycogen synthesis before transfection. Conclusion: the h-TERT-chang liver hepatocytes constructed by h-TERT gene have the function of expressing and secreting ALB and synthesizing glycogen. It provides a cheap, safe and reliable source of hepatocytes for the wide application of bioartificial liver in clinic, and establishes a new experimental basis and biological model for hepatocyte transplantation and drug screening in vitro.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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