Rbm24在心肌细胞中的重要作用及相关信号分子初筛

发布时间：2018-03-24 06:12

  本文选题：Rbm24　切入点：心肌细胞　出处：《厦门大学》2014年硕士论文

【摘要】：目的研究Rbm24在心肌分化和心肌细胞中作用的分子机制,寻找Rbm24的靶标mRNA及与其相互结合的蛋白,揭示Rbm24在干细胞心肌分化过程中的作用,为探讨Rbm24敲低而导致心肌收缩受损的心肌病的相关信号通路提供一定的研究依据,并为心肌病的治疗提供一个重要的新靶点。 方法首先,通过qRT-PCR检测Rbm24在ESC分化过程的表达情况。同时,采用Western blot免疫印迹分析结合细胞免疫荧光染色检测Rbm24在原代心肌细胞、H9C2细胞系、C2C12细胞系的表达情况。然后,利用RNA干扰技术构建的Rbm24敲低细胞siRNA-Rbm24,分析Rbm24敲低对心肌结构蛋白的表达及心脏收缩的影响。最后,利用基因克隆和脂质体转染技术构建稳定过表达Rbm24-Flag的心肌母细胞H9C2-23,采用RNA蛋白免疫沉淀芯片(RIP-CHIP)技术对Rbm24相结合的靶标mRNA进行系统地分析,并结合RIP-PCR进行验证。同时,采用蛋白免疫共沉淀(CO-IP).质谱分析技术对与Rbm24相互结合的蛋白进行系统地分析,并通过基因克隆技术和CO-IP进行验证。 结果在ESC分化成心肌细胞的第三天,Rbm24开始表达,心肌分化启动,Rbm24的表达一直上调到第六天。在原代心肌细胞中,a-actinin标记的心肌细胞均显示Rbm24表达阳性,且在核质中均匀分布。在心肌母细胞H9C2中,Rbm24在核质中均匀分布,诱导H9C2分化后,Rbm24蛋白表达量上调,且细胞质中的蛋白表达量上调明显。在肌原细胞系C2C12中,Rbm24不表达,利用马血清诱导C2C12分化成肌细胞的过程中,Rbm24表达量逐渐升高。这些数据表明Rbm24是早期心肌分化的标志物,在心肌组织中特异性表达。在心肌细胞中,Rbm24敲低将导致心肌结构蛋白TNNT2、TPM、MYH6(α-MHC)和ACTN2的表达减少,并且使得细胞内肌丝的形态变得不清晰,细胞的自发性收缩也减少了。这些数据表明,Rbm24在心肌功能的维持中有重要作用。RIP-CHIP技术获得了与Rbm24相结合的靶标mRNA,并通过RIP-PCR验证Rbm24与MYOG和Stat3mRNA相结合。CO-IP结合质谱分析获得可能与Rbm24相互结合的蛋白,并进一步通过基因克隆与CO-IP验证与Rbm24相互结合的蛋白。 结论Rbm24在ESC分化成心肌细胞及心肌组织中特异性表达,表明Rbm24是早期心肌分化的标志物,且在心肌细胞功能和心肌收缩力的维持中有重要作用,很可能作为临床心肌病的一个指征。Rbm24可能通过调节其下游的靶标mRNA以及与相关蛋白的相互作用,在心肌分化和心肌细胞功能中起重要作用。
[Abstract]:Objective to study the molecular mechanism of the role of Rbm24 in myocardial differentiation and cardiomyocytes, to search for the target mRNA of Rbm24 and its binding proteins, and to reveal the role of Rbm24 in the process of myocardial differentiation of stem cells. In order to study the signal pathway related to cardiomyopathy caused by Rbm24 knockdown and myocardial contraction damage, it provides a new important target for the treatment of cardiomyopathy. Methods first, the expression of Rbm24 in the differentiation of ESC was detected by qRT-PCR. Meanwhile, the expression of Rbm24 in the primary cardiomyocytes was detected by Western blot immunoblotting and cellular immunofluorescence staining. RNA interference technique was used to construct Rbm24 knock low cell siRNA-Rbm24. The effects of Rbm24 knockout on the expression of myocardial structural protein and cardiac contraction were analyzed. Using gene cloning and liposome transfection techniques to construct stable cardiomyoblast H9C2-23 expressing Rbm24-Flag stably, RNA protein immunoprecipitation microarray RIP-CHIPP was used to analyze the target mRNA binding to Rbm24, and it was verified with RIP-PCR. The proteins interacting with Rbm24 were systematically analyzed by protein co-immunoprecipitation and mass spectrometry, and verified by gene cloning and CO-IP. Results on the third day after ESC differentiation into cardiomyocytes, the expression of Rbm24 began to express, and the expression of Rbm24 was up-regulated to the sixth day. The positive expression of Rbm24 was found in the primary cardiomyocytes labeled with a-actinin. Rbm24 was homogeneously distributed in the nucleus and cytoplasm of cardiomyoblastoma H9C2, the expression of Rbm24 protein was up-regulated after H9C2 differentiation, and the expression of Rbm24 in cytoplasm was obviously up-regulated, but not in the myogenic cell line C2C12. The expression of Rbm24 was gradually increased during the differentiation of C2C12 into myocytes induced by horse serum. These data suggest that Rbm24 is a marker of early myocardial differentiation. Specific expression in myocardial tissue. Low Rbm24 knockout in cardiomyocytes resulted in a decrease in the expression of myocardial structural protein TNNT2TPMTPMPMMYH6 (伪 -MHC6) and ACTN2, and the morphology of myofilament in the cells became unclear. These data indicate that Rbm24 plays an important role in the maintenance of myocardial function. RIP-CHIP technique has obtained target mRNAs combined with Rbm24, and verified by RIP-PCR the combination of Rbm24 with MYOG and Stat3mRNA. CO-IP binding mass spectrometry analysis. To obtain proteins that may interact with Rbm24, Furthermore, the protein binding to Rbm24 was identified by gene cloning and CO-IP. Conclusion the specific expression of Rbm24 in cardiomyocytes and myocardial tissues induced by ESC suggests that Rbm24 is a marker of early myocardial differentiation and plays an important role in the maintenance of myocardial function and contractility. As an indication of clinical cardiomyopathy, Rbm24 may play an important role in myocardial differentiation and cardiomyocyte function by regulating its downstream target mRNA and its interaction with related proteins.
【学位授予单位】：厦门大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R54

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