依托泊苷通过内质网应激诱导活化肝星状细胞的凋亡研究
发布时间：2018-08-20 14:27
【摘要】：背景和目的:肝纤维化是各类损伤因素累及肝脏后的一种损伤修复反应,肝纤维化甚至肝硬化能否成功逆转是目前纤维化研究领域的焦点。肝星状细胞的活化在肝纤维化形成过程中起着至关重要的作用,静息的肝星状细胞活化后获得成纤维细胞表型,大量增殖并分泌过量的胶原,从而导致细胞外基质的过度沉积,因此,清除活化的肝星状细胞成为逆转纤维化的关键。在肝纤维化逆转过程中活化的星状细胞的去向主要包括凋亡、衰老及转换为静息表型,其中诱导活化的星状细胞凋亡被证实可减弱乃至逆转纤维化。依托泊苷(etoposide,VP-16)是广泛应用的化疗药物,在诸多肿瘤中可以表达出抗癌作用,然而其对肝星状细胞及肝纤维化的逆转作用尚不明确。我们的实验旨在探索依托泊苷对人肝星状细胞株LX-2增殖、凋亡的影响及可能的作用机制。方法:不同浓度的依托泊苷处理LX-2细胞,采用CCK-8法检测细胞增殖活性,JC-1染色检测线粒体膜电位,流式细胞术检测细胞周期、细胞凋亡水平,caspase-3活性检测试剂盒检测细胞caspase-3活性,蛋白质印迹法检测相关蛋白的表达水平,逆转录-聚合酶链反应检测纤维化相关因子RNA的表达水平。依托泊苷单独或联合caspase抑制剂、JNK抑制剂预处理LX-2细胞,采用CCK-8法检测细胞活性,流式细胞术检测细胞凋亡水平,蛋白质印迹法检测相关蛋白的表达水平。将正常肝细胞株LO-2和QSG-7701处以相同浓度的依托泊苷,CCK-8法和流式细胞术检测细胞的增殖抑制率及凋亡率。结果:依托泊苷明显抑制LX-2细胞的增殖,使细胞周期停滞在G2/M期,且导致高水平的细胞凋亡。依托泊苷是通过抑制ERK通路发挥其抗增殖作用的,且其诱导的凋亡率可通过caspase抑制剂预处理得到部分逆转。经过依托泊苷处理后,内质网应激的标志性蛋白如CHOP,BIP,Caspase12,JNK的表达被激活,而JNK进一步激活促凋亡蛋白Bim和Bax,并同时抑制抗凋亡蛋白Bcl-2的活性。应用JNK抑制剂后可以逆转依托泊苷抗增殖和促凋亡的作用。依托泊苷处理后减弱α-SMA和Type I collagen的表达水平,且增加了基质金属蛋白酶(MMPs)与其抑制剂(TIMPs)的比值。更重要的是,与正常肝细胞株相比,依托泊苷对肝星状细胞有更显著的抗增殖和促凋亡效果。结论:依托泊苷通过激活内质网应激从而诱导LX-2细胞的凋亡,并通过抑制胶原合成和促进胶原降解表现出抗纤维化的作用。因此,依托泊苷可成为治疗肝纤维化一个潜在药物靶点。
[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 伪 -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.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575.2
，
本文编号：2193944
[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 伪 -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.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575.2
，
本文编号：2193944
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