發(fā)布時(shí)間：2018-06-24 22:07

  本文選題：線粒體融合蛋白2 + 凋亡�。� 參考：《浙江大學(xué)》2015年博士論文

【摘要】：肝癌是我國常見的惡性腫瘤之一,目前占我國惡性腫瘤死亡原因的第二位,肝癌早期往往不易發(fā)現(xiàn),一旦發(fā)現(xiàn)多數(shù)已屬中晚期,如果不能徹底手術(shù)切除,目前仍缺乏其他有效的治療方法。通過各種手段干預(yù)腫瘤細(xì)胞的活化增殖,是目前治療肝癌的研究方向和研究熱點(diǎn)。腫瘤的形成多與失控的細(xì)胞增殖相關(guān),而細(xì)胞凋亡在這種病態(tài)的增殖密切相關(guān)。細(xì)胞增殖抑制基因(hyperplasia suppressor gene, HSG)是利用差異顯示技術(shù)得到的一個(gè)與高血壓相關(guān)的新基因,最近研究發(fā)現(xiàn)HSG與線粒體融合相關(guān),因此被稱為線粒體融合蛋白2(Mfn2)。研究表明,Mfn2通過與ras基因結(jié)合,抑制Ras-Erk通路的活性,從而上調(diào)周期調(diào)控蛋白P27和P21Cipl的表達(dá)、下調(diào)PCNA的表達(dá)、阻滯細(xì)胞于GO/G1期,從而抑制細(xì)胞增殖,提示Mfn2可能與腫瘤發(fā)生發(fā)展密切相關(guān)。 第一部分線粒體融合蛋白2(Mfn2)通過線粒體凋亡途徑誘導(dǎo)肝癌細(xì)胞凋亡 目的： Mfn2顯著抑制血管內(nèi)皮細(xì)胞(VSMCs)增殖,并具有潛在的誘導(dǎo)細(xì)胞凋亡效應(yīng)。本文證實(shí)Mfn2誘導(dǎo)肝癌細(xì)胞凋亡并探討其可能的分子機(jī)制。 方法： 運(yùn)用QRT-PRC和免疫組化分析肝癌與癌旁正常肝組織Mfn2基因與蛋白表達(dá)水平。本研究采用肝癌細(xì)胞株HepG2與Huh7細(xì)胞,運(yùn)用腺病毒載體外源性加入重組的Mfn2,建立過表達(dá)Mfn2的細(xì)胞模型。應(yīng)用流式細(xì)胞術(shù)分析Mfn2對細(xì)胞凋亡、線粒體膜電位、活性氧水平的影響,運(yùn)用Western blot方法檢測細(xì)胞色素c釋放、Bax移位和活化的Caspase3表達(dá),并探討其誘導(dǎo)細(xì)胞凋亡的分子機(jī)制。 結(jié)果： 定量PCR和免疫組化顯示,與癌旁正常肝組織相比,癌組織中Mfn2在基因與蛋白水平均低表達(dá)(P0.01)。免疫組化顯示,Mfn2的低表達(dá)與腫瘤的大小及分期密切相關(guān)(P=0.038和0.040)。生存曲線表明低表達(dá)Mfn2的肝癌病人預(yù)后較差(P0.01)。 肝癌細(xì)胞株HepG2與Huh7過表達(dá)Mfn2,發(fā)現(xiàn)細(xì)胞凋亡、線粒體膜電位(△Ψm)下降、細(xì)胞內(nèi)活性氧水平(ROS)增加、Bax由細(xì)胞質(zhì)易位于線粒體而細(xì)胞色素c由線粒體釋放入細(xì)胞質(zhì)。 結(jié)論： Mfn2在癌組織中低表達(dá),與腫瘤大小、分期及病人生存率密切相關(guān)。發(fā)現(xiàn)Mfn2可能通過線粒體凋亡途徑誘導(dǎo)肝癌細(xì)胞凋亡。 第二部分線粒體融合蛋白2(Mfn2)通過激發(fā)內(nèi)質(zhì)網(wǎng)(ER)內(nèi)鈣離子(Ca2+)流入線粒體從而誘導(dǎo)肝癌細(xì)胞凋亡 目的： Mfn2顯著抑制細(xì)胞增殖并具有誘導(dǎo)肝癌細(xì)胞凋亡效應(yīng)。另外,Mfn2不但促進(jìn)線粒體融合并維持線粒體的網(wǎng)絡(luò)結(jié)構(gòu),而且是維持線粒體與內(nèi)質(zhì)網(wǎng)(ER)緊密聯(lián)系的橋梁蛋白。本研究探討Mfn2可能通過激發(fā)內(nèi)質(zhì)網(wǎng)內(nèi)鈣離子流入線粒體從而誘導(dǎo)肝癌細(xì)胞凋亡。 方法： 應(yīng)用肝癌細(xì)胞株HepG2,通過腺病毒載體外源性加入Mfn2,建立過表達(dá)Mfn2的細(xì)胞模型。