长牡蛎内源性细胞凋亡通路及VDAC2基因功能的研究

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	长牡蛎内源性细胞凋亡通路及VDAC2基因功能的研究
其他题名	Elucidation of Mitochondrial Apoptosis Pathway and Functional Characterization of Voltage-Dependent Anion Channel 2 (VDAC2) in Pacific Oyster
	李颖翔
学位类型	博士
导师	张国范
	2016-05-21
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	海洋生物学理学博士
关键词	长牡蛎细胞凋亡线粒体 Bcl-2 Vdac2
其他摘要	牡蛎是一种非常重要的水产养殖动物，与牡蛎相关的产业在全世界范围内广泛分布。长牡蛎（Crassostrea gigas）是一种固着生活的海洋动物，主要生长于陆海交界的潮间带。潮间带复杂多变的环境，加之环境中充斥着的包括病原生物和重金属在内的多种胁迫因子，时刻威胁着长牡蛎的生存。为了更好地适应潮间带环境，长牡蛎进化出了一套复杂的逆境适应机制。近些年来，陆续有证据表明，细胞凋亡过程很可能作为逆境适应机制的重要组成部分，在长牡蛎的逆境响应过程中发挥着重要的作用。然而，迄今为止，人们对于长牡蛎，乃至整个软体动物门生物的细胞凋亡机制都知之甚少。我们迫切需要相关的研究为科学研究者揭开长牡蛎细胞凋亡机制的神秘面纱，从而为更好地理解长牡蛎的潮间带环境适应机制打下基础。而与此同时，长牡蛎基因组的问世为我们全面解析长牡蛎细胞凋亡的分子机制提供了基础。内源性细胞凋亡通路是细胞凋亡中的一个非常重要也是研究相对比较多的类型。内源性细胞凋亡已被证实能够参与生物体对多种逆境胁迫的响应，从而保护机体在不利条件下更好地生存。内源性细胞凋亡在脊椎动物中的研究相对较多，且目前认为脊椎动物中该通路较为保守。而在无脊椎动物中，关于内源性细胞凋亡分子机制的深入研究非常匮乏（目前只有节肢动物、线虫动物和扁形动物中有较为深入的研究），仅根据已有的证据来看，该通路在无脊椎动物中似乎表现出了一定的分化。软体动物中，关于该通路分子调控机制的研究寥寥无几，长牡蛎基因组及帽贝基因组的测序工作中发现了不少内源性细胞凋亡相关基因，但并没有进一步的功能研究。其他的研究基本都是局限于对某个或某几个内源性凋亡相关同源基因进行表达模式研究以及基因功能的简单探索，所得到的研究成果太过零散。因此，亟需更多的深入性研究来帮助我们更好地了解和认识无脊椎动物中，内源性细胞凋亡通路的进化情况。本研究选取长牡蛎的内源性细胞凋亡通路做为研究对象，通过反向遗传学手段对该信号通路的调控机制进行了系统性的分析。我们根据紫外照射能够有效诱导长牡蛎血淋巴细胞发生凋亡这一实验事实，确定选用紫外照射做为本研究中的凋亡诱导因子。我们首先发现紫外照射很可能会引起长牡蛎细胞线粒体外膜通透性的上升。随后我们发现，紫外照射还能诱导细胞色素C从长牡蛎细胞线粒体内外膜间隙释放到细胞质中。由此我们认为，长牡蛎的内源性细胞凋亡过程很可能也像脊椎动物一样，涉及到了线粒体外膜通透性上升及细胞色素C释放。进一步，我们发现紫外照射能够引起长牡蛎血淋巴细胞中凋亡酶Cgcaspase 9和Cgcaspase 3的活性上升。而在向长牡蛎细胞胞质提取液中加入细胞色素C后，无论是Cgcaspase 9还是 Cgcaspase 3，二者的酶活也都显著上升，提示凋亡酶Cgcaspase 9 和 Cgcaspase 3均可能参与了长牡蛎内源性细胞凋亡过程，且二者的激活与细胞色素C的释放很可能存在关联。同时我们发现，一旦Cgcaspase 9的活性受到抑制，Cgcaspase 3的酶活水平也随之降低，因而我们认为caspase 9 很可能在长牡蛎内源性细胞凋亡通路中位于caspase 3的上游，并能激活后者。不过，由于我们没有在长牡蛎基因组中找到编码结构域完整的APAF-1同源蛋白的基因（事实上整个软体动物门中都没有发现该同源基因的存在），因此细胞色素C激活caspase 9的分子机制尚不明确，仍需进一步的研究。同时，我们选取Bcl-2家族做为研究对象，深入研究了该家族的功能。我们发现长牡蛎的Bcl-2家族缺少了BH3-only亚家族成员，于是从抗凋亡亚家族和促凋亡亚家族中各自选取了两个基因（抗凋亡亚家族中选取了CgBcl-2和CgBcl-xL，促凋亡亚家族中选取了CgBak和CgBax），获得了它们的全长并对其所编码蛋白质进行了结构域分析。随后，我们借助qPCR技术分析了这4个基因在不同组织及几种逆境胁迫刺激（包括病毒刺激、弧菌刺激、干露刺激及高温刺激等）下的表达模式，发现上述基因都能在不同程度上参与长牡蛎对多种环境胁迫刺激的响应。此外，用紫外照射长牡蛎血淋巴细胞后，发现这4个基因的表达量均发生了显著的变化，提示它们很可能参与了长牡蛎的内源性细胞凋亡过程。进一步，我们通过体外验证的方式，发现长牡蛎Bcl-2家族成员能够诱导或阻止酿酒酵母（Saccharomyces cerevisiae）的死亡，并能引发或抑制人HEK293T细胞的凋亡。我们又利用RNA干扰技术成功抑制了长牡蛎细胞中CgBak基因的表达，并观察到CgBak表达量下调的细胞在紫外照射后的细胞凋亡水平也显著下降。上述结果说明长牡蛎Bcl-2家族很可能参与了长牡蛎内源性细胞凋亡的调控过程。随后，针对长牡蛎Bcl-2家族具体如何参与细胞凋亡调控过程的问题，我们又进行了一系列的研究。我们在HeLa细胞中过表达CgBak蛋白和CgBax蛋白，发现紫外照射处理后，有一部分的HeLa细胞中CgBak蛋白和CgBax蛋白会发生从细胞质到线粒体的转位。而在HEK293T细胞中，利用免疫印迹技术，我们也观察到了相似的结果。该结果提示CgBak和CgBax可能会在发生细胞凋亡时从细胞质转位到线粒体。进一步我们发现，从长牡蛎细胞中分离得到的线粒体，在与CgBak蛋白或CgBax蛋白（均由原核表达获得）共同孵育一段时间，均能够释放细胞色素C。结合上述结果，我们认为长牡蛎Bcl-2家族中，促凋亡亚家族的CgBak和CgBax蛋白可能会在发生细胞凋亡的时候从细胞质转位到线粒体，并引起线粒体释放细胞色素C。同时，我们利用酵母双杂交和免疫共沉淀技术，证明抗凋亡亚家族的CgBcl-2和CgBcl-xL蛋白，很可能与促凋亡亚家族的CgBak和CgBax蛋白之间存在着蛋白间直接相互作用关系，且该相互作用很可能是由四个蛋白均含有的BH3结构域所介导的。我们推测，抗凋亡亚家族蛋白正是通过这种直接相互作用抑制促凋亡亚家族蛋白的功能的。我们随后将研究目标放在了Bcl-2家族的上游。