重组人肝再生增强因子基因转染肝细胞系的生物学活性研究

发布时间：2018-04-21 08:20

  本文选题：肝再生增强因子 + HepG2细胞　； 参考：《中国人民解放军军医进修学院》2010年硕士论文

【摘要】： 人肝再生增强因子(human augmenter of liver regeneration, hALR)是目前已知唯一可以特异性刺激肝源性细胞增殖的细胞因子,是早期表达的肝再生增强因子之一。它能促进肝细胞增殖和肝脏再生,还能够保护肝脏、预防肝纤维化以及提高肝移植的成功率。 目的：研究人肝再生增强因子(hALR)对HepG2细胞增殖的影响,探讨其促进肝细胞增殖的机制,为生物人工肝细胞反应器提供合适的细胞材料。 方法：本研究采用转染重组质粒pcDNA3.1(-)hALR的HepG2细胞(前期研究构建),经G418筛选,在体外培养、传代后,进行Western blot免疫印迹试验及免疫荧光实验,检测细胞中hALR的表达,并与未转染重组质粒的HepG2细胞进行比较;采用蛋白免疫印迹和ELISA方法，检测两组细胞培养液中hALR的分泌情况;使用流式细胞仪检测细胞中增殖细胞相关核抗原Ki-67,了解重组质粒转染前后HepG2细胞的增殖状况;以蛋白免疫印迹试验检测两组肝细胞及培养液中转化生长因子α(TGFα)、表皮生长因子受体(EGFR)的表达情况,研究hALR是否通过影响TGFα、EGFR的表达促进肝细胞的增殖。 结果：转染重组质粒pcDNA3.1(-)hALR的HepG2细胞功能稳定,形态良好,细胞胞浆中有大量hALR表达,较未转染重组质粒的HepG2细胞为多,细胞培养液中亦有hALR分泌,且随着培养时间的延长,细胞培养液中的hALR含量迅速升高,优于未转染重组质粒的HepG2细胞;转染重组质粒的HepG2细胞中Ki-67阳性细胞计数明显高于未转染重组质粒的HepG2细胞，说明转染重组质粒使肝细胞增殖活跃。转染重组质粒pcDNA3.1(-)hALR使得肝细胞及培养液中TGFα含量升高,肝细胞中EGFR含量升高。 结论：本研究结果表明,前期研究构建的转染了hALR基因的肝细胞系能较高表达hALR,且细胞增殖活跃;根据结果推测,hALR可能通过上调TGFα及其受体EGFR的表达来促进肝细胞的再生;生物人工肝细胞反应器中应用该细胞系,在完成肝细胞的合成、代谢和解毒功能的同时,还可补充hALR、TGFα等可促进肝细胞生长的细胞因子,为研制生物活性更强的生物人工肝细胞反应器提供物质基础。
[Abstract]:Human augmenter of liver Regeneration (hALR) is the only known cytokine that can specifically stimulate the proliferation of hepatogenic cells and is one of the early expressed augmenters of liver regeneration. It can promote hepatocyte proliferation and liver regeneration, protect liver, prevent liver fibrosis and improve the success rate of liver transplantation. Aim: to study the effect of human augmenter of liver regeneration (hALR) on the proliferation of HepG2 cells and to explore the mechanism of promoting the proliferation of hepatocytes. Methods: in this study, HepG2 cells transfected with recombinant plasmid pcDNA3.1(-)hALR were used to detect the expression of hALR by Western blot immunoblotting assay and immunofluorescence assay. The secretion of hALR in the culture medium of the two groups was detected by Western blot and ELISA. The proliferative cell associated nuclear antigen Ki-67 was detected by flow cytometry to investigate the proliferation of HepG2 cells before and after transfection of recombinant plasmid. The expression of transforming growth factor- 伪 (TGF- 伪) and epidermal growth factor receptor (EGFR) in hepatocytes and culture media were detected by Western blot assay. The effect of hALR on the expression of TGF 伪 -EGFR was studied. Results: the HepG2 cells transfected with recombinant plasmid pcDNA3.1(-)hALR had stable function and good morphology. There was a large amount of hALR expression in the cytoplasm of the cells, which was more than that in the HepG2 cells without the recombinant plasmid transfection. HALR was secreted in the cell culture medium, and with the prolongation of the culture time. The hALR content in cell culture medium increased rapidly, which was superior to that in HepG2 cells without recombinant plasmid, and the number of Ki-67 positive cells in HepG2 cells transfected with recombinant plasmid was significantly higher than that in HepG2 cells without recombinant plasmid. The results showed that the proliferation of hepatocytes was active after transfection of recombinant plasmids. Transfection of recombinant plasmid pcDNA3.1(-)hALR increased the content of TGF 伪 in hepatocytes and culture medium, and the content of EGFR in hepatocytes. Conclusion: the results of this study indicate that the liver cell lines transfected with hALR gene can express hALRhighly, and the proliferation of HALR cells is active. It is speculated that HALR may promote the regeneration of hepatocytes by up-regulating the expression of TGF 伪 and its receptor EGFR. When the cell line was used in the biological artificial hepatocyte reactor, the synthesis, metabolism and detoxification function of the hepatocytes could be completed, and the cytokines such as hALRGF-a could promote the growth of hepatocytes. It provides a material basis for the development of bioactive bioartificial hepatocyte reactor.
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

【参考文献】

相关期刊论文 前10条

1 唐琳;孙航;张林;郭晖;张玲;刘杞;;阻断肝再生增强因子的表达对人肝癌细胞株HepG2增殖的抑制作用[J];癌症;2006年06期

2 王志毅;张艳;石小枫;刘杞;;肝细胞移植联合肝再生增强因子治疗大鼠急性肝功能衰竭的研究[J];第三军医大学学报;2007年10期

3 王新国;刘杞;孙航;;人肝再生增强因子对体外单个核细胞PKCα-NF-κB及IL-2的影响[J];第三军医大学学报;2009年21期

4 王明国;表皮生长因子受体[J];国外医学(分子生物学分册);2002年02期

5 林莹;佟明华;汪嵘;孔祥平;梁世中;;Na~+,K~+-ATPase调节肝再生增强因子促HepG2细胞增殖[J];广西农业生物科学;2006年01期

6 张勇,宋良文;肝再生增强因子研究进展[J];军事医学科学院院刊;2005年05期

7 石小枫;孙航;唐琳;陈学华;刘杞;;肝再生增强因子对外原性抗原引起机体免疫应答影响的研究[J];免疫学杂志;2007年06期

8 张艳;王志毅;石小枫;孙航;李红;刘杞;;同种肝细胞移植细胞排斥反应机理及肝再生增强因子的作用[J];免疫学杂志;2007年06期

9 林莹;孔祥平;梁世中;;肝再生增强因子生物功能和作用机理研究进展[J];中国生化药物杂志;2006年02期

10 章波,王清明,陈惠鹏;肝再生增强因子研究进展[J];生理科学进展;2001年01期

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