Wnt对Connexin43表达及其功能的影响

【作者】 刘晓宇；

【导师】 左伋； 李笑天； 刘雯；

【作者基本信息】 复旦大学 ， 遗传学， 2007， 博士

【摘要】 缝隙连接蛋白43(connexin43，Cx43)是连接蛋白家族的成员之一，在心脏发育中具有重要作用，其表达水平的异常增高和减低均会导致小鼠心脏畸形，并且这种畸形主要位于右室流出道区域，即Cx43缺陷可能是先天性心脏畸形(Congenital Malformation of Heart，CHM)的重要病因之一。研究表明Cx43可能通过影响细胞迁移产生作用，其缺陷引起心脏畸形的可能机制：神经嵴细胞异常及心肌细胞异常。Wnt家族参与了细胞增殖、分化、凋亡和细胞定位控制等过程。Wnt-frizzled信号转导通路是在无脊椎动物和脊椎动物的发育过程中的一条重要通路，对体轴、中枢神经系统以及肢体的成型过程尤为重要。在脊椎动物的心脏发育过程中，也发现了该信号转导途径的几个成员的表达。有学者提出，Wnt1(Wnt1家族成员)信号能够调节Cx43，这种信号转导路径可能涉及wnt1／β-catenin信号通路。Wnt1信号的变化可以改变Cx43，导致缝隙连接(Gap junction；GJ)通道构造的改变，直接或间接干扰GJ通道的活性；而对该家族中另一成员Wnt-3a的研究则鲜有报道。本研究拟在细胞水平初步探讨Wnt1家族另一成员Wnt-3a信号通路对Cx43的表达调控及相关细胞生物学行为的影响，从而为进一步研究其与在心脏发育中的作用奠定基础。我们首先构建Wnt-3a高表达和低表达的真核表达载体。在此基础上，分别以PC12和H9c2细胞进行研究。大鼠肾上腺嗜铬细胞瘤细胞(PC12)是最常用的神经生物和神经化学研究的细胞模型；同时建立大鼠心肌细胞系H9c2细胞模型，探讨转染后相应细胞模型中Cx43的表达变化及其意义。应用RT-PCR、Western blot、免疫细胞化学等方法研究其表达变化，并用Transwell小室实验对不同细胞模型迁移能力的变化进行检测。结果表明：与对照相比，Wnt-3a高表达模型中Cx43 mRNA和蛋白表达水平明显升高，与此相对应，细胞迁移率也高于对照组；两组低表达模型中，Cx43 mRNA和蛋白表达显著降低，细胞迁移率相应降低，表明Cx43表达的异常引发了细胞迁移率的改变。进一步检测Wnt通路对H9c2相应细胞模型Cx43的表达调控作用下相应细胞生物学行为的变化，通过激光共聚焦显微镜观察胞内钙离子浓度的改变，并用RT-PCR方法对心脏发育相关的两个基因GATA4、Nkx2.5的表达变化进行研究。结果表明：MTT以及流式细胞仪检测说明稳定转染Wnt-3a后细胞增殖率明显高于正常对照细胞，进入G2期的细胞增多，细胞增殖分裂能力增强；与对照相比，Wnt-3a高表达细胞模型中激光共聚焦结果揭示胞内钙离子浓度明显升高；两组低表达模型中，胞内钙离子浓度低于正常对照组。此外，在过表达Wnt-3a的H9c2细胞中，与心脏发育相关的GATA4、Nkx2.5基因的表达明显提高，而在低表达细胞中明显降低。作为Wnt信号通路中的重要分子，β-catenin在胞内的表达水平被认为与细胞的增殖和迁移密切相关。另外，在心肌细胞连接处缺乏β-catenin将导致心脏结构完整性的损害；且研究发现其与胞膜亦有联系，发挥一定的胞间连接功能。探讨β-catenin与Cx43的关系将有助于揭示Wnt-3a对Cx43调控及细胞迁移能力改变的分子机制。本文在H9c2细胞模型中，通过免疫共沉淀实验证实Cx43与β-catenin存在一定的相互作用。免疫荧光观察显示在Cx43高表达细胞中，β-catenin广泛分布于胞质、细胞核中，以及胞膜附近；而低表达Cx43细胞β-catenin表达量低，而且主要定位于胞膜附近。这一结果表明Cx43表达的改变可能涉及β-catenin的定位，多方面的协同作用共同影响了相应细胞的迁移。综上，本研究在体外证实了Wnt经典信号通路对Cx43的表达调控及由之引起的细胞生物学行为的改变，为将来可能的一些临床干预提供了理论依据。更多还原

