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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。
OBJECTIVE: To construct a eukaryotic expression plasmid of short hairpin RNA (shRNA) of rat p300 gene and to observe the effect of silencing p300 gene on the expression of subunit C5b-9 induced rat mesangial cells glomerular messangial cell (GMC) and the activation of activating transcription factor 3 (ATF3). Methods: Three shRNA sequences were designed according to the different sites of p300 gene by DNA recombination technique, then cloned into eukaryotic expression vector pGCsi-U6 / Neo / GFP / shRNA respectively and electroporated into GMC. Sublytic C5b-9 stimulate. The p300 shRNA with the best interference effect was screened by Western blot. The apoptosis rate of GMC was detected by flow cytometry. The ATF3 acetylation level was detected by immunoprecipitation (IP) and Western blot. Results: Nucleic acid sequencing showed that p300 shRNA recombinant plasmid was successfully constructed. Western blot showed p300 shRNA-2 had the best efficiency of silencing. After treated with p300 shRNA, the apoptosis rate of GMC induced by sublytic C5b-9 was significantly decreased, and the ATF3 acetylation level was also significantly decreased. CONCLUSION: The eukaryotic expression plasmid of p300 shRNA was successfully constructed and p300 can preliminarily confirm that p300 can promote the apoptosis of GMC induced by sublytic C5b-9. The mechanism may be related to p300 acetylation modification of ATF3.