PYK_2小干扰RNA对成年大鼠心脏成纤维细胞增殖的影响

Effect of PYK_2 gene siRNA on proliferation of adult rat cardiac fibroblasts

目的以富含脯氨酸的酪氨酸激酶2(PYK2)为靶基因,设计并构建短发夹状小干扰RNA表达载体,探讨PYK2小干扰RNA表达载体对心脏成纤维细胞(cardiac fibroblasts,CFs)增殖的影响。方法以pAVU6+27质粒为载体,以PYK2为靶基因,设计并构建短发夹状小干扰RNA表达载体,行酶切和测序鉴定。用PYK2短发夹状小干扰RNA表达载体转染体外培养的成年大鼠心脏成纤维细胞,反转录聚合酶链反应和Westernblot法检测PYK2基因和蛋白表达的改变,四唑盐比色法和3H-TdR掺入率检测转染后细胞的增殖和DNA合成情况。结果酶切鉴定和测序分析表明靶向PYK2的RNA干扰重组体构建成功;重组体转染CFs后,PYK2的mRNA和蛋白水平较空载体转染的对照组显著下降;重组体转染CFs后MTT比色A值和3H-TdR掺入率均明显低于对照组。结论PYK2小干扰RNA表达载体是抑制心脏成纤维细胞增殖和DNA合成的有效手段,有利于深入探讨PYK2在心肌纤维化发生发展中的作用。

Objective To design and construct the vector of protein-rich tyrosine kinase 2 small RNA interference and investigate the effect of the recombinant plasmid on the proliferation of adult rat cardiac fibroblasts. Methods The pAVU6 ＋ 27-PYK2 siRNA expression vector was constructed by gene recombination, then transfected into the cultured adult rat cardiac fibroblasts by DOTAP method. PYK2 mRNA and protein in cardiac fibroblasts were determined by RT-PCR and Western blotting respectively. Cell proliferation was tested by MTT and 3 H-TdR incorporation. Results The pAVU6 ＋ 27-PYK2 siRNA expression vector was successfully constructed. The recombinant plasmid can inhibit the proliferation of adult rat cardiac fibroblasts, as compared with control. Conclusion pAVU6 ＋27-PYK2 siRNA expression vector is efficient to suppress DNA synthesis of cardiac fibroblasts, which will be beneficial to further study of myocardial fibrosis.