RNA编辑酶ADAR1对小鼠淋巴细胞周期和凋亡的影响

发布时间：2018-04-19 19:36

本文选题：siRNA + RNA干扰　；参考：《第四军医大学》2008年硕士论文

【摘要】： RNA编辑(RNA editing)是DNA转录成RNA后, RNA编辑酶(RNA editing enzyme)对前体mRNA中的核苷酸进行删除、添加或重新修饰的过程。它改变了基因的序列以及遗传信息,从而产生广泛的生理效应。RNA编辑酶以脱氨方式使得RNA特异位点的腺嘌呤脱氨转变为次黄嘌呤,或者是特异位点的胞嘧啶变为尿嘧啶,而次黄嘌呤在碱基配对时被识别为鸟嘌呤。这些替换使得原有遗传密码或者剪切位点发生转变,从而使其编码蛋白质的结构和功能发生改变。ADAR1 (RNA依赖性腺嘌呤脱氨酶,adenosine deaminase acting on RNA)是研究最为广泛的一种RNA编辑酶。研究显示淋巴细胞增殖时ADAR1表达显著升高,而降低ADAR1表达后淋巴细胞杀伤功能显著降低。提示ADAR1可能通过调控细胞功能进而影响排斥反应的发生。据此我们前期通过小鼠双向淋巴细胞的培养实验已经证实降低ADAR1的表达能够抑制淋巴细胞的增殖,本实验借助排斥反应模型即双向淋巴细胞混合培养,通过ADAR1特异siRNA作用,抑制ADAR1的表达,观察淋巴细胞的细胞周期各期的变化,以及细胞凋亡的差异。进一步揭示ADAR1影响淋巴细胞增殖的机制。目的:探讨RNA编辑酶ADAR1的表达受抑制后,小鼠淋巴细胞的细胞周期和细胞凋亡的变化。方法:用电转法将ADAR1特异性siRNA转入到处于混合培养的小鼠淋巴细胞中,培养48h,用RT-PCR检测转染效率;而后用流式细胞仪检测,观察细胞的周期和凋亡变化;最后通过RT-PCR检验周期蛋白E(cyclin E)基因和周期蛋白A1(cyclin A1)基因表达量的变化。结果:转染ADAR1特异性siRNA 48h后,小鼠淋巴细胞G1期细胞量增加,S期细胞量减少,G2细胞量保持恒定。另外,cyclin E基因的表达量降低而cyclin A1基因表达保持恒定。结论:处于经典混合培养体系下的小鼠淋巴细胞,在其RNA编辑酶ADAR1受到特异siRNA抑制后,小鼠淋巴细胞的增殖受到明显抑制,发生G1期到S期细胞的转化受阻,细胞凋亡增加。
[Abstract]:RNA editing is a process in which DNA is transcribed into RNA, and RNA editing enzyme editing enzyme removes, adds or remodifies nucleotides from precursor mRNA. It changes the sequence of genes and genetic information, which produces a wide range of physiological effects. RNA-editing enzymes deaminize adenine to Hypoxanthine at RNA specific sites, or cytosine from specific sites to uracil. Hypoxanthine is identified as guanine in base pairs. These substitutions change the original genetic code or the splicing site, and change the structure and function of the encoded protein. Adenosine deaminase acting on RNA-dependent adenosine acting is one of the most widely studied RNA editing enzymes. The results showed that the expression of ADAR1 increased significantly during lymphocyte proliferation, but the cytotoxicity of lymphocytes decreased after decreasing the expression of ADAR1. The results suggest that ADAR1 may affect the occurrence of rejection by regulating cell function. It has been proved that the reduction of the expression of ADAR1 can inhibit the proliferation of lymphocytes through the bidirectional lymphocyte culture of mice in the early stage. In this experiment, the effect of ADAR1 specific siRNA was obtained by using the model of rejection, i.e., the mixed culture of bidirectional lymphocytes. The expression of ADAR1 was inhibited and the changes of cell cycle and apoptosis were observed. Furthermore, the mechanism of ADAR1 affecting lymphocyte proliferation was revealed. Aim: to investigate the changes of cell cycle and apoptosis of mouse lymphocytes after the expression of RNA editing enzyme ADAR1 was inhibited. Methods: ADAR1 specific siRNA was transfered into murine lymphocytes cultured in mixed culture for 48h, then RT-PCR was used to detect transfection efficiency, and flow cytometry was used to detect the changes of cell cycle and apoptosis. Finally, the expression of cyclin E(cyclin E) gene and cyclin A1(cyclin A 1 gene were detected by RT-PCR. Results: after transfection of ADAR1 specific siRNA for 48 h, the number of lymphocytes in G 1 phase increased and the number of G 2 cells decreased in S phase. In addition, the expression of cyclin E gene decreased while the expression of cyclin A1 gene remained constant. Conclusion: after the RNA editing enzyme ADAR1 was inhibited by specific siRNA, the proliferation of mouse lymphocytes was significantly inhibited, the transformation from G1 phase to S phase was blocked, and apoptosis was increased.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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