【摘要】：背景和目的:肝纖維化是各類損傷因素累及肝臟后的一種損傷修復(fù)反應(yīng),肝纖維化甚至肝硬化能否成功逆轉(zhuǎn)是目前纖維化研究領(lǐng)域的焦點(diǎn)。肝星狀細(xì)胞的活化在肝纖維化形成過程中起著至關(guān)重要的作用,靜息的肝星狀細(xì)胞活化后獲得成纖維細(xì)胞表型,大量增殖并分泌過量的膠原,從而導(dǎo)致細(xì)胞外基質(zhì)的過度沉積,因此,清除活化的肝星狀細(xì)胞成為逆轉(zhuǎn)纖維化的關(guān)鍵。在肝纖維化逆轉(zhuǎn)過程中活化的星狀細(xì)胞的去向主要包括凋亡、衰老及轉(zhuǎn)換為靜息表型,其中誘導(dǎo)活化的星狀細(xì)胞凋亡被證實(shí)可減弱乃至逆轉(zhuǎn)纖維化。依托泊苷(etoposide,VP-16)是廣泛應(yīng)用的化療藥物,在諸多腫瘤中可以表達(dá)出抗癌作用,然而其對(duì)肝星狀細(xì)胞及肝纖維化的逆轉(zhuǎn)作用尚不明確。我們的實(shí)驗(yàn)旨在探索依托泊苷對(duì)人肝星狀細(xì)胞株LX-2增殖、凋亡的影響及可能的作用機(jī)制。方法:不同濃度的依托泊苷處理LX-2細(xì)胞,采用CCK-8法檢測(cè)細(xì)胞增殖活性,JC-1染色檢測(cè)線粒體膜電位,流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期、細(xì)胞凋亡水平,caspase-3活性檢測(cè)試劑盒檢測(cè)細(xì)胞caspase-3活性,蛋白質(zhì)印跡法檢測(cè)相關(guān)蛋白的表達(dá)水平,逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)檢測(cè)纖維化相關(guān)因子RNA的表達(dá)水平。依托泊苷單獨(dú)或聯(lián)合caspase抑制劑、JNK抑制劑預(yù)處理LX-2細(xì)胞,采用CCK-8法檢測(cè)細(xì)胞活性,流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡水平,蛋白質(zhì)印跡法檢測(cè)相關(guān)蛋白的表達(dá)水平。將正常肝細(xì)胞株LO-2和QSG-7701處以相同濃度的依托泊苷,CCK-8法和流式細(xì)胞術(shù)檢測(cè)細(xì)胞的增殖抑制率及凋亡率。結(jié)果:依托泊苷明顯抑制LX-2細(xì)胞的增殖,使細(xì)胞周期停滯在G2/M期,且導(dǎo)致高水平的細(xì)胞凋亡。依托泊苷是通過抑制ERK通路發(fā)揮其抗增殖作用的,且其誘導(dǎo)的凋亡率可通過caspase抑制劑預(yù)處理得到部分逆轉(zhuǎn)。經(jīng)過依托泊苷處理后,內(nèi)質(zhì)網(wǎng)應(yīng)激的標(biāo)志性蛋白如CHOP,BIP,Caspase12,JNK的表達(dá)被激活,而JNK進(jìn)一步激活促凋亡蛋白Bim和Bax,并同時(shí)抑制抗凋亡蛋白Bcl-2的活性。應(yīng)用JNK抑制劑后可以逆轉(zhuǎn)依托泊苷抗增殖和促凋亡的作用。依托泊苷處理后減弱α-SMA和Type I collagen的表達(dá)水平,且增加了基質(zhì)金屬蛋白酶(MMPs)與其抑制劑(TIMPs)的比值。更重要的是,與正常肝細(xì)胞株相比,依托泊苷對(duì)肝星狀細(xì)胞有更顯著的抗增殖和促凋亡效果。結(jié)論:依托泊苷通過激活內(nèi)質(zhì)網(wǎng)應(yīng)激從而誘導(dǎo)LX-2細(xì)胞的凋亡,并通過抑制膠原合成和促進(jìn)膠原降解表現(xiàn)出抗纖維化的作用。因此,依托泊苷可成為治療肝纖維化一個(gè)潛在藥物靶點(diǎn)。
[Abstract]:Background and objective: liver fibrosis is a kind of damage repair response after various injury factors involve the liver. Whether liver fibrosis or even liver cirrhosis can be successfully reversed is the focus of current fibrosis research field. The activation of hepatic stellate cells plays an important role in the formation of hepatic fibrosis. After the activation of resting hepatic stellate cells, fibroblast phenotypes are obtained, and a large number of proliferation and excessive collagen secretion lead to excessive deposition of extracellular matrix. Therefore, the clearance of activated hepatic stellate cells is the key to reverse fibrosis. Apoptosis, aging and conversion to resting phenotype are the main directions of activated stellate cells in the process of hepatic fibrosis reversal, among which the induction of activated stellate cell apoptosis has been proved to attenuate or even reverse fibrosis. Etoposideside VP-16 is a widely used chemotherapeutic drug, which can express anticancer effect in many tumors. However, its reverse effect on hepatic stellate cells and hepatic fibrosis is unclear. Our experiment was to explore the effect of etoposide on the proliferation and apoptosis of human hepatic stellate cell line LX-2 and its possible mechanism. Methods: LX-2 cells were treated with etoposide at different concentrations. The proliferative activity of LX-2 cells was detected by CCK-8 assay and the mitochondrial membrane potential was detected by JC-1 staining. The cell cycle was detected by flow cytometry, and the activity of caspase-3 was detected by the assay kit of apoptosis level and caspase-3 activity. Western blotting was used to detect the expression of related protein, and reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of fibrosis related factor (RNA). The LX-2 cells were pretreated with etoposide alone or in combination with caspase inhibitor, and the cell activity was detected by CCK-8 assay, apoptosis level was detected by flow cytometry, and the expression level of related proteins was detected by Western blotting. The normal hepatocyte lines LO-2 and QSG-7701 were treated with etoposide CCK-8 method and flow cytometry to detect the cell proliferation inhibition and apoptosis rate. Results: etoposide significantly inhibited the proliferation of LX-2 cells and resulted in cell cycle arrest in G 2 / M phase and high level of apoptosis. Etoposide exerts its antiproliferative effect by inhibiting ERK pathway, and the apoptosis rate induced by etoposide can be partially reversed by pretreatment with caspase inhibitor. After treatment with etoposide, the expression of iconic protein of endoplasmic reticulum stress, such as CHOP-BIPP-Caspase12, JNK, was activated, while JNK further activated apoptosis-promoting proteins Bim and Bax, and inhibited the activity of anti-apoptotic protein Bcl-2 at the same time. The antiproliferative and apoptotic effects of etoposide could be reversed by JNK inhibitor. Etoposide reduced the expression of 偽 -SMA and Type I collagen and increased the ratio of matrix metalloproteinase (MMPs) to its inhibitor (TIMPs). More importantly, etoposide has more antiproliferative and apoptotic effects on hepatic stellate cells than normal hepatocytes. Conclusion: etoposide induces apoptosis of LX-2 cells by activating endoplasmic reticulum stress and inhibits collagen synthesis and collagen degradation. Therefore, etoposide may be a potential drug target for the treatment of liver fibrosis.
【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R575.2
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本文編號(hào)：2193944