我们选取Cgp53基因做为潜在的上游调控基因，并围绕该基因进行了一系列的功能验证。首先，借助qPCR技术，我们发现Cgp53基因的表达量在紫外照射后发生显著变化且表现出明显的上升趋势。此外，我们发现当细胞中Cgp53基因的表达受到抑制时，这些细胞在紫外照射后的细胞凋亡率也会降低。上述结果提示Cgp53很可能参与了长牡蛎内源性细胞凋亡的调控且发挥了促凋亡的功能。进一步，我们在HEK293T细胞中进行了双荧光素酶报告基因检测实验，发现Cgp53能够在体外有效地且剂量依赖性地激活CgBak和CgBax基因的转录。此外，我们发现Cgp53表达受抑制的细胞，紫外照射后CgBak基因表达量上升的时间点显著推迟，提示Cgp53可能通过依赖转录的方式，在上游激活CgBak和CgBax的表达，从而引起长牡蛎的细胞凋亡。我们随后通过免疫共沉淀技术证明Cgp53蛋白与CgBcl-2、CgBcl-xL、CgBak和CgBax四个蛋白间均可能存在蛋白间直接相互作用关系，提示Cgp53还可能通过不依赖转录的方式（可能是依赖蛋白间相互作用的方式）调控Bcl-2家族的功能，从而实现对细胞凋亡的调控。综合以上结果和推测，我们认为Cgp53很可能具有通过依赖转录和不依赖转录两种途径，在Bcl-2家族的上游调控后者功能，借此调控长牡蛎细胞凋亡水平的能力。不过，我们没有在长牡蛎的基因组中发现BH3-only亚家族同源基因的存在，且迄今为止该亚家族在软体动物中也未见报道，该亚家族的缺失将会对长牡蛎的逆境适应造成什么正面和负面的影响，将会是一个很有意思的研究方向。此外，我们还研究了长牡蛎线粒体外膜孔蛋白编码基因CgVDAC2的功能及其与内源性细胞凋亡的关系。我们获得了该基因的全长并分析了其在不同组织、不同发育时期及病毒刺激下的表达模式，发现该基因可能参与了长牡蛎对病毒刺激的响应。随后我们发现，CgVDAC2在HEK293T细胞中的过表达，能够有效降低该细胞在紫外照射后的细胞凋亡率。进一步我们发现，通过RNA干扰手段抑制了CgVDAC2基因表达的长牡蛎血淋巴细胞，在紫外照射后凋亡率会上升。我们又借助酵母双杂交和免疫共沉淀技术，证明CgVDAC2蛋白和CgBak蛋白间很可能存在着蛋白间直接相互作用关系。上述结果提示CgVDAC2可能参与了长牡蛎细胞凋亡调控，并可能通过结合和抑制CgBak蛋白的方式发挥抗凋亡活性。综上所述，本研究基本阐明了由p53、Bcl-2家族、线粒体、细胞色素C、caspase 9和caspase 3构成的长牡蛎内源性细胞凋亡通路的分子机制。该研究作为软体动物中首次对细胞凋亡机制的全面系统研究，填补了相关研究领域的许多空白，为我们更好地理解内源性细胞凋亡在无脊椎动物中的进化和起源提供了新的证据。同时也为我们更好地理解长牡蛎是如何适应复杂多变的潮间带环境的这一问题提供了理论支持。同时，对CgVDAC2基因功能的研究，也是软体动物中首次针对VDAC家族蛋白功能的研究。实验结果为更好地理解VDAC2蛋白与内源性细胞凋亡的调控关系提供了新证据，同时也为理解OsHV-1病毒感染机制提供了新思路。; Industries related to oyster, an important aquaculture species, are widely distributed over the world. Pacific oyster (Crassostrea gigas) is an estuarine and intertidal zone animal with sessile behavior, exposed to variable salinity, fluctuating temperature, toxic metals, desiccation and microbial pathogens, which are all adversity stresses that threaten the survival of this sedentary organism. Pacific oyster has evolved complex mechanisms to adapt the complex and inconstant circumstance. In recent years, it has been reported that apoptosis, as a vital part of the environmental adaptation system, gets involved in response of oyster to various stresses. So far, however, little is known about the exact mechanism of apoptosis in oyster, and even in all the mollusks. Studies in this field are urgently required to help us reveal the mechanism of mitochondrial apoptosis in C. gigas, which will further be helpful to understand how oyster acclimates to the fickle environment. With the publication of genome information of Pacific oyster, it is time to comprehensively investigate the molecular regulation mechanism of C. gigas. Several forms of apoptosis have been found up to now. Among them, mitochondrial apoptosis pathway is a very major one and has received more attention. The mitochondrial apoptosis has been demonstrated to be involved in the response of organisms to various environmental stresses, making organisms surviving. There are much more studies on the exact mechanisms of mitochondrial apoptosis in vertebrate than invertebrate. Base on the existing evidences, it