發(fā)布時(shí)間：2018-06-11 19:09

  本文選題：Hic-5 + 細(xì)胞外基質(zhì)　； 參考：《西南醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:探討肝星狀細(xì)胞中克隆蛋白Hydrogen peroxide-inducible clone 5在細(xì)胞學(xué)層面對肝細(xì)胞增殖的作用。方法:以購買的人肝星狀細(xì)胞株LX-2及人肝細(xì)胞株HL-7702為研究對象;將實(shí)驗(yàn)分為三組:A.人肝細(xì)胞株單獨(dú)培養(yǎng)組;B.人肝星狀細(xì)胞LX-2細(xì)胞株與對照組人肝細(xì)胞HL-7702細(xì)胞株共培養(yǎng)組;C.Hic-5 si RNA人肝星狀細(xì)胞HL-7702細(xì)胞株與對照組人肝細(xì)胞LX-2細(xì)胞株共培養(yǎng)組。沉默實(shí)驗(yàn)組人肝星狀細(xì)胞株中Hic-5基因,將實(shí)驗(yàn)組B,C肝星狀細(xì)胞株與對照組A肝胞株建立體外共培養(yǎng)體系。反復(fù)試驗(yàn)選取培養(yǎng)時(shí)間節(jié)點(diǎn)0 h,24 h,48 h,72 h。采用免疫熒光方法(Immunofluorescence technique,IF)檢測Hic-5,α-SMA蛋白的表達(dá)進(jìn)行活化肝星狀細(xì)胞的鑒定及對Hic-5表達(dá)及分布的鑒定。以蛋白質(zhì)免疫印跡(Western Blot,WB)方法檢測不同時(shí)間點(diǎn)Hic-5和Collagen I(細(xì)胞外基質(zhì)成分膠原蛋白I),cyclin D1蛋白(細(xì)胞增殖周期蛋白)表達(dá)情況以檢測Hic-5基因隨培養(yǎng)時(shí)間的變化及對Collagen I合成及肝細(xì)胞增殖的影響。采用免疫組化(Immunohistochemistry,IHC)方法檢測各組既定時(shí)間點(diǎn)內(nèi)人肝細(xì)胞株HL-7702中ki 67的表達(dá)變化,采用Cell Counting Kit 8(CCK-8)方法檢驗(yàn)肝細(xì)胞株存活率并繪制細(xì)胞增殖曲線,以探究Hic-5對肝細(xì)胞增殖的影響。結(jié)果:(1)IF法測得Hic-5在培養(yǎng)的人HSCs表達(dá),且與α-SMA共表達(dá)于HSCs,隨著HSCs的活化,Hic-5的表達(dá)逐漸增強(qiáng)。(2)WB法測得在同一實(shí)驗(yàn)組內(nèi),隨著設(shè)置的時(shí)間點(diǎn)內(nèi)培養(yǎng)時(shí)間的遞增,Cyclin D1,Collagen I兩者表達(dá)水平均逐漸提升。但在不同時(shí)間點(diǎn)內(nèi)Hic-5 siRNA人肝星狀細(xì)胞LX-2細(xì)胞株與人肝細(xì)胞HL-7702細(xì)胞株共培養(yǎng)組較人肝星狀細(xì)胞株LX-2細(xì)胞株與人肝細(xì)胞HL-7702細(xì)胞株共培養(yǎng)組表達(dá)Cyclin D1的水平顯著升高,差異有統(tǒng)計(jì)學(xué)意義,Hic-5 siRNA人肝星狀細(xì)胞LX-2細(xì)胞株與人肝細(xì)胞HL-7702細(xì)胞株共培養(yǎng)組較人肝星狀細(xì)胞LX-2細(xì)胞株與人肝細(xì)胞HL-7702細(xì)胞株共培養(yǎng)組表達(dá)Collagen I的水平顯著降低,差異有統(tǒng)計(jì)學(xué)意義。(3)CCK-8法測得Hic-5 siRNA人肝星狀細(xì)胞LX-2細(xì)胞株與人肝細(xì)胞HL-7702細(xì)胞株共培養(yǎng)組增殖曲線較人肝星狀細(xì)胞LX-2細(xì)胞株與人肝細(xì)胞HL-7702細(xì)胞株共培養(yǎng)組顯著升高,差異有統(tǒng)計(jì)學(xué)意義。免疫組化法測得,同人肝星狀細(xì)胞與人肝細(xì)胞株共培養(yǎng)組比較,各組Hic-5siRNA人肝星狀細(xì)胞株與人肝細(xì)胞株共培養(yǎng)組表達(dá)Ki-67的陽性細(xì)胞數(shù)較顯著升高,差異有統(tǒng)計(jì)學(xué)意義,且隨著培養(yǎng)時(shí)間遞增,Ki-67的陽性細(xì)胞數(shù)亦呈遞增。結(jié)論:(1)培養(yǎng)的細(xì)胞為人肝星狀細(xì)胞,且Hic-5,α-SMA共表達(dá)于人HSCs,隨著HSCs的活化,Hic-5的表達(dá)逐漸增強(qiáng);(2)培養(yǎng)人肝細(xì)胞為活細(xì)胞,隨培養(yǎng)時(shí)間遞增,細(xì)胞周期蛋白Cyclin D1合成增加。(3)活化的肝星狀細(xì)胞促進(jìn)HGF分泌,且可能受到Hic-5調(diào)節(jié)。(4)Hic-5基因缺失可能減少collagenⅠ的合成或促其分解,同時(shí)Hic-5基因缺失可能促進(jìn)人肝細(xì)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 偽 -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 偽 -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 偽 -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，

本文編號：2006343