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  本文關(guān)鍵詞： 線粒體通透性轉(zhuǎn)換孔道 大黃酸 HepG2細(xì)胞 細(xì)胞凋亡　出處：《復(fù)旦大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：肝癌是常見的惡性腫瘤,位于男性腫瘤致死率第二位,女性腫瘤致死率第三位。傳統(tǒng)的腫瘤治療方式效果有限,5年的生存率僅為3%-67%,因此尋找肝癌有效藥物治療方法十分必要。而中醫(yī)藥對改善肝癌患者癥狀、減少放化療毒性、提高生存質(zhì)量、延長生存期等方面恰恰發(fā)揮著獨特的優(yōu)勢。大黃酸(4,5-二羥基蒽醌-2-羧酸)是提取自我國傳統(tǒng)中藥大黃和虎杖根莖中的有效成分,其抗癌作用機制己被大量研究證實。鑒于其對不同的腫瘤細(xì)胞(乳腺癌,宮頸癌,胃癌以及肝癌)的增殖抑制作用,推測其具有重要的臨床藥用價值,同時相關(guān)分子機制研究結(jié)果提示,線粒體依賴的內(nèi)源性凋亡通路有可能參與大黃酸誘導(dǎo)的細(xì)胞凋亡。Ca2+超載,氧化應(yīng)激,缺氧,細(xì)胞毒性藥物均可導(dǎo)致線粒體通透性孔道開放與跨膜電位的消失,進而誘導(dǎo)細(xì)胞凋亡。研究表明：線粒體通透性轉(zhuǎn)換孔道在腫瘤細(xì)胞凋亡中起關(guān)鍵作用。目前國內(nèi)外對大黃酸的研究雖多有報道,但未見其對線粒體生理性作用與凋亡誘導(dǎo)作用相關(guān)性的分析,因此進一步探索大黃酸誘導(dǎo)人肝癌HepG2細(xì)胞的凋亡作用,并研究線粒體通透轉(zhuǎn)換孔道在大黃酸誘導(dǎo)細(xì)胞凋亡中的地位,具有極為重要藥理學(xué)意義。本研究采用HepG2細(xì)胞系及大鼠肝臟離體線粒體作為實驗?zāi)Ｐ?通過測定HepG2細(xì)胞系線粒體膜電位及ATP水平變化、線粒體腫脹程度、Ca2+外泄程度分析大黃酸誘導(dǎo)HepG2細(xì)胞線粒體損傷作用及機制,采用流式細(xì)胞儀、Westernblot、熒光檢測以及TUNEL檢測技術(shù),系統(tǒng)研究線粒體通透性轉(zhuǎn)換孔道在大黃酸誘導(dǎo)肝癌HepG2細(xì)胞凋亡過程中的重要作用機制。結(jié)果表明：大黃酸可導(dǎo)致線粒體功能障礙、腫脹以及Ca2+外泄,這些作用呈劑量依賴性,并且可以被線粒體通透性轉(zhuǎn)換孔道抑制劑CsA完全抑制,提示大黃酸可直接作用于線粒體,導(dǎo)致線粒體通透性轉(zhuǎn)換孔道開放以及線粒體功能障礙；大黃酸誘導(dǎo)的HepG2細(xì)胞凋亡作用可被CsA所阻斷,提示線粒體通透性轉(zhuǎn)換孔道在大黃酸介導(dǎo)肝癌細(xì)胞株凋亡中起著重要作用。綜上,本研究在國內(nèi)外率先闡明線粒體通透性轉(zhuǎn)換孔道在大黃酸誘導(dǎo)的HepG2細(xì)胞凋亡中起著關(guān)鍵性作用,HepG2細(xì)胞凋亡與線粒體依賴的內(nèi)源性凋亡通路有關(guān)。從而為大黃酸的輔助腫瘤治療提供細(xì)胞分子生物學(xué)理論基礎(chǔ),為發(fā)現(xiàn)大黃酸輔助肝癌治療的新靶標(biāo)和尋找腫瘤輔助治療同類新藥提供生物學(xué)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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R96

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