干预G蛋白βγ亚基介导的信号转导途径对心肌细胞肥大的影响

Effects of inhibiting G-protein βγ dimers on signal transduction pathway and cardiac myocyte hypertrophy

目的研究G蛋白!"亚基在心肌细胞肥大中的作用。方法采用细菌内同源重组方法制备重组腺病毒载体rAd-!ARKct50MOI(multiplicityofinfection)感染经去甲肾上腺素处理肥大乳鼠心肌细胞,荧光倒置显微镜下观察心肌细胞形态及绿色荧光蛋白的表达,EMAIL图像分析软件测定心肌细胞的表面积,RT-PCR检测!ARKct、#-MHC和!-MHC的mRNA表达水平。结果经测序鉴定证实rAd-!ARKct构建成功,对心肌细胞的感染率40%～50%,与去甲肾上腺素组相比,重组腺病毒组#-MHC定量比值显著上升,!-MHC定量比值显著降低。结论编码!ARKct的重组腺病毒表达载体可以体外感染乳鼠心肌细胞并在心肌细胞中表达!ARKct;抑制G!"蛋白的功能,可显著逆转去甲肾上腺素介导的肌球蛋白重链表型的改变(#-MHC/!-MHC),逆转心肌细胞肥大。

Objectives To evaluate the potential function of Gβγ in the onset of cardiac muscle cell hypertrophy induced by norepinephrine(NE). Methods The gene of βARKct was cloned into a shuttle vector, pAdTrack-CMV. Transfecting hypertrophy Neonatal cardiomyocytes induced by NE with 50 MOI recombinant adenoviruses. Measured area of single cell with EMAIL pathologic picture analysis software. Total RNA was extracted from cells by using the TRIzol reagent. RT-PCR was performed to amplify the following genes fragments:βARKct ?琢-MHC,β-MHC,and GAPDH. Results Recombinant adenoviruses were confirmed by PCR with βARKct-specific primers. The diameter and area of infection group were significantly lower than NE group. The levels of β-MHC and ANF mRNA in infection group were significantly higher than those of control group (P<0.05). Conclusions Inhibition of Gβγ signaling pathway by overexpression of βARKct can significantly reverse cardiomyocyte hypertrophy in vitro.