RNA干扰沉默p300基因对亚溶解型C5b-9诱导大鼠肾小球系膜细胞凋亡和ATF3乙酰化的影响

发布时间：2019-01-27 17:31

【摘要】：目的:构建大鼠p300基因短发夹状小干扰RNA(short hairpin RNA,shRNA)真核表达质粒,观察沉默p300基因对亚溶解型(sublytic)C5b-9诱导大鼠肾小球系膜细胞(glomerular messangial cell,GMC)凋亡和激活转录因子3(activating transcription factor 3,ATF3)乙酰化水平的影响。方法:用DNA重组技术针对p300基因不同位点设计3个shRNA序列,然后分别克隆到真核表达载体pGCsi-U6/Neo/GFP/shRNA中,经电转染入GMC,再给予sublytic C5b-9刺激。Western blot筛选干涉效果最佳的p300 shRNA,流式细胞术检测GMC凋亡率,免疫沉淀(immunoprecipitation,IP)和Western blot联合检查ATF3乙酰化水平。结果:核酸测序表明p300 shRNA重组质粒构建成功。Western blot显示p300 shRNA-2具有最佳沉默效率。p300 shRNA处理GMC后,由sublytic C5b-9诱导的GMC凋亡率显著下降,同时ATF3乙酰化水平也明显降低。结论:成功构建了大鼠p300 shRNA真核表达质粒,并初步证实了p300能够促进sublytic C5b-9诱导的GMC凋亡,其机制可能与p300乙酰化修饰ATF3有关。
[Abstract]:Objective: to construct the eukaryotic expression plasmid of rat p300 gene short hairpin small interfering RNA (short hairpin RNA,shRNA and to observe the effect of silencing p300 gene on (glomerular messangial cell, of rat glomerular Mesangial cells induced by sublytic (sublytic) C5b-9. GMC) apoptosis and activation of transcription factor 3 (activating transcription factor 3 ATF 3) acetylation level. Methods: three shRNA sequences were designed for different sites of p300 gene by DNA recombination technique, and then were cloned into eukaryotic expression vector pGCsi-U6/Neo/GFP/shRNA and transfected into GMC, by electrotransfection. Then sublytic C5b-9 stimulated. Western blot was used to screen out the best interference effect. The apoptosis rate of GMC was detected by flow cytometry with p300 shRNA, and the ATF3 acetylation level was detected by combined immunoprecipitation (immunoprecipitation,IP) and Western blot. Results: nucleic acid sequencing showed that p300 shRNA-2 had the best silencing efficiency when the p300 shRNA recombinant plasmid was successfully constructed, the GMC apoptosis rate induced by sublytic C5b-9 was significantly decreased and the level of ATF3 acetylation was significantly decreased after GMC was treated with p300 shRNA. Conclusion: rat p300 shRNA eukaryotic expression plasmid was successfully constructed, and it was preliminarily confirmed that p300 can promote GMC apoptosis induced by sublytic C5b-9. The mechanism may be related to p300 acetylation modified ATF3.
【作者单位】：南京医科大学免疫学系;
【基金】：江苏省高校自然科学研究面上项目(14KJB310006) 南京医科大学基础医学院优势学科教师培养基金(JX10131802/007)
【分类号】：R692

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