運(yùn)用鈣離子示蹤劑,檢測內(nèi)質(zhì)網(wǎng)與線粒體內(nèi)鈣離子的變化。另外,分別應(yīng)用藥物肝素(Heparin)(特異性抑制內(nèi)質(zhì)網(wǎng)上IP3激活的鈣離子釋放通道)和RU360(特異性阻斷線粒體鈣離子攝入)處理過表達(dá)Mfn2的細(xì)胞,分析細(xì)胞內(nèi)細(xì)胞器的鈣離子流向并檢測細(xì)胞凋亡、線粒體膜電位、活性氧水平和細(xì)胞色素c釋放。 結(jié)果： 在Mfn2的外源性處理下,內(nèi)質(zhì)網(wǎng)(ER)內(nèi)鈣離子下降、線粒體內(nèi)鈣離子增加。然而,運(yùn)用Heparin和RU360處理過表達(dá)Mfn2的HepG2細(xì)胞,發(fā)現(xiàn)細(xì)胞不發(fā)生凋亡、線粒體膜電位(△Ψm)不下降、活性氧水平(ROS)不升高、內(nèi)質(zhì)網(wǎng)(ER)內(nèi)鈣離子不下降和線粒體內(nèi)鈣離子水平不升高。而且我們發(fā)現(xiàn),外源性Mfn2下調(diào)線粒體鈣離子內(nèi)流的“守門員”蛋白MICUs的表達(dá)。 結(jié)論： 本研究表明,Mfn2通過激發(fā)內(nèi)質(zhì)網(wǎng)內(nèi)鈣離子流入線粒體從而誘導(dǎo)肝癌細(xì)胞凋亡。內(nèi)質(zhì)網(wǎng)內(nèi)鈣離子通過IP3激活的鈣離子釋放通道釋放,線粒體通過線粒體鈣離子單向傳遞體攝入內(nèi)質(zhì)網(wǎng)釋放的鈣離子。
[Abstract]:Liver cancer is one of the most common malignant tumors in China. It now accounts for second of the causes of the death of malignant tumors in China. It is often difficult to find the early stage of liver cancer. If most of them are in the middle and late stages, there is still a lack of other effective treatment methods if they are not completely removed. Hyperplasia suppressor gene (HSG) is a new gene related to high blood pressure using differential display technology (gene, HSG), and the recent study of HSG and line It is known as mitochondrial fusion protein 2 (Mfn2). Studies have shown that Mfn2 can inhibit the expression of P27 and P21Cipl by binding to the ras gene, up regulate the expression of P27 and P21Cipl, down regulate the expression of PCNA, block the cell in GO/G1 period and inhibit the proliferation of cells, suggesting that Mfn2 may be closely related to the development of the tumor. Relevant.
Part one, mitochondrial fusion protein 2 (Mfn2) induces apoptosis in hepatocellular carcinoma cells through mitochondrial apoptosis pathway.
