本文選題：Rbm24　切入點：心肌細胞　出處：《廈門大學(xué)》2014年碩士論文

【摘要】：目的研究Rbm24在心肌分化和心肌細胞中作用的分子機制,尋找Rbm24的靶標(biāo)mRNA及與其相互結(jié)合的蛋白,揭示Rbm24在干細胞心肌分化過程中的作用,為探討Rbm24敲低而導(dǎo)致心肌收縮受損的心肌病的相關(guān)信號通路提供一定的研究依據(jù),并為心肌病的治療提供一個重要的新靶點。 方法首先,通過qRT-PCR檢測Rbm24在ESC分化過程的表達情況。同時,采用Western blot免疫印跡分析結(jié)合細胞免疫熒光染色檢測Rbm24在原代心肌細胞、H9C2細胞系、C2C12細胞系的表達情況。然后,利用RNA干擾技術(shù)構(gòu)建的Rbm24敲低細胞siRNA-Rbm24,分析Rbm24敲低對心肌結(jié)構(gòu)蛋白的表達及心臟收縮的影響。最后,利用基因克隆和脂質(zhì)體轉(zhuǎn)染技術(shù)構(gòu)建穩(wěn)定過表達Rbm24-Flag的心肌母細胞H9C2-23,采用RNA蛋白免疫沉淀芯片(RIP-CHIP)技術(shù)對Rbm24相結(jié)合的靶標(biāo)mRNA進行系統(tǒng)地分析,并結(jié)合RIP-PCR進行驗證。同時,采用蛋白免疫共沉淀(CO-IP).質(zhì)譜分析技術(shù)對與Rbm24相互結(jié)合的蛋白進行系統(tǒng)地分析,并通過基因克隆技術(shù)和CO-IP進行驗證。 結(jié)果在ESC分化成心肌細胞的第三天,Rbm24開始表達,心肌分化啟動,Rbm24的表達一直上調(diào)到第六天。在原代心肌細胞中,a-actinin標(biāo)記的心肌細胞均顯示Rbm24表達陽性,且在核質(zhì)中均勻分布。在心肌母細胞H9C2中,Rbm24在核質(zhì)中均勻分布,誘導(dǎo)H9C2分化后,Rbm24蛋白表達量上調(diào),且細胞質(zhì)中的蛋白表達量上調(diào)明顯。在肌原細胞系C2C12中,Rbm24不表達,利用馬血清誘導(dǎo)C2C12分化成肌細胞的過程中,Rbm24表達量逐漸升高。這些數(shù)據(jù)表明Rbm24是早期心肌分化的標(biāo)志物,在心肌組織中特異性表達。在心肌細胞中,Rbm24敲低將導(dǎo)致心肌結(jié)構(gòu)蛋白TNNT2、TPM、MYH6(α-MHC)和ACTN2的表達減少,并且使得細胞內(nèi)肌絲的形態(tài)變得不清晰,細胞的自發(fā)性收縮也減少了。這些數(shù)據(jù)表明,Rbm24在心肌功能的維持中有重要作用。RIP-CHIP技術(shù)獲得了與Rbm24相結(jié)合的靶標(biāo)mRNA,并通過RIP-PCR驗證Rbm24與MYOG和Stat3mRNA相結(jié)合。CO-IP結(jié)合質(zhì)譜分析獲得可能與Rbm24相互結(jié)合的蛋白,并進一步通過基因克隆與CO-IP驗證與Rbm24相互結(jié)合的蛋白。 結(jié)論Rbm24在ESC分化成心肌細胞及心肌組織中特異性表達,表明Rbm24是早期心肌分化的標(biāo)志物,且在心肌細胞功能和心肌收縮力的維持中有重要作用,很可能作為臨床心肌病的一個指征。Rbm24可能通過調(diào)節(jié)其下游的靶標(biāo)mRNA以及與相關(guān)蛋白的相互作用,在心肌分化和心肌細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 (偽 -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.
【學(xué)位授予單位】：廈門大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R54

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本文編號：1657052