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题名	穿梭蛋白symplekin在细胞内的功能和调控机制
作者	张晨
学位类别	博士
答辩日期	2014-05
授予单位	中国科学院研究生院
授予地点	北京
导师	曹毅
关键词	symplekin 紧密连接 细胞分化 细胞周期 启动子 miR-124
其他题名	Functions of shuttle protein symplekin and the regulation mechanisms
学位专业	细胞生物学
中文摘要	Symplekin是细胞紧密连接膜下相关蛋白，在极化的上皮细胞内具有细胞膜及核内的双重定位。以往的研究报导显示，symplekin参与调节mRNA腺苷酸化、上皮细胞增殖、肠上皮细胞分化及肿瘤细胞有丝分裂等。我们在前期工作中发现，symplekin参与紧密连接的形成，通过与ZO-1相互作用或调控ZO-1的表达进而调节上皮屏障通透性及细胞极性。另外，symplekin在癌前病变及癌变的肝细胞中，其在紧密连接处的表达下调。然而symplekin在细胞核内的功能、所参与的信号通路及其在细胞内分布的调控机制依然缺乏系统研究，关于symplekin的表达调控机制目前也未见报道。 在本研究中，我们建立了上皮细胞体外的去分化培养模型，并构建了symplekin的稳定干扰细胞系，通过一系列实验证实，在去分化细胞中symplekin的细胞核定位增加，膜定位减少，细胞增殖速率上升。表达谱芯片结果显示symplekin在核中主要参与细胞周期调控，并能够影响许多细胞周期蛋白和相关激酶的表达。此外我们发现，symplekin能够与ERK1/2相互结合，且核内磷酸化程度增加，提示symplekin可作为ERK1/2的底物被磷酸化后入核调控基因表达。在symplekin的干扰细胞中，我们检测到一定的EMT特征，说明了symplekin对维持上皮细胞连接和极性的重要性。 另外我们发现在肝细胞癌组织和肝癌细胞系中symplekin表达呈现下调趋势，测序发现其启动子区多出18 bp序列，进一步分析发现此序列在癌组织中出现频率较高，且含有此序列的启动子转录活性降低。另外我们发现miR-124的表达在肝组织和细胞系中与symplekin呈相反趋势，miR-124 mimics的处理能够降低symplekin的表达。这些结果提示symplekin在肝癌中的表达受到启动子的变异和microRNA的调控。 此外我们利用Boyden chamber构建了一系列新的体外模型研究了幽门螺旋杆菌(H. pylori)与胃上皮细胞的作用，发现在H. pylori感染细胞中许多细胞连接分子表达下降，并且H. pylori与细胞基顶面直接接触能够引起紧密连接最明显的下调，H. pylori的感染也能够影响MUC1的表达和糖基化。这些结果证实了寄居于胃肠上皮基顶面的H. pylori能够严重破坏上皮粘膜屏障的结构与功能。 关键词：symplekin，紧密连接，细胞分化，细胞周期，启动子，miR-124， H. pylori，粘膜屏障
英文摘要	Symplekin is multifunctional protein localized to both the tight junction and the nucleus in polarized epithelial cells with known roles in mRNA polyadenylation, proliferation, differentiation and tumorigenesis. Our previous studies confirmed that symplekin participates in the assembly of tight junctions and determines the epithelial monolayers permeability as well as cellular polarity. We also observed deceased expression of symplekin in premalignant and malignant hepatocytes. However, the nuclear functions symplekin and its regulation mechanisms, including related signal pathways have not been systematically investigated. Besides, the expression regulation of symplekin has not been studied yet. In the present study, by the use of dedifferentiated culture of epithelial cells and symplekin depleted stable cell lines, we have demonstrated that nuclear accumulation of symplekin increased in dedifferentiated cells with up-regulated proliferation rate. The mRNA profile analysis suggested symplekin predominantly participated in cell cycle progression and regulated expression of many cyclins and associated CDKs. Moreover, symplekin could be co-immunoprecipitated with ERK1/2 and its phosphorylated level was higher in the nuclear, indicating symplekin might be a substrate for ERK1/2 kinase. Partial EMT features could be detected in symplekin depleted cells, which also verified the significance of symplekin in maintaining epithelial junctions and cell polarity. Furthermore, decreased expression of symplekin was observed in hepatocellular carcinomas. An 18 bp AT-rich sequence was newly found in the promoter with reduced transcriptional activity, and its heterozygous or homozygous status was related to the expressions of symplekin in cultured cells and hepatic tumor tissues. We also found opposite expression pattern of symplekin and miR-124 in vivo and in vitro, and the treatment of miR-124 mimics impaired symplekin expression in hepatic cells. These results suggest that expression of symplekin in hepatocellular carcinomas was regulated by the alternation of its promoter and microRNAs. In another aspect, we developed several new in vitro methods to study the relationships between H. pylori and the gastric epithelial cells using Boyden Chambers. We found that H. pylori infection could repress expression of different adhesion molecules and MUC1, and direct contact of H. pylori with the apical membrane of the cells resulted in the greatest increase in permeability compared to basal membrane binding or non-binding of H. pylori to the cells according to the degree of damage at the tight junctions. These results strongly indicate that H. Pylori dwelling on the apical surface of the gastrointestinal epithelium could directly induce serious injury of the mucosal barrier. Key words: symplekin, tight junction, cell differentiation, cell cycle, promoter, miR-124, H. pylori, mucosal barrier
语种	中文
公开日期	2014-07-01
内容类型	学位论文
源URL	[http://159.226.149.42:8088/handle/152453/7921]
专题	昆明动物研究所_分子病理学
推荐引用方式 GB/T 7714	张晨. 穿梭蛋白symplekin在细胞内的功能和调控机制[D]. 北京. 中国科学院研究生院. 2014.

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