【Abstract】 Being a member of connexin family, Connexin43 plays a crucial role in cardiac development. Studies in mouse pointed out the abnormal expression of Cx43 could result in cardiac anomalies, mainly with right outflow tract. The impairment of Cx43 may be an important reason for Congenital Malformation of Heart.Cx43 may be one of the leading causes of cardiac anomalies. It has been suggestted that the abnormal migration of cells, including neural crest cells and cardiac myocytes, may contribute to these processes.The Wnt signaling pathways is though to be functionally conserved in vertebrates and invertebrates and plays an important role in cell proliferation, differentiation, organogenesis and oncogenesis. And several members of the Wnt-frizzled signal transduction pathways were found to be expressed during cardiac development in vertebrates.Wnt 1 of the Wnt 1 family member signaling has been shown to be an important modulator of Cx43-dependent intercellular coupling in the heart. The Wnt-1/β-catenin signaling pathway could change the expression of Cx43, which affected the structure and function of gap junctional intercellular communication. Few reports are concerned about Wnt-3a, which is another member of the family.To clarify the molecular mechanism that the expression of Cx43 may control the growth and several phenotypes of neural crest cells and cardiac myocytes by regulation of Wn-3at, and my be involved in the CHM, we designed in vitro experiments.In first part, we constructed high-expressed and low-expressed eukaryotic vector of Wnt-3a.Then we chose PC12 and H9c2 cells to take further research. Rat adrenal pheochromocytoma（PC12） cell line is a well established model for the nerve system research. Rat heart cell-line H9c2 cells were also good models. Then we detected the expression profile of Cx43 in cell models. The Reverse transcription-polymerase chain action （RT-PCR）, Western blot and immunocytochemistry were performed, and the method of Transwell migration assay was taken in the experiments. The results indicated that mRNA and protein level of Cx43 were increased in the cells high expressed Wnt-3a.Ferthermore, The migration ability of those cells were also improved compared to the non-transfected cells As for the situation in the cells low expressed Wnt-3a group , mRNA,protein level of Cx43 and the migration ability were reduced in siWnt-3a-l and siWnt-3a-2. Thus, the dysfunction of Cx43 expression could induce the change for migration ability of those cells.Then we investigated the regulation of Wnt signaling pathway to Cx43 and severalphenotypes of Rat heart cell-line H9c2 cells. To investigate the change of the intracellular [Ca²⁺], we examined it by LSCM with fluorescent dye Fluo-3/AM labeling cells, and [Ca²⁺] changes were represented by fluorescent intensities. The expression levels of two genes related to cardiac development were also detected by RT-PCR. The results of MTT assay and FCM analysis indicated that more Wnt-3a⁺-H9c2 cells enter into G2-phase and the proliferation rate of the cells increased significantly compared to the non-transfected cells..Ferthermore, The intracellular [Ca²⁺] of those cells were also improved compared to the non-transfected cells As for the situation in the cells low expressed Wnt-3a group, intracellular [Ca²⁺] were reduced in pSilencer/siWnt-3a-1 and pSilencer/siWnt-3a-2. The two genes （GATA4,Nkx2.5） related to cardiac development were even increased in the Wnt-3a⁺-H9c2 cells, while those of siWnt-3a-1-H9c2 and siWnt-3a-2-H9c2 were all reduced compared to the control.Being the important part in Wnt signaling pathway, the expression ofβ-catenin took responsibility for the proliferation and migration of the cell. Furthermore, the lack ofβ-catenin in the intercellular junction of cardiac myocytes would result in the impairment of the whole structure in heart. It’s reported that the relationship betweenβ-catenin and cytoplasmic membrane was so important that it was responsible for the intercellular junction. The relation ofβ-catenin and Cx43 could reveal the assumption that the regulation of Cx43 and the change of cell migration by Wnt-3a. In this paper we detected Cx43-β-catenin interaction by coimmunoprecipitaton, and observed the location of them both in Wnt-3a overexpressed and low expressed H9c2 cells by immunofluorescence analysis. The results showed that.β-catenin existed widely in cytoplasm, nuclera, even near the cytoplasmic membrane in Cx43 high expressed H9c2 cells. The expression ofβ-catenin were reduced greatly and located only near the cytoplasmic membrane in Cx43 low expressed H9c2 cells. There was direct interaction between them by coimmunoprecipitaton assay. All these indicated that the change of Cx43 may affect the location ofβ-catenin. Cooperation effect of several aspects changed the migration of the cell together.For all the above, this research work verified the regulation of Wnt-3a on Cx43 and the corresponding change of cell phenotypes. And thus offers potential opportunities for further therapeutic interventions.更多还原