could be suggested that mitochondrial apoptosis pathway is quite conserved throughout the entire vertebrate. But in invertebrate, although numerous DNA sequences of apoptosis-related homologous genes have been found in recent years, only a few have been done on the study of mitochondrial apoptosis mechanism in invertebrate as yet (less has been done in mollusks). Besides, the pathways seemed to be diverse in different invertebrates, according to the few existing evidence. Thus, more studies are needed to reveal the diversity of mitochondrial apoptosis in invertebrates and further understand the evolution of the pathway in animals. In this study, we systematically investigated the molecular regulatory mechanism of mitochondrial apoptosis in C. gigas, using the reverse genetics. UV irradiation was chosen to be used as the apoptosis inducer as we found UV irradiation could induce the apoptosis in the hemocytes of C. gigas. We found that UV irradiation lead to the increase of mitochondrial outer membrane permeabilization (MOMP) and the release of cytochrome c from mitochondria to cytosol in C. gigas. Based on this result, we considered that mitochondrial apoptosis pathway might involve MOMP and release of cytochrome c from mitochondria to cytosol, which was consistent with the pathway in vertebrate. Thereafter, UV irradiation was found to activate Cgcaspase 9 and Cgcaspase 3 in hemocytes of C. gigas. In addition, cytochrome c was also found to activate Cgcaspase 9 and Cgcaspase 3 in cytoplasmic extract of C. gigas. The results here suggested that Cgcaspase 9 and Cgcaspase 3 were both involved in mitochondrial apoptosis and the activation of them might be associated with the release of cytochrome c. The activity level of Cgcaspase 3 decreased once Cgcaspase 9 was inhibited, implying that Cgcaspase 9 could activate Cgcaspase 3 in mitochondrial apoptosis pathway. However, as we didn’t find any APAF-1 homologue containing all the major domains in C. gigas, the mechanism of cytochrome c-mediated Cgcaspase 9 activation is still unclear. Additionally, we did some work to investigate the role of Pacific oyster Bcl-2 family. Due to the missing of BH3-only subfamily, only members of anti-apoptotic subfamily and pro-apoptotic subfamily were studied herein. They are CgBcl-2 and CgBcl-xL from the anti-apoptotic subfamily, and CgBak and CgBax from the pro-apoptotic subfamily. We obtained the full-length cDNA sequence of them and performed domain analysis on the predicted proteins encoded by the four genes. Then, the expression patterns of the genes under several types of stresses, including virus infection, vibrio infection, air exposure stimulation and heat shock treatment were analyzed using qPCR. The results showed that the genes responded to all types of stresses in various degrees. Besides, the expression levels of the four genes in