线粒体融合蛋白2诱导肝癌细胞凋亡的分子机制的研究

发布时间：2018-06-24 22:07

本文选题：线粒体融合蛋白2 + 凋亡　；参考：《浙江大学》2015年博士论文

【摘要】：肝癌是我国常见的恶性肿瘤之一,目前占我国恶性肿瘤死亡原因的第二位,肝癌早期往往不易发现,一旦发现多数已属中晚期,如果不能彻底手术切除,目前仍缺乏其他有效的治疗方法。通过各种手段干预肿瘤细胞的活化增殖,是目前治疗肝癌的研究方向和研究热点。肿瘤的形成多与失控的细胞增殖相关,而细胞凋亡在这种病态的增殖密切相关。细胞增殖抑制基因(hyperplasia suppressor gene, HSG)是利用差异显示技术得到的一个与高血压相关的新基因,最近研究发现HSG与线粒体融合相关,因此被称为线粒体融合蛋白2(Mfn2)。研究表明,Mfn2通过与ras基因结合,抑制Ras-Erk通路的活性,从而上调周期调控蛋白P27和P21Cipl的表达、下调PCNA的表达、阻滞细胞于GO/G1期,从而抑制细胞增殖,提示Mfn2可能与肿瘤发生发展密切相关。第一部分线粒体融合蛋白2(Mfn2)通过线粒体凋亡途径诱导肝癌细胞凋亡目的： Mfn2显著抑制血管内皮细胞(VSMCs)增殖,并具有潜在的诱导细胞凋亡效应。本文证实Mfn2诱导肝癌细胞凋亡并探讨其可能的分子机制。方法：运用QRT-PRC和免疫组化分析肝癌与癌旁正常肝组织Mfn2基因与蛋白表达水平。本研究采用肝癌细胞株HepG2与Huh7细胞,运用腺病毒载体外源性加入重组的Mfn2,建立过表达Mfn2的细胞模型。应用流式细胞术分析Mfn2对细胞凋亡、线粒体膜电位、活性氧水平的影响,运用Western blot方法检测细胞色素c释放、Bax移位和活化的Caspase3表达,并探讨其诱导细胞凋亡的分子机制。结果：定量PCR和免疫组化显示,与癌旁正常肝组织相比,癌组织中Mfn2在基因与蛋白水平均低表达(P0.01)。免疫组化显示,Mfn2的低表达与肿瘤的大小及分期密切相关(P=0.038和0.040)。生存曲线表明低表达Mfn2的肝癌病人预后较差(P0.01)。肝癌细胞株HepG2与Huh7过表达Mfn2,发现细胞凋亡、线粒体膜电位(△Ψm)下降、细胞内活性氧水平(ROS)增加、Bax由细胞质易位于线粒体而细胞色素c由线粒体释放入细胞质。结论： Mfn2在癌组织中低表达,与肿瘤大小、分期及病人生存率密切相关。发现Mfn2可能通过线粒体凋亡途径诱导肝癌细胞凋亡。第二部分线粒体融合蛋白2(Mfn2)通过激发内质网(ER)内钙离子(Ca2+)流入线粒体从而诱导肝癌细胞凋亡目的： Mfn2显著抑制细胞增殖并具有诱导肝癌细胞凋亡效应。另外,Mfn2不但促进线粒体融合并维持线粒体的网络结构,而且是维持线粒体与内质网(ER)紧密联系的桥梁蛋白。本研究探讨Mfn2可能通过激发内质网内钙离子流入线粒体从而诱导肝癌细胞凋亡。方法：应用肝癌细胞株HepG2,通过腺病毒载体外源性加入Mfn2,建立过表达Mfn2的细胞模型。运用钙离子示踪剂,检测内质网与线粒体内钙离子的变化。另外,分别应用药物肝素(Heparin)(特异性抑制内质网上IP3激活的钙离子释放通道)和RU360(特异性阻断线粒体钙离子摄入)处理过表达Mfn2的细胞,分析细胞内细胞器的钙离子流向并检测细胞凋亡、线粒体膜电位、活性氧水平和细胞色素c释放。结果：在Mfn2的外源性处理下,内质网(ER)内钙离子下降、线粒体内钙离子增加。然而,运用Heparin和RU360处理过表达Mfn2的HepG2细胞,发现细胞不发生凋亡、线粒体膜电位(△Ψm)不下降、活性氧水平(ROS)不升高、内质网(ER)内钙离子不下降和线粒体内钙离子水平不升高。而且我们发现,外源性Mfn2下调线粒体钙离子内流的“守门员”蛋白MICUs的表达。结论：本研究表明,Mfn2通过激发内质网内钙离子流入线粒体从而诱导肝癌细胞凋亡。内质网内钙离子通过IP3激活的钙离子释放通道释放,线粒体通过线粒体钙离子单向传递体摄入内质网释放的钙离子。
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.7

【共引文献】