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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 investigate the effect of CAMKⅡδ on cardiomyocyte hypertrophy and intracellular calcium concentration by down-regulating the expression of CAMKⅡδ in neonatal rat cardiomyocytes by RNAi technique. Methods: The plasmid vector pCAMKⅡδshRNA-1 and pCAMKδshRNA-2 encoding the specific short hairpin RNA (shRNA) coding sequence of CAMKⅡδ gene was constructed and transfected with plasmid pGenesil-HK (pHK) containing non-specific shRNA coding sequence as control Primary cultured neonatal rat cardiomyocytes were stained with GAPDH by semi-quantitative RT-PCR and Western blot. The concentration of calcium ion in myocardial cells of each group was analyzed by Fura-2 / AM combined with calcium fluorescence indicator. 3H-LEU incorporation was used to test the cardiomyocyte hypertrophy induced by Ang Ⅱ Impact. Results: ① pCAMKⅡδshRNA-1 reduced CAMKⅡδ mRNA and protein expression by 62% and 50% respectively compared with the control group, while pCAMKⅡδshRNA-2 reduced by 40% and 35% respectively. The difference was statistically significant. There was no significant difference in the expression of CAMKⅡδ between pHK and blank control group. ② 3H-LEU incorporation and intracellular calcium concentration in pCAMKⅡδshRNA-1 and pCAMKⅡδshRNA-2 groups were significantly lower than those in blank control group and control plasmid (P <0.01) after AngⅡ (10-7mol / L) stimulation for 48h. Conclusion: RNAi can selectively down-regulate the expression of CAMKⅡδ in primary cultured neonatal rat cardiomyocytes, and significantly inhibit the effect of AngⅡ on cell hypertrophy and intracellular calcium overload.