hemocytes all changed significantly upon UV irradiation, implying their involvement in mitochondrial apoptosis of C. gigas. Then we conducted some more experiments and found that Pacific oyster members regulated the death or survival of the yeast (Saccharomyces cerevisiae) and influenced the apoptotic level of HEK293T cells. Moreover, it was observed that the hemocytes in which CgBak was silenced by siRNA, displayed a lower apoptotic percentage after UV irradiation than the normal ones. It was suggested by the results here that Bcl-2 family of C. gigas might probably take charge in the regulation of mitochondrial apoptosis. Investigations on how Pacific oyster regulated mitochondrial apoptosis were then performed. We overexpressed CgBak and CgBax in HeLa cells before irradiated the cells with UV light. It turned out that CgBak protein and CgBax protein in some cells translocated from cytoplasm to mitochondria after the irradiation. Similar results were observed in HEK293T cells overexpressing CgBak and CgBax, indicating the translocation of CgBak and CgBax from cytoplasm to mitochondria upon the occurrence of apoptosis. Thereafter, we incubated the mitochondria isolated from Pacific oyster hemocytes with CgBak protein or CgBax protein, and detected the release of cytochrome c from mitochondria. Based on these results, it was suggested that once mitochondrial apoptosis occurred, CgBak and CgBax would translocated from cytoplasm to mitochondria and induced the mitochondria to release cytochrome c to cytoplasm. In addition, we demonstrated the direct interaction between the proteins from pro-apoptotic subfamily and the ones from anti-apoptotic subfamily using yeast two hybrid and co-immunoprecipitation. The results obtained by the two assays also indicated that, it was the BH3 domain that mediated the direct interaction. CgBcl-2 and CgBcl-xL, the proteins from anti-apoptotic subfamily, seemed to inhibit the pro-apoptotic activities of CgBak and CgBax through the direct interaction. Subsequently, we focused on the regulatory factors at the upstream part of Bcl-2 family. The Cgp53 was chosen and investigated to reveal its role in mitochondrial apoptosis. We conducted qPCR analysis and found that the mRNA level of Cgp53 displayed a significant uptrend upon UV irradiation. Besides, once the expression of Cgp53 was inhibited, the hemocytes showed a significant lower apoptotic percentage upon UV irradiation, signifying the involvement and probable pro-apoptotic role of Cgp53 in mitochondrial apoptosis of C. gigas. A dual-luciferase reporter assay was performed and revealed that Cgp53 might activate the transcription of CgBak and CgBax in a dose-dependent manner. Moreover, the expression level of CgBak increased later upon UV irradiation in Cgp53-inhibited hemocytes than normal hemocytes, confirming the activation of Cgp53 to the transcription