肝星状细胞中Hic-5对肝细胞增殖的实验研究

发布时间：2018-06-11 19:09

  本文选题：Hic-5 + 细胞外基质　； 参考：《西南医科大学》2017年硕士论文

【摘要】：目的:探讨肝星状细胞中克隆蛋白Hydrogen peroxide-inducible clone 5在细胞学层面对肝细胞增殖的作用。方法:以购买的人肝星状细胞株LX-2及人肝细胞株HL-7702为研究对象;将实验分为三组:A.人肝细胞株单独培养组;B.人肝星状细胞LX-2细胞株与对照组人肝细胞HL-7702细胞株共培养组;C.Hic-5 si RNA人肝星状细胞HL-7702细胞株与对照组人肝细胞LX-2细胞株共培养组。沉默实验组人肝星状细胞株中Hic-5基因,将实验组B,C肝星状细胞株与对照组A肝胞株建立体外共培养体系。反复试验选取培养时间节点0 h,24 h,48 h,72 h。采用免疫荧光方法(Immunofluorescence technique,IF)检测Hic-5,α-SMA蛋白的表达进行活化肝星状细胞的鉴定及对Hic-5表达及分布的鉴定。以蛋白质免疫印迹(Western Blot,WB)方法检测不同时间点Hic-5和Collagen I(细胞外基质成分胶原蛋白I),cyclin D1蛋白(细胞增殖周期蛋白)表达情况以检测Hic-5基因随培养时间的变化及对Collagen I合成及肝细胞增殖的影响。采用免疫组化(Immunohistochemistry,IHC)方法检测各组既定时间点内人肝细胞株HL-7702中ki 67的表达变化,采用Cell Counting Kit 8(CCK-8)方法检验肝细胞株存活率并绘制细胞增殖曲线,以探究Hic-5对肝细胞增殖的影响。结果:(1)IF法测得Hic-5在培养的人HSCs表达,且与α-SMA共表达于HSCs,随着HSCs的活化,Hic-5的表达逐渐增强。(2)WB法测得在同一实验组内,随着设置的时间点内培养时间的递增,Cyclin D1,Collagen I两者表达水平均逐渐提升。但在不同时间点内Hic-5 siRNA人肝星状细胞LX-2细胞株与人肝细胞HL-7702细胞株共培养组较人肝星状细胞株LX-2细胞株与人肝细胞HL-7702细胞株共培养组表达Cyclin D1的水平显著升高,差异有统计学意义,Hic-5 siRNA人肝星状细胞LX-2细胞株与人肝细胞HL-7702细胞株共培养组较人肝星状细胞LX-2细胞株与人肝细胞HL-7702细胞株共培养组表达Collagen I的水平显著降低,差异有统计学意义。(3)CCK-8法测得Hic-5 siRNA人肝星状细胞LX-2细胞株与人肝细胞HL-7702细胞株共培养组增殖曲线较人肝星状细胞LX-2细胞株与人肝细胞HL-7702细胞株共培养组显著升高,差异有统计学意义。免疫组化法测得,同人肝星状细胞与人肝细胞株共培养组比较,各组Hic-5siRNA人肝星状细胞株与人肝细胞株共培养组表达Ki-67的阳性细胞数较显著升高,差异有统计学意义,且随着培养时间递增,Ki-67的阳性细胞数亦呈递增。结论:(1)培养的细胞为人肝星状细胞,且Hic-5,α-SMA共表达于人HSCs,随着HSCs的活化,Hic-5的表达逐渐增强;(2)培养人肝细胞为活细胞,随培养时间递增,细胞周期蛋白Cyclin D1合成增加。(3)活化的肝星状细胞促进HGF分泌,且可能受到Hic-5调节。(4)Hic-5基因缺失可能减少collagenⅠ的合成或促其分解,同时Hic-5基因缺失可能促进人肝细胞增殖。
[Abstract]:Aim: to investigate the effects of Hydrogen peroxide-inducible clone 5, a cloned protein in hepatic stellate cells, on the proliferation of hepatocytes at cytological level. Methods: the human hepatic stellate cell line LX-2 and the human hepatic cell line HL-7702 were used as the research objects, and the experiment was divided into three groups. Human hepatocytes were cultured alone. Human hepatic stellate cell line (LX-2) and human hepatocyte HL-7702 cell line were co-cultured with HL-7702 cell line and human hepatic stellate cell line (HL-7702) were co-cultured with HL-7702 cells. The Hic-5 gene was silenced in the human hepatic stellate cell line of the experimental group, and the co-culture system was established between the experimental group BHSC cell line and the control group A hepatocyte strain in vitro. Repeated experiments were carried out to select the culture time node 0 h ~ 24 h ~ 48 h ~ 72 h. The expression of Hic-5 and 伪 -SMA proteins was detected by immunofluorescence technique and the expression and distribution of Hic-5 in activated hepatic stellate cells were identified. The expression of Hic-5 and Collagen I (extracellular matrix component collagen I) cyclin D1 protein (cyclin D1) was detected by Western blotblotWB method in order to detect the change of Hic-5 gene with culture time and the expression of Collagen I. Synthesis and effect of hepatocyte proliferation. The expression of Ki-67 in human hepatocyte line HL-7702 was detected by immunohistochemical method. Cell Counting Kit 8 (CCK-8) was used to test the survival rate of hepatocytes and draw the cell proliferation curve to explore the effect of Hic-5 on the proliferation of hepatocytes. Results the expression of Hic-5 in cultured human HSCs was detected by 1: 1 if method, and co-expressed in HSCswith 伪 -SMA. With the activation of HSCs, the expression of Hic-5 was gradually increased. The expression of Hic-5 was detected in the same experimental group. The expression level of Cyclin D _ 1 and Collagen I increased gradually with the increase of culture time. However, the expression of Cyclin D1 in Hic-5 siRNA human hepatic stellate cell LX-2 cell line and human hepatocyte HL-7702 cell line co-culture group was significantly higher than that in human hepatic stellate cell line LX-2 cell line and human hepatocyte HL-7702 cell line co-culture group at different time points. The expression of Collagen I in Hic-5 siRNA LX-2 cell line and HL-7702 cell line co-cultured group was significantly lower than that in human hepatic stellate cell LX-2 cell line and human hepatocyte HL-7702 cell line co-culture group, and the expression of Collagen I in HL-7702 cell line was significantly lower than that in human hepatic stellate cell LX-2 cell line and human hepatocyte HL-7702 cell line co-culture group. The proliferation curve of Hic-5 siRNA human hepatic stellate cell LX-2 cell line and human hepatocyte HL-7702 cell line co-cultured was significantly higher than that of human hepatic stellate cell LX-2 cell line and human hepatocyte HL-7702 cell coculture group. The difference is statistically significant. Compared with the co-culture group of human hepatic stellate cell and human hepatic cell line, the number of Ki-67 positive cells in Hic-5 siRNA co-cultured group was significantly higher than that in human hepatic cell line co-culture group, and the difference was statistically significant, the immunohistochemical method showed that the number of Ki-67 positive cells in Hic-5 siRNA co-cultured group was significantly higher than that in human hepatic cell line co-culture group. The number of Ki-67 positive cells increased with culture time. Conclusion the Hic-5 and 伪 -SMA co-expressed in human HSCs, and the expression of HSCS was increased gradually with the activation of HSCs. Hepatic stellate cells activated by cyclin D1) promoted HGF secretion, and the deletion of Hic-5 Hic-5 gene may reduce the synthesis or promote the decomposition of collagen 鈪，

本文编号：2006343