發(fā)布時(shí)間：2018-06-29 22:47

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

【摘要】：目的:構(gòu)建并鑒定大鼠野生型激活轉(zhuǎn)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(dá)質(zhì)粒,并觀察其在大鼠腎小球系膜細(xì)胞(glomerular mesangial cells, GMCs)中過表達(dá)及沉默ATF3基因的情況。方法:(1)用DNA重組技術(shù)構(gòu)建大鼠ATF3基因真核表達(dá)質(zhì)粒pcDNA3.1/ATF3和構(gòu)建針對ATF3基因不同位點(diǎn)的五個(gè)shRNA序列的真核表達(dá)質(zhì)粒ATF3 shRNA1-5。(2)體外分別將pcDNA3.1/ATF3質(zhì)�；駻TF3 shRNA1-5質(zhì)粒轉(zhuǎn)染GMCs,篩選pcDNA3.1/ATF3和ATF3 shRNA轉(zhuǎn)染最佳時(shí)間,并將GMCs作相應(yīng)的分組處理。Western blot檢測ATF3蛋白的相對表達(dá)量來鑒定pcDNA3.1/ATF3的表達(dá)及篩選最佳沉默效率shRNA。結(jié)果:(1)核酸序列測定表明,pcDNA3.1/ATF3質(zhì)粒及ATF3 shRNA質(zhì)粒構(gòu)建正確。Western blot證實(shí),pcDNA3.1/ATF3質(zhì)粒成功表達(dá)ATF3蛋白,ATF3 shRNA-1具有最佳沉默效率。結(jié)論:(1)成功構(gòu)建了大鼠野生型ATF3基因真核表達(dá)質(zhì)粒pcDNA3.1/ATF3。(2)成功構(gòu)建并篩選出最佳沉默效率的大鼠ATF3 shRNA-1。該結(jié)果為進(jìn)一步研究ATF3基因在亞溶解型補(bǔ)體C5b-9(sublytic C5b-9)誘導(dǎo)GMCs凋亡病變中的作用奠定了基礎(chǔ)。 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(dá)水平的變化,探討過表達(dá)或靶向沉默ATF3基因?qū)ublytic C5b-9誘導(dǎo)GMCs凋亡病變的影響。方法:(1)以sublytic C5b-9刺激GMCs為實(shí)驗(yàn)?zāi)Ｐ?應(yīng)用RT-PCR、Real-time PCR、Western blot和免疫組化技術(shù)檢查ATF3 mRNA豐度和蛋白表達(dá)水平,用Annexin V(AV)- propidium iodide (PI)雙染后流式細(xì)胞儀定量分析GMCs凋亡數(shù)量。(2)體外將pcDNA3.1/ATF3質(zhì)�；駻TF3 shRNA質(zhì)粒瞬時(shí)轉(zhuǎn)染GMCs,并將GMCs作相應(yīng)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(xì)胞儀定量分析GMCs凋亡數(shù)量。結(jié)果:(1)Sublytic C5b-9刺激GMCs后,ATF3 mRNA及蛋白表達(dá)能迅速增高,至3h達(dá)到峰值;ATF3蛋白主要分布在GMCs細(xì)胞核及核周;Sublytic C5b-9刺激的GMCs,細(xì)胞凋亡率較對照組顯著增高。(2)Sublytic C5b-9刺激的GMCs和轉(zhuǎn)染pcDNA3.1/ATF3質(zhì)粒的GMCs均呈現(xiàn)核Hoechst 33342染色致密增強(qiáng)或碎裂等凋亡形態(tài)學(xué)的改變;同時(shí),GMCs凋亡百分率也明顯增高。pcDNA3.1/ATF3質(zhì)粒轉(zhuǎn)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(zhuǎn)染ATF3 shRNA質(zhì)粒的GMCs再給予sublytic C5b-9刺激后,其GMCs凋亡率則顯著降低。結(jié)論:Sublytic C5b-9刺激GMCs能誘導(dǎo)GMCs中ATF3表達(dá)上調(diào)和細(xì)胞凋亡,而ATF3表達(dá)增加對GMCs的凋亡有促進(jìn)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(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R363

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王迎偉,何球藻,秦慧蓮,高豐光,湯仁仙;補(bǔ)體C5b-9抗血清對抗胸腺細(xì)胞血清性腎炎大鼠腎組織一氧化氮合成及其病變的影響[J];中國病理生理雜志;2000年09期

相關(guān)博士學(xué)位論文 前1條

1 王慧;Sublytic C5b-9誘導(dǎo)Thy-1腎炎GMCs凋亡的機(jī)制研究[D];南京醫(yī)科大學(xué);2008年

，

本文編號：2083678