ATF3在sublyticC5b-9复合物诱导GMCs凋亡中的作用

发布时间：2018-06-29 22:47

  本文选题：激活转录因子3(ATF3) + shRNA　； 参考：《南京医科大学》2009年硕士论文

【摘要】：目的:构建并鉴定大鼠野生型激活转录因子3(activating transcription factor 3, ATF3)基因及大鼠ATF3特异性小发夹RNA(small hairpin RNA, shRNA)真核表达质粒,并观察其在大鼠肾小球系膜细胞(glomerular mesangial cells, GMCs)中过表达及沉默ATF3基因的情况。方法:(1)用DNA重组技术构建大鼠ATF3基因真核表达质粒pcDNA3.1/ATF3和构建针对ATF3基因不同位点的五个shRNA序列的真核表达质粒ATF3 shRNA1-5。(2)体外分别将pcDNA3.1/ATF3质粒或ATF3 shRNA1-5质粒转染GMCs,筛选pcDNA3.1/ATF3和ATF3 shRNA转染最佳时间,并将GMCs作相应的分组处理。Western blot检测ATF3蛋白的相对表达量来鉴定pcDNA3.1/ATF3的表达及筛选最佳沉默效率shRNA。结果:(1)核酸序列测定表明,pcDNA3.1/ATF3质粒及ATF3 shRNA质粒构建正确。Western blot证实,pcDNA3.1/ATF3质粒成功表达ATF3蛋白,ATF3 shRNA-1具有最佳沉默效率。结论:(1)成功构建了大鼠野生型ATF3基因真核表达质粒pcDNA3.1/ATF3。(2)成功构建并筛选出最佳沉默效率的大鼠ATF3 shRNA-1。该结果为进一步研究ATF3基因在亚溶解型补体C5b-9(sublytic C5b-9)诱导GMCs凋亡病变中的作用奠定了基础。 目的:检查大鼠GMCs受sublytic C5b-9刺激后,其ATF3基因mRNA丰度和蛋白表达水平的变化,探讨过表达或靶向沉默ATF3基因对sublytic C5b-9诱导GMCs凋亡病变的影响。方法:(1)以sublytic C5b-9刺激GMCs为实验模型,应用RT-PCR、Real-time PCR、Western blot和免疫组化技术检查ATF3 mRNA丰度和蛋白表达水平,用Annexin V(AV)- propidium iodide (PI)双染后流式细胞仪定量分析GMCs凋亡数量。(2)体外将pcDNA3.1/ATF3质粒或ATF3 shRNA质粒瞬时转染GMCs,并将GMCs作相应的分组处理,再用Hoechst 33342染色检查各组GMCs的凋亡形态,并用AV-PI双染后流式细胞仪定量分析GMCs凋亡数量。结果:(1)Sublytic C5b-9刺激GMCs后,ATF3 mRNA及蛋白表达能迅速增高,至3h达到峰值;ATF3蛋白主要分布在GMCs细胞核及核周;Sublytic C5b-9刺激的GMCs,细胞凋亡率较对照组显著增高。(2)Sublytic C5b-9刺激的GMCs和转染pcDNA3.1/ATF3质粒的GMCs均呈现核Hoechst 33342染色致密增强或碎裂等凋亡形态学的改变;同时,GMCs凋亡百分率也明显增高。pcDNA3.1/ATF3质粒转染的GMCs再给予sublytic C5b-9刺激后,上述现象更为明显。与之相反,转染ATF3 shRNA质粒的GMCs再给予sublytic C5b-9刺激后,其GMCs凋亡率则显著降低。结论:Sublytic C5b-9刺激GMCs能诱导GMCs中ATF3表达上调和细胞凋亡,而ATF3表达增加对GMCs的凋亡有促进作用。
[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.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R363

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