Objective:
Mfn2 significantly inhibits the proliferation of vascular endothelial cells (VSMCs) and has a potential effect on inducing apoptosis. This paper has confirmed that Mfn2 induces apoptosis in hepatoma cells and discusses its possible molecular mechanism.
Method:
The expression of Mfn2 gene and protein expression level in liver cancer and normal liver tissue was analyzed by QRT-PRC and immunohistochemistry. The study adopted the hepatoma cell line HepG2 and Huh7 cells, using adenovirus vector to add recombinant Mfn2 to the recombinant Mfn2, and set up a cell model to express Mfn2. The flow cytometry was used to analyze the apoptosis, the mitochondrial membrane potential and the survival of Mfn2. The Western blot method was used to detect the expression of cytochrome c release, Bax shift and activated Caspase3, and to explore the molecular mechanism of cell apoptosis induced by the effect of sex oxygen level.
Result:
The quantitative PCR and immunohistochemistry showed that Mfn2 was low in the gene and protein level in the cancerous tissues (P0.01). The low expression of Mfn2 was closely related to the size and staging of the tumor (P=0.038 and 0.040). The survival curve showed that the prognosis of liver cancer patients with low expression of Mfn2 was poor (P0.01).
The hepatoma cell line HepG2 and Huh7 overexpressed Mfn2, and the apoptosis was found, the mitochondrial membrane potential (delta m) decreased, the intracellular reactive oxygen level (ROS) increased, and the cytoplasm of the cytoplasm was easily located in the mitochondria, and the cytochrome C was released into the cytoplasm from mitochondria.
Conclusion:
The low expression of Mfn2 in cancer tissues is closely related to tumor size, staging and survival rate. It is found that Mfn2 may induce apoptosis of hepatocellular carcinoma cells through mitochondrial apoptosis pathway.
The second part, mitochondrial fusion protein 2 (Mfn2), induces the apoptosis of hepatoma cells by stimulating calcium (Ca2+) into the mitochondria in the endoplasmic reticulum (ER).
Objective:
Mfn2 significantly inhibits cell proliferation and induces apoptosis in hepatoma cells. In addition, Mfn2 not only promotes mitochondrial fusion and maintains mitochondrial network structure, but also maintains a close link between mitochondria and endoplasmic reticulum (ER). This study explores that Mfn2 may induce liver cancer by stimulating the inflow of calcium ions into mitochondria in the endoplasmic reticulum Cell apoptosis.
Method:
The hepatoma cell line HepG2 was used to establish a cell model to express Mfn2 by adding exogenous Mfn2 to the adenovirus vector. Calcium ion tracer was used to detect the changes of calcium ions in the endoplasmic reticulum and mitochondria. In addition, the drug heparin (Heparin) (specific inhibition of the calcium release channel activated by the endoplasmic reticulum IP3) and RU360 (specificity) were used respectively. Cells that overexpress Mfn2 are blocked by blocking mitochondrial calcium intake, and the flow of calcium ions in cell organelles is analyzed and apoptosis, mitochondrial membrane potential, reactive oxygen species (ROS) and cytochrome c release are detected.
Result:
Under the exogenous treatment of Mfn2, calcium ions in the endoplasmic reticulum (ER) decreased and the calcium ions in mitochondria increased. However, Heparin and RU360 were used to treat HepG2 cells expressing Mfn2, the cells were not apoptotic, the mitochondrial membrane potential (delta m) did not decrease, the activity oxygen level (ROS) did not increase, the calcium ions in the endoplasmic reticulum (ER) did not decrease and the intracellular calcium was in the mitochondria. The level of ions did not increase. Moreover, we found that exogenous Mfn2 reduced the expression of "goalkeeper" protein MICUs in mitochondrial calcium influx.
Conclusion:
This study shows that Mfn2 induces the apoptosis of hepatoma cells by stimulating the inflow of calcium ions into mitochondria and induces the release of calcium ions in the endoplasmic reticulum through the release of calcium ions activated by IP3 and the intake of calcium ions released by endoplasmic reticulum through mitochondrial calcium ions.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R735.7

【共引文獻(xiàn)】

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