文章摘要
张 婧,何风霞,邱 文,赵 聃,卢燕来,王迎伟.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[J].南京医科大学学报,2017,(3):275~280
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
Effect of silencing p300 gene by RNA interference on apoptosis and ATF3 acetylation in rat glomerular messangial cells stimulated by sublytic C5b-9
投稿时间:2016-07-08  
DOI:10.7655/NYDXBNS20170304
中文关键词: RNA干扰  p300  亚溶解型C5b-9  凋亡  乙酰化
英文关键词: RNA interference  p300  sublytic C5b-9  apoptosis  acetylation
基金项目: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)
作者单位
张 婧 南京医科大学免疫学系江苏 南京 211166 
何风霞 南京医科大学免疫学系江苏 南京 211166 
邱 文 南京医科大学免疫学系江苏 南京 211166 
赵 聃 南京医科大学免疫学系江苏 南京 211166 
卢燕来 南京医科大学免疫学系江苏 南京 211166 
王迎伟 南京医科大学免疫学系江苏 南京 211166 
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中文摘要:
      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。
英文摘要:
      Objective:To construct rat p300 short hairpin RNA (shRNA) eukaryotic expression vector,and investigate the effect of silencing p300 gene on the apoptosis and activating transcription factor 3(ATF3) acetylation in rat glomerular messangial cells (GMCs) stimulated by sublytic C5b-9. Methods:Three kinds of shRNAs targeting p300 gene were synthesized and cloned into eukaryotic expression vector pGCsi-U6/Neo/GFP/shRNA. The recombinant plasmids were transfected into cultured GMCs by NeonTM transfection system. P300 protein in the transfected cells was detected by Western blot to find out the optimal shRNA against p300 gene. The cell apoptosis was measured by flow cytometry and the acetylation of ATF3 was evaluated by Western blot combined with immunoprecipitation (IP) assays. Results:It was verified by nucleotide sequencing that the constructed p300 shRNAs were correct. Western blot assay showed that the p300 shRNA-2 was able to silence the target gene most effectively. Knockdown of p300 by shRNA in the GMCs reduced the number of GMC apoptosis as well as the level of ATF3 acetylation induced by sublytic C5b-9. Conclusion:The rat eukaryotic expression vector p300 shRNA was successfully constructed. It was preliminarily confirmed that p300 could promote GMC apoptosis triggered by sublytic C5b-9 through p300-regulated ATF3 acetylation.
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