线粒体通透性转换孔道在大黄酸诱导的人肝肿瘤HepG2细胞系凋亡中作用的研究

发布时间：2018-01-30 00:36

本文关键词： 线粒体通透性转换孔道大黄酸 HepG2细胞细胞凋亡　出处：《复旦大学》2014年硕士论文　论文类型：学位论文

【摘要】：肝癌是常见的恶性肿瘤,位于男性肿瘤致死率第二位,女性肿瘤致死率第三位。传统的肿瘤治疗方式效果有限,5年的生存率仅为3%-67%,因此寻找肝癌有效药物治疗方法十分必要。而中医药对改善肝癌患者症状、减少放化疗毒性、提高生存质量、延长生存期等方面恰恰发挥着独特的优势。大黄酸(4,5-二羟基蒽醌-2-羧酸)是提取自我国传统中药大黄和虎杖根茎中的有效成分,其抗癌作用机制己被大量研究证实。鉴于其对不同的肿瘤细胞(乳腺癌,宫颈癌,胃癌以及肝癌)的增殖抑制作用,推测其具有重要的临床药用价值,同时相关分子机制研究结果提示,线粒体依赖的内源性凋亡通路有可能参与大黄酸诱导的细胞凋亡。Ca2+超载,氧化应激,缺氧,细胞毒性药物均可导致线粒体通透性孔道开放与跨膜电位的消失,进而诱导细胞凋亡。研究表明：线粒体通透性转换孔道在肿瘤细胞凋亡中起关键作用。目前国内外对大黄酸的研究虽多有报道,但未见其对线粒体生理性作用与凋亡诱导作用相关性的分析,因此进一步探索大黄酸诱导人肝癌HepG2细胞的凋亡作用,并研究线粒体通透转换孔道在大黄酸诱导细胞凋亡中的地位,具有极为重要药理学意义。本研究采用HepG2细胞系及大鼠肝脏离体线粒体作为实验模型,通过测定HepG2细胞系线粒体膜电位及ATP水平变化、线粒体肿胀程度、Ca2+外泄程度分析大黄酸诱导HepG2细胞线粒体损伤作用及机制,采用流式细胞仪、Westernblot、荧光检测以及TUNEL检测技术,系统研究线粒体通透性转换孔道在大黄酸诱导肝癌HepG2细胞凋亡过程中的重要作用机制。结果表明：大黄酸可导致线粒体功能障碍、肿胀以及Ca2+外泄,这些作用呈剂量依赖性,并且可以被线粒体通透性转换孔道抑制剂CsA完全抑制,提示大黄酸可直接作用于线粒体,导致线粒体通透性转换孔道开放以及线粒体功能障碍；大黄酸诱导的HepG2细胞凋亡作用可被CsA所阻断,提示线粒体通透性转换孔道在大黄酸介导肝癌细胞株凋亡中起着重要作用。综上,本研究在国内外率先阐明线粒体通透性转换孔道在大黄酸诱导的HepG2细胞凋亡中起着关键性作用,HepG2细胞凋亡与线粒体依赖的内源性凋亡通路有关。从而为大黄酸的辅助肿瘤治疗提供细胞分子生物学理论基础,为发现大黄酸辅助肝癌治疗的新靶标和寻找肿瘤辅助治疗同类新药提供生物学信息。
[Abstract]:Hepatocellular carcinoma (HCC) is a common malignant tumor, which is the second leading cause of tumor mortality in males and the third in females. The traditional tumor therapy has a limited effect, and the 5-year survival rate is only 3-67%. Therefore, it is necessary to find effective drug therapy for liver cancer. Traditional Chinese medicine can improve the symptoms of patients with liver cancer, reduce the toxicity of radiotherapy and chemotherapy, and improve the quality of life. The prolongation of survival time and other aspects play a unique advantage. Rhein 4- 5- dihydroxyanthraquinone-2-carboxylic acid is extracted from the traditional Chinese traditional Chinese medicine rhubarb and Polygonum cuspidatum rhizome active components. Its anti-cancer mechanism has been confirmed by a large number of studies. In view of its different tumor cells (breast cancer, cervical cancer, gastric cancer and liver cancer) proliferation inhibition, it is assumed that it has important clinical medicinal value. At the same time, the related molecular mechanism suggests that mitochondrial dependent endogenous apoptosis pathway may be involved in the apoptosis induced by Rhein, Ca 2 overload, oxidative stress, hypoxia. Cytotoxic drugs can lead to the opening of mitochondrial permeability channels and the disappearance of transmembrane potential. Studies show that mitochondrial permeability transition pore plays a key role in apoptosis of tumor cells. There are many reports about Rhein at home and abroad. However, there was no correlation between mitochondrial physiological and apoptosis-inducing effects, so the apoptosis of human hepatoma HepG2 cells induced by Rhein was further explored. It is of great pharmacological significance to study the role of mitochondrial permeability transition pore in the apoptosis induced by Rhein. In this study, HepG2 cell line and isolated rat liver mitochondria were used as the experimental model. The mitochondrial membrane potential and the level of ATP in HepG2 cell line were measured. The extent of mitochondrial swelling and the extent of Ca 2 release were analyzed to analyze the mechanism of mitochondrial damage induced by Rhein in HepG2 cells. Flow cytometry, fluorescence detection and TUNEL detection were used. The mechanism of mitochondrial permeability transition pore in the apoptosis of HepG2 cells induced by Rhein was systematically studied. The results showed that Rhein could lead to mitochondrial dysfunction. Swelling and Ca2 leakage, these effects are dose-dependent, and can be completely inhibited by mitochondrial permeability conversion pore inhibitor CsA, suggesting that Rhein can act directly on mitochondria. It leads to the opening of mitochondrial permeability transition channels and mitochondrial dysfunction. Rhein induced apoptosis of HepG2 cells can be blocked by CsA, suggesting that mitochondrial permeability transition pore plays an important role in Rhein mediated apoptosis of hepatoma cells. This study was the first to clarify that mitochondrial permeability transition channels play a key role in the apoptosis of HepG2 cells induced by Rhein. Apoptosis of HepG2 cells is related to mitochondrial dependent endogenous apoptotic pathway, which provides a theoretical basis of cellular molecular biology for the adjuvant tumor therapy of Rhein. It provides biological information for the discovery of new targets for Rhein-assisted liver cancer therapy and the search for similar new drugs for tumor adjuvant therapy.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

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