RNA干扰下调钙调蛋白激酶Ⅱ_δ表达对血管紧张素Ⅱ诱导心肌细胞肥大的影响  

Effect on RNA interference targeting CAMKⅡ_δ on cardiomyocyte hypertrophic responses induced by angiotensin Ⅱ

作　　者：王岚[1] 吕家高[1] 张存泰[1] 孙萌[1] 刘念[1] 阮燕菲[1] 王琳[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院心内科,武汉430030

出　　处：《临床心血管病杂志》2008年第5期378-382,共5页Journal of Clinical Cardiology

基　　金：国家自然科学基金项目(No:30470714)

摘　　要：目的:用RNA干扰技术下调乳鼠心肌细胞钙调蛋白激酶Ⅱδ(CAMKⅡδ)的表达,观察CAMKⅡδ对心肌细胞肥厚及细胞内钙浓度的影响。方法:构建针对CAMKⅡδ基因的特异性短发夹RNA(shRNA)编码序列的质粒载体pCAMKⅡδshRNA-1,pCAMKδshRNA-2,以含非特异性shRNA编码序列的质粒载体pGenesil-HK(pHK)为对照,转染原代培养的乳鼠心肌细胞,通过半定量RT-PCR和Western blot法检测CAMKⅡδ表达情况,以内参照GAPDH进行标化。用钙荧光指示剂Fura-2/AM结合图像分析各组心肌细胞内钙离子浓度,用3H-LEU掺入量来检验各组心肌细胞在血管紧张素Ⅱ(AngⅡ)刺激下促心肌细胞肥大效应的影响。结果:①pCAMKⅡδshRNA-1使CAMKⅡδ的mRNA和蛋白质表达较对照组分别降低62%和50%,pCAMKⅡδshRNA-2使之分别降低40%和35%,差异有统计学意义。pHK与空白对照组CAMKⅡδ表达差异无统计学意义。②给予AngⅡ(10-7mol/L)刺激48h后,pCAMKⅡδshRNA-1,pCAMKⅡδshRNA-2组的3H-LEU掺入量和细胞内钙浓度显著低于空白对照组和对照质粒(P<0.01)。结论:RNA干扰技术能选择性下调原代培养的乳鼠心肌细胞CAMKⅡδ的表达,并显著抑制AngⅡ的促细胞肥大和促细胞内钙超载的效应。Objective： To knockdown the expression of delta isform of Ca^2＋/calmodulin-dependent protein kinase Ⅱ （CAMK Ⅱδ） in neonatal rat cadiocytes by RNA interference, and to observe the changes of hypertrophic response and intracellular calcium concentration after down-regulation of CAMK Ⅱδ. Method： Primary cultured neonatal rat cadiocytes were transfected by plasmids pCAMK ⅡδshRNA-1 and pCAMK Ⅱδ shRNA-2 carrying a CAMK Ⅱδ-specific shRNA-coding template sequence, or by a control plasmid pGenesil-HK（pHK） carrying a nonspecific shRNA-coding sequence. The mRNA and protein expression of CAMK Ⅱδ were analyzed by RT-PCR and Western blot, respectively and standardized by the internal control gene GAPDH. Intracellular calcium concentration was measured by using the fluorecent Ca^2＋ indicator Fluo-2/AM. Neonatal rat cadiocytes＇ hypertrophic response was assayed by 3H-leucine incorporation. Result： ①CAMK Ⅱδ mRNA and protein were significantly reduced to 62% and 50% by pCAMK ⅡδshRNA-1, 40% and 35% by pCAMK ⅡδshRNA-2, respectively while no change was found in pHK treated myocytes. ②Intracellular free Ca^2＋ content and 3 H-leucine incorporation were significantly increased in the pCAMK ⅡδshRNA-1 and pCAMK ⅡδshRNA-2 group respectively in comparison to normal cell control and pHK group. Conclusion： RNA interference can selectively knockdown CAMK Ⅱδ expression in cultured cadiocytes and attenuate the effect Ang Ⅱ induced cadiocyte hypertrophic and calcium overload.

关 键 词：心肌肥厚 钙调蛋白激酶Ⅱ RNA干扰 质粒 转染 

分 类 号：R541.3[医药卫生—心血管疾病]