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目的:研究沉默大鼠赖氨酸乙酰基转移酶7(lysine acetyltransferase 7,KAT7)基因对亚溶解型(sublytic)C5b-9复合物刺激大鼠肾小球系膜细胞(glomerular messangial cell,GMC)诱导单核趋化蛋白-1(monocyte chemotactic protein-l,MCP-1)生成的影响.方法:针对KAT7基因不同位点,用DNA重组技术设计4个小发夹RNA(short hairpin RNA,shRNA),分别克隆到真核表达载体pGCsi-U6/Neo/GFP/shRNA中.之后,用NeonTM电转仪将上述质粒转染至体外培养的GMC中,再给予sublytic C5b-9刺激.行Western blot实验检查筛选出针对KAT7基因且有最佳沉默效率的shRNA.此外,用real-time PCR和ELISA法分别检测GMC行不同分组处理后MCP-1的mRNA和蛋白水平.结果:核酸测序表明,重组的shKAT7质粒构建成功.Western blot显示,shKAT7-2是具备最佳沉默效率的shRNA.用shKAT7转染GMC后,由sublytic C5b-9刺激GMC诱导的MCP-1基因表达水平显著下降.结论:成功构建了大鼠KAT7 shRNA真核表达质粒,并初步证实KAT7基因的表达能够促进sublytic C5b-9刺激大鼠GMC诱导趋化因子MCP-1的合成与分泌.“,”Objective:To explore the effect of silencing rat(lysine acetyltransferase 7)KAT7 gene on the production of monocyte chemotactic protein-1(MCP-1)in rat glomerular messangial cells(GMC)by sublytic C5b-9 stimulation. Methods:Four kinds of short hairpin RNA(shRNA)stargeting KAT7 gene were synthesized and cloned into eukaryotic expression vector pGCsi-U6/Neo/GFP/ shRNA. The recombinant plasmids were transfected into cultured rat GMC by NeonTM transfection system, and KAT7 protein in the transfected cells was detected by Western blot to find out the optimal shRNA against KAT7 gene. Moreover, the expression of MCP-1 both in GMC and in the supernatant was measured by real-time PCR and ELISA assays. Results:Nucleotide sequencing demonstrated that the constructed KAT7 shRNAs were correct. Western blot experiment showed that the shKAT7-2 could effectively silence the target gene. Meanwhile, after the knockdown of KAT7 by shRNA in the GMC, the production of MCP-1 was significantly decreased upon sublytic C5b-9 stimulation. Conclusion:The rat eukaryotic expression vector shKAT7 was successfully constructed. It is preliminarily confirmed that the expression of KAT7 could obviously promote the production of MCP-1 in rat GMC treated by sublytic C5b-9.