本文選題：原代肝星狀細(xì)胞 + HSC-T6��； 參考：《西南交通大學(xué)》2017年碩士論文

【摘要】：肝纖維化是肝臟損傷后的病理性修復(fù)反應(yīng),其特征是肝臟中肝星狀細(xì)胞(hepatic Stellacells,HSCs)激活、增殖和細(xì)胞外基質(zhì)蛋白過度沉積,本研究檢測了葉酸攝取相關(guān)基因在原代HSCs活化過程中的mRNA表達(dá),發(fā)現(xiàn)Folr1基因的表達(dá)在HSC活化過程中持續(xù)性增強(qiáng),而Slc19a1和Slc46a1的表達(dá)則表現(xiàn)出下降趨勢,這表明FR-α在HSCs攝取葉酸方面占據(jù)主導(dǎo)作用;葉酸是人體自身不能合成的必需物質(zhì),在細(xì)胞的生長、分化、損傷修復(fù)等方面起著重要作用。葉酸缺乏可影響多種細(xì)胞的增殖、凋亡和侵襲以及細(xì)胞表型改變。HSCs的活化是肝纖維化的關(guān)鍵,本研究采用不同濃度葉酸(Omg/L葉酸缺乏組、1 mg/L中濃度組、5mg/L高濃度組)處理HSC-T6細(xì)胞,研究葉酸對HSC-T6細(xì)胞的作用,探索葉酸能否抑制HSCs活化或者逆轉(zhuǎn)已活化HSCs至靜息態(tài)。首先,通過CCK-8實(shí)驗(yàn),觀察到葉酸缺乏可以抑制HSC-T6細(xì)胞的生長;BrdU,AO/PI雙染色和流式細(xì)胞儀檢測細(xì)胞凋亡實(shí)驗(yàn)結(jié)果顯示葉酸缺乏抑制HSC-T6細(xì)胞生長是通過抑制HSC-T6細(xì)胞增殖和促進(jìn)HSC-T6細(xì)胞凋亡實(shí)現(xiàn)的。細(xì)胞劃痕實(shí)驗(yàn)顯示:葉酸缺乏可以抑制HSC-T6細(xì)胞遷移,這表明葉酸參與調(diào)控T6細(xì)胞的表型改變。其次,通過QPCR和Western blotting實(shí)驗(yàn),本研究發(fā)現(xiàn)葉酸缺乏作用下HSC-T6細(xì)胞內(nèi)p-ERK1/2的表達(dá)顯著低于中濃度組和高濃度組,而ERK1/2的表達(dá)無顯著差異,這表明葉酸缺乏可通過下調(diào)ERK1/2途徑中p-ER1/2的表達(dá)抑制HSC-T6細(xì)胞增殖;葉酸缺乏組中p-p53和p-p21的表達(dá)顯著高于其余兩組,而p53和p21的在不同濃度葉酸作用HSC-T6細(xì)胞內(nèi)的表達(dá)無顯著性差異。這表明葉酸缺乏通過上調(diào)p53信號通路中p-p53和p-p21的表達(dá)促進(jìn)HSC-T6凋亡;葉酸缺乏組中細(xì)胞代謝相關(guān)基因a-SMA和Vimentin的表達(dá)顯著低于其余兩組,而E-cadherin顯著高于其余兩組,隨后我們研究了細(xì)胞代謝途徑中的關(guān)鍵蛋白PKM2的表達(dá),發(fā)現(xiàn)葉酸缺乏組中PKM2表達(dá)同樣顯著低于其余兩組,這表明葉酸缺乏上調(diào)上皮細(xì)胞標(biāo)志物E-cadherin的表達(dá)及下調(diào)間葉細(xì)胞標(biāo)志α-SMA、Vimentin和PKM2等蛋白表達(dá)而表現(xiàn)出MET轉(zhuǎn)變。CCN蛋白因子是具信號傳遞功能的細(xì)胞基質(zhì)蛋白,參與組織修復(fù)反應(yīng)并涉及肝纖維化發(fā)生發(fā)展。本研究在制備了高純度的大鼠原代HSCs并通過體外培養(yǎng)進(jìn)行激活的基礎(chǔ)上,同步定量分析了 6個(gè)CCN因子在激活過程中的表達(dá),對CCN因子在HSCs激活過程中的作用進(jìn)行更全面的評價(jià)。
[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- 偽 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 偽 -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.
【學(xué)位授予單位】：西南交通大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R575.2

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本文編號：2057007