of CgBak, which further lead to apoptosis. We then assayed the direct interaction between Cgp53 and the four Bcl-2 family proteins studied in our work, using co-immunoprecipitation. The demonstration of the direct interaction implied the possibility that Cgp53 might regulate Bcl-2 family in a transcription-independent way. Taken together, a conclusion could be drawn that Cgp53 might regulate the activities of Pacific oyster Bcl-2 family in two distinct ways, the transcription-dependent way and the transcription- independent way, and thus regulated (induced) mitochondrial apoptosis of C. gigas. However, we could not find any member of BH3-only subfamily in Pacific oyster and even the whole mollusks. It will definitely be quite interesting to study how the missing of this crucial subfamily would impact on the adaptation of oyster to the intertidal circumstance. In addition, we did some research on Pacific oyster voltage dependent anion channel 2 (VDAC2), revealing its function and association with mitochondrial apoptosis. We firstly obtained the full-length cDNA sequence of CgVDAC2 and analyzed its expression pattern in distinct tissues, at different developmental stages and upon virus infection, the result of which suggested the response of CgVDAC2 to OsHV-1 infection. Then we found that the overexpression of CgVDAC2 in HEK293T cells reduced the UV-irradiation-induced apoptosis level. Moreover, Pacific oyster hemocytes in which CgVDAC2 was silenced by siRNA displayed a higher apoptotic percentage upon UV irradiation. Besides, the direct interaction between CgVDAC2 and CgBak was confirmed using yeast hybrid assay and co-immunoprecipitation assay. The results above indicated that CgVDAC2 might involve in regulation of mitochondrial apoptosis and inhibit the pro-apoptotic activity of CgBak. In summary, we elucidated the mechanism of mitochondrial apoptosis in C. gigas, which included p53, Bcl-2 family, mitochondria, cytochrome c, caspase 9, caspase 3 and so on. This is the first work systematically studying the mechanism of apoptosis in mollusks, which filled in a gap in this field and provided new evidence for better understanding the evolution and origin of mitochondrial apoptosis in invertebrate. This work would also be helpful for better understanding how oyster adapted to the complex and changeable circumstance. In addition, the investigation on the function of CgVDAC2 is the first one in mollusks. The results updated our knowledge on the relationship between VDAC2 and mitochondrial apoptosis, and provided a new idea in understanding the infection process of OsHV-1 to oyster.
学科领域	海洋生物学
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/112518
专题	实验海洋生物学重点实验室
作者单位	中国科学院海洋研究所
第一作者单位	中国科学院海洋研究所
推荐引用方式 GB/T 7714	李颖翔. 长牡蛎内源性细胞凋亡通路及VDAC2基因功能的研究[D]. 北京. 中国科学院大学,2016.

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