本文選題：siRNA + RNA干擾　； 參考：《第四軍醫(yī)大學(xué)》2008年碩士論文

【摘要】： RNA編輯(RNA editing)是DNA轉(zhuǎn)錄成RNA后, RNA編輯酶(RNA editing enzyme)對前體mRNA中的核苷酸進(jìn)行刪除、添加或重新修飾的過程。它改變了基因的序列以及遺傳信息,從而產(chǎn)生廣泛的生理效應(yīng)。RNA編輯酶以脫氨方式使得RNA特異位點(diǎn)的腺嘌呤脫氨轉(zhuǎn)變?yōu)榇吸S嘌呤,或者是特異位點(diǎn)的胞嘧啶變?yōu)槟蜞奏?而次黃嘌呤在堿基配對時(shí)被識別為鳥嘌呤。這些替換使得原有遺傳密碼或者剪切位點(diǎn)發(fā)生轉(zhuǎn)變,從而使其編碼蛋白質(zhì)的結(jié)構(gòu)和功能發(fā)生改變。ADAR1 (RNA依賴性腺嘌呤脫氨酶,adenosine deaminase acting on RNA)是研究最為廣泛的一種RNA編輯酶。研究顯示淋巴細(xì)胞增殖時(shí)ADAR1表達(dá)顯著升高,而降低ADAR1表達(dá)后淋巴細(xì)胞殺傷功能顯著降低。提示ADAR1可能通過調(diào)控細(xì)胞功能進(jìn)而影響排斥反應(yīng)的發(fā)生。據(jù)此我們前期通過小鼠雙向淋巴細(xì)胞的培養(yǎng)實(shí)驗(yàn)已經(jīng)證實(shí)降低ADAR1的表達(dá)能夠抑制淋巴細(xì)胞的增殖,本實(shí)驗(yàn)借助排斥反應(yīng)模型即雙向淋巴細(xì)胞混合培養(yǎng),通過ADAR1特異siRNA作用,抑制ADAR1的表達(dá),觀察淋巴細(xì)胞的細(xì)胞周期各期的變化,以及細(xì)胞凋亡的差異。進(jìn)一步揭示ADAR1影響淋巴細(xì)胞增殖的機(jī)制。 目的:探討RNA編輯酶ADAR1的表達(dá)受抑制后,小鼠淋巴細(xì)胞的細(xì)胞周期和細(xì)胞凋亡的變化。 方法:用電轉(zhuǎn)法將ADAR1特異性siRNA轉(zhuǎn)入到處于混合培養(yǎng)的小鼠淋巴細(xì)胞中,培養(yǎng)48h,用RT-PCR檢測轉(zhuǎn)染效率;而后用流式細(xì)胞儀檢測,觀察細(xì)胞的周期和凋亡變化;最后通過RT-PCR檢驗(yàn)周期蛋白E(cyclin E)基因和周期蛋白A1(cyclin A1)基因表達(dá)量的變化。 結(jié)果:轉(zhuǎn)染ADAR1特異性siRNA 48h后,小鼠淋巴細(xì)胞G1期細(xì)胞量增加,S期細(xì)胞量減少,G2細(xì)胞量保持恒定。另外,cyclin E基因的表達(dá)量降低而cyclin A1基因表達(dá)保持恒定。 結(jié)論:處于經(jīng)典混合培養(yǎng)體系下的小鼠淋巴細(xì)胞,在其RNA編輯酶ADAR1受到特異siRNA抑制后,小鼠淋巴細(xì)胞的增殖受到明顯抑制,發(fā)生G1期到S期細(xì)胞的轉(zhuǎn)化受阻,細(xì)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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R392

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