本文選題：激活轉錄因子3(ATF3) + shRNA�。� 參考：《南京醫(yī)科大學》2009年碩士論文

【摘要】：目的:構建并鑒定大鼠野生型激活轉錄因子3(activating transcription factor 3, ATF3)基因及大鼠ATF3特異性小發(fā)夾RNA(small hairpin RNA, shRNA)真核表達質粒,并觀察其在大鼠腎小球系膜細胞(glomerular mesangial cells, GMCs)中過表達及沉默ATF3基因的情況。方法:(1)用DNA重組技術構建大鼠ATF3基因真核表達質粒pcDNA3.1/ATF3和構建針對ATF3基因不同位點的五個shRNA序列的真核表達質粒ATF3 shRNA1-5。(2)體外分別將pcDNA3.1/ATF3質�；駻TF3 shRNA1-5質粒轉染GMCs,篩選pcDNA3.1/ATF3和ATF3 shRNA轉染最佳時間,并將GMCs作相應的分組處理。Western blot檢測ATF3蛋白的相對表達量來鑒定pcDNA3.1/ATF3的表達及篩選最佳沉默效率shRNA。結果:(1)核酸序列測定表明,pcDNA3.1/ATF3質粒及ATF3 shRNA質粒構建正確。Western blot證實,pcDNA3.1/ATF3質粒成功表達ATF3蛋白,ATF3 shRNA-1具有最佳沉默效率。結論:(1)成功構建了大鼠野生型ATF3基因真核表達質粒pcDNA3.1/ATF3。(2)成功構建并篩選出最佳沉默效率的大鼠ATF3 shRNA-1。該結果為進一步研究ATF3基因在亞溶解型補體C5b-9(sublytic C5b-9)誘導GMCs凋亡病變中的作用奠定了基礎。 目的:檢查大鼠GMCs受sublytic C5b-9刺激后,其ATF3基因mRNA豐度和蛋白表達水平的變化,探討過表達或靶向沉默ATF3基因對sublytic C5b-9誘導GMCs凋亡病變的影響。方法:(1)以sublytic C5b-9刺激GMCs為實驗模型,應用RT-PCR、Real-time PCR、Western blot和免疫組化技術檢查ATF3 mRNA豐度和蛋白表達水平,用Annexin V(AV)- propidium iodide (PI)雙染后流式細胞儀定量分析GMCs凋亡數(shù)量。(2)體外將pcDNA3.1/ATF3質�；駻TF3 shRNA質粒瞬時轉染GMCs,并將GMCs作相應的分組處理,再用Hoechst 33342染色檢查各組GMCs的凋亡形態(tài),并用AV-PI雙染后流式細胞儀定量分析GMCs凋亡數(shù)量。結果:(1)Sublytic C5b-9刺激GMCs后,ATF3 mRNA及蛋白表達能迅速增高,至3h達到峰值;ATF3蛋白主要分布在GMCs細胞核及核周;Sublytic C5b-9刺激的GMCs,細胞凋亡率較對照組顯著增高。(2)Sublytic C5b-9刺激的GMCs和轉染pcDNA3.1/ATF3質粒的GMCs均呈現(xiàn)核Hoechst 33342染色致密增強或碎裂等凋亡形態(tài)學的改變;同時,GMCs凋亡百分率也明顯增高。pcDNA3.1/ATF3質粒轉染的GMCs再給予sublytic C5b-9刺激后,上述現(xiàn)象更為明顯。與之相反,轉染ATF3 shRNA質粒的GMCs再給予sublytic C5b-9刺激后,其GMCs凋亡率則顯著降低。結論:Sublytic C5b-9刺激GMCs能誘導GMCs中ATF3表達上調和細胞凋亡,而ATF3表達增加對GMCs的凋亡有促進作用。
[Abstract]:Objective: to construct and identify the rat wild type activated transcription factor 3 (activating transcription factor 3 (ATF3) gene and the rat ATF3 specific small hairpin (shRNA) eukaryotic expression plasmid. The overexpression and silencing of ATF3 gene in rat glomerular Mesangial cells (glomerular mesangial cells, GMCs) were observed. Methods: (1) construct rat ATF3 eukaryotic expression plasmid pcDNA3.1% ATF3 by DNA recombination technique and construct ATF3 shRNA1-5based eukaryotic expression plasmid targeting five shRNA sequences of ATF3 gene. (2) transfect pcDNA3.1% ATF3 plasmid or ATF3 shRNA1-5 plasmid into GMCsin vitro, screen pcDNA3.1% ATF3 and pcDNA3.1pATF3. The best time for ATF3 shRNA transfection, The relative expression of ATF3 protein was detected by Western blot to identify the expression of pcDNA3.1% ATF3 and to screen the best silencing efficiency shRNA. Results: (1) nucleic acid sequence analysis showed that pcDNA3.1% ATF3 plasmid and ATF3 shRNA plasmid were constructed correctly. Western blot confirmed that pcDNA3.1% ATF3 plasmid successfully expressed ATF3 shRNA-1 had the best silencing efficiency. Conclusion: (1) the rat wild-type ATF3 gene eukaryotic expression plasmid pcDNA3.1% ATF3 / ATF3 was successfully constructed and the rat ATF3 shRNA-1 with optimal silencing efficiency was successfully constructed and screened. These results provide a basis for further study on the role of ATF3 gene in sublytic complement C5b-9 (sublytic C5b-9) -induced apoptosis of GMCs. Aim: to investigate the changes of ATF3 mRNA abundance and protein expression in rat GMCs stimulated by sublytic C5b-9, and to investigate the effect of overexpression or targeted silencing of ATF3 gene on sublytic C5b-9 induced apoptosis in GMCs. Methods: (1) using sublytic C5b-9 stimulated GMCs as the experimental model, RT-PCR real-time PCR blot and immunohistochemical technique were used to detect the abundance of ATF3 mRNA and the expression of ATF3 protein. Annexin V (AV) propidium iodide (Pi was used to analyze the apoptosis of GMCs by flow cytometry. (2) pcDNA3.1% ATF3 or ATF3 shRNA plasmids were transiently transfected into GMCs, then treated with corresponding groups. The apoptosis morphology of GMCs in each group was detected by Hoechst 33342 staining. The apoptosis of GMCs was analyzed by flow cytometry with double staining of AV-PI. Results: (1) the expression of ATF3 mRNA and protein in GMCs stimulated by Sublytic C5b-9 was increased rapidly. The peak value of ATF3 protein was mainly distributed in the GMCs nucleus and the GMCs stimulated by Sublytic C5b-9 at 3 h, and the apoptosis rate was significantly higher than that in the control group. (2) both the GMCs stimulated by Sublytic C5b-9 and those transfected with pcDNA3.1% ATF3 plasmid showed dense enhancement or fragmentation by Hoechst 33342 staining. Morphological changes of apoptosis; At the same time, the percentage of apoptosis of GMCs was also significantly increased. The above phenomenon was more obvious after GMCs transfected with pcDNA3.1% ATF3 plasmid were stimulated with sublytic C5b-9. In contrast, GMCs transfected with ATF3 shRNA plasmid were stimulated with sublytic C5b-9, and the apoptosis rate of GMCs decreased significantly. Conclusion the upregulation of ATF3 expression and apoptosis in GMCs can be induced by the stimulation of GMCs by 1: Sublytic C5b-9, and the increase of ATF3 expression can promote the apoptosis of GMCs.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R363

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