钙调神经磷酸酶Aβ小干扰RNA筛选及对心肌细胞肥大的抑制  

Sieving CaN AβsiRNA Sequence and Its Relative Inhibition of Myocardial Hypertrophy on the Cellular Level

作　　者：齐迎春[1] 谢晓华[1] 陈雯[1] 李朝晖 

机构地区：[1]解放军总医院南楼综合外一科,北京100853

出　　处：《中华保健医学杂志》2012年第2期94-97,共4页Chinese Journal of Health Care and Medicine

基　　金：军队"十五"医药卫生科研基金重点课题(02Z0010);军队"十一五"科技攻关课题(06G117)

摘　　要：目的在细胞水平上筛选钙调神经磷酸酶(CaN)Aβ基因(mRNA)的小干扰RNA(siRNA)的干扰序列,为研究抑制在体心肌肥大的方法提供理论基础。方法针对CaNAβmRNA,参照siRNA设计原则,应用软件设计候选干扰序列,构建相应的小发夹RNA表达载体(si1053,si1280,si1539)。培养乳鼠心肌细胞,分为6组:空白对照组,肥大模型组(醛固酮诱导),肥大+si1280组,肥大+si1539组,肥大+si1053组,肥大+siGFP阴性对照组(转染针对GFP特定序列的shRNA质粒)。48h后显微镜下测量心肌细胞直径、CaNAβ及心房利钠因子(ANF)、β-重链肌球蛋白(β-MHC)mRNA水平。结果肥大模型组心肌细胞直径以及心肌组织CaNAβ、ANF、β-MHCmRNA表达水平较空白对照组明显增加(P<0.05)。肥大+si1053、肥大+si1280、肥大+si1539组细胞直径以及CaNAβ、ANFmRNA水平均较肥大模型组及肥大+siGFP阴性对照组降低(P<0.05),3组之间的CaNAβmRNA水平无明显差异(P>0.05),其中肥大+si1280组的细胞直径、ANF及β-MHCmRNA水平较肥大+si1053组、肥大+si1539组有明显减低(P<0.05)。结论成功构建CaNAβmRNA的小发夹RNA表达质粒,siRNA抑制CaNAβmRNA的表达,可抑制细胞水平醛固酮诱导的心肌细胞肥大;其中1280(21bp)为RNA干扰的相对更有效片段。Objective This study was designed to sieve small interfering RNA sequence targeting calcineurin Aβ,and to study the effect of CaN Aβ gene silencing on myocardial hypertrophy on the cellular level.Methods We designed and constructed shRNA expression plasmid targeting 3 different sites of CaN Aβ gene,that is,si1280,si1053,si1539.Cultured myocardial cells were divided into 6 groups：Blank group;hypertrophic model group;hypertrophy＋si1280 group,hypertrophy＋si1053 group,hypertrophy＋si1539 group,and hypertrophy＋siGFP group（negative control）.Diameters of myocytes was measured and mRNA levels of CaN Aβ,ANF,β-MHC gene were detected 48 hours after transfection.Results The mRNA level of CaN Aβ,ANF and β-MHC gene,and cellular diameters increased of model group compared to blank group（P0.05）.The cellular diameter and CaN Aβ,ANF mRNA level of si1053,si1280,si1539 groups decreased（P0.05）.There was no difference between three positive transfection group in CaN Aβ mRNA levels（P0.05）.While,of si1280 group,the diameter,the mRNA level of ANF and β-MHC were lower than that from other two groups.Conclusions The shRNA expressing plasmid was constructed successfully.CaN Aβ gene silencing could inhibit myocardial hypertrophy on cultured cellular level.Si1280（21bp）of CaNAβ was one of the most effective RNAi target site.

关 键 词：RNA干扰 钙调神经磷酸酶Aβ 转染 心肌细胞 

分 类 号：R542.2[医药卫生—心血管疾病] R342[医药卫生—内科学]