叶酸对肝星状细胞再程序化作用的研究

发布时间：2018-06-23 11:42

本文选题：原代肝星状细胞 + HSC-T6　；参考：《西南交通大学》2017年硕士论文

【摘要】：肝纤维化是肝脏损伤后的病理性修复反应,其特征是肝脏中肝星状细胞(hepatic Stellacells,HSCs)激活、增殖和细胞外基质蛋白过度沉积,本研究检测了叶酸摄取相关基因在原代HSCs活化过程中的mRNA表达,发现Folr1基因的表达在HSC活化过程中持续性增强,而Slc19a1和Slc46a1的表达则表现出下降趋势,这表明FR-α在HSCs摄取叶酸方面占据主导作用;叶酸是人体自身不能合成的必需物质,在细胞的生长、分化、损伤修复等方面起着重要作用。叶酸缺乏可影响多种细胞的增殖、凋亡和侵袭以及细胞表型改变。HSCs的活化是肝纤维化的关键,本研究采用不同浓度叶酸(Omg/L叶酸缺乏组、1 mg/L中浓度组、5mg/L高浓度组)处理HSC-T6细胞,研究叶酸对HSC-T6细胞的作用,探索叶酸能否抑制HSCs活化或者逆转已活化HSCs至静息态。首先,通过CCK-8实验,观察到叶酸缺乏可以抑制HSC-T6细胞的生长;BrdU,AO/PI双染色和流式细胞仪检测细胞凋亡实验结果显示叶酸缺乏抑制HSC-T6细胞生长是通过抑制HSC-T6细胞增殖和促进HSC-T6细胞凋亡实现的。细胞划痕实验显示:叶酸缺乏可以抑制HSC-T6细胞迁移,这表明叶酸参与调控T6细胞的表型改变。其次,通过QPCR和Western blotting实验,本研究发现叶酸缺乏作用下HSC-T6细胞内p-ERK1/2的表达显著低于中浓度组和高浓度组,而ERK1/2的表达无显著差异,这表明叶酸缺乏可通过下调ERK1/2途径中p-ER1/2的表达抑制HSC-T6细胞增殖;叶酸缺乏组中p-p53和p-p21的表达显著高于其余两组,而p53和p21的在不同浓度叶酸作用HSC-T6细胞内的表达无显著性差异。这表明叶酸缺乏通过上调p53信号通路中p-p53和p-p21的表达促进HSC-T6凋亡;叶酸缺乏组中细胞代谢相关基因a-SMA和Vimentin的表达显著低于其余两组,而E-cadherin显著高于其余两组,随后我们研究了细胞代谢途径中的关键蛋白PKM2的表达,发现叶酸缺乏组中PKM2表达同样显著低于其余两组,这表明叶酸缺乏上调上皮细胞标志物E-cadherin的表达及下调间叶细胞标志α-SMA、Vimentin和PKM2等蛋白表达而表现出MET转变。CCN蛋白因子是具信号传递功能的细胞基质蛋白,参与组织修复反应并涉及肝纤维化发生发展。本研究在制备了高纯度的大鼠原代HSCs并通过体外培养进行激活的基础上,同步定量分析了 6个CCN因子在激活过程中的表达,对CCN因子在HSCs激活过程中的作用进行更全面的评价。
[Abstract]:Hepatic fibrosis is a pathological repair response after liver injury, which is characterized by the activation, proliferation and excessive deposition of extracellular matrix proteins in the liver of hepatic stellate cells. In this study, the mRNA expression of folate uptake related genes in primary HSCs was detected. It was found that the expression of Folr1 gene continued to increase during HSC activation, while the expression of Slc19a1 and Slc46a1 decreased. This indicates that FR- 伪 plays a leading role in the uptake of folic acid by HSCs, and folic acid is an essential substance which cannot be synthesized by human body. It plays an important role in cell growth, differentiation, injury repair and so on. Folic acid deficiency can affect the proliferation, apoptosis and invasion of many kinds of cells, and the activation of HSCs is the key to hepatic fibrosis. In this study, HSC-T6 cells were treated with different concentrations of folic acid (Omg / L folic acid deficiency group) (1 mg / L medium concentration group, 5 mg / L high concentration group). To study the effect of folic acid on HSC-T6 cells and to explore whether folic acid can inhibit the activation of HSCs or reverse the activation of HSCs to resting state. First, through the CCK-8 experiment, It was observed that folic acid deficiency could inhibit the growth of HSC-T6 cells by BrdUU AOP / Pi double staining and flow cytometry. The results showed that folic acid deficiency inhibited HSC-T6 cell growth by inhibiting the proliferation of HSC-T6 cells and promoting HSC-T6 cell apoptosis. Cell scratch assay showed that folic acid deficiency could inhibit the migration of HSC-T6 cells, which suggested that folic acid was involved in regulating the phenotypic changes of T6 cells. Secondly, by QPCR and Western blotting, we found that the expression of p-ERK1 / 2 in HSC-T6 cells with folic acid deficiency was significantly lower than that in medium and high concentration groups, but there was no significant difference in ERK1 / 2 expression in HSC-T6 cells. These results suggested that folic acid deficiency could inhibit the proliferation of HSC-T6 cells by down-regulating the expression of p-ER1 / 2 in ERK1 / 2 pathway, and the expression of p-p53 and p-p21 in folic acid deficiency group was significantly higher than that in the other two groups, but the expression of p53 and p21 in HSC-T6 cells was not significantly different at different concentrations of folic acid. These results indicated that folic acid deficiency promoted HSC-T6 apoptosis by up-regulating the expression of p-p53 and p-p21 in p53 signaling pathway, and the expression of metabolism-related genes a-SMA and vimentin in folic acid deficiency group was significantly lower than that in the other two groups, but E-cadherin was significantly higher than that in the other two groups. Then we studied the expression of PKM2, a key protein in cell metabolism pathway, and found that PKM2 expression in folic acid deficiency group was significantly lower than that in the other two groups. These results suggest that folic acid is deficient in up-regulating the expression of E-cadherin and down-regulating the expression of 伪 -SMAVimentin and PKM2 proteins in mesenchymal cells, which indicates that met transformation. CCN protein factor is a cellular matrix protein with signal transduction function. Participate in tissue repair response and involve the development of liver fibrosis. On the basis of the preparation of high purity rat primary HSCs and activation in vitro, the expression of 6 CCN factors in the activation process was analyzed. The role of CCN factor in the activation of HSCs was evaluated more comprehensively.
【学位授予单位】：西南交通大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575.2

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