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目的:用RNA干扰技术(RNA interference,RNAi)选择性下调大鼠心肌细胞上乌苷酸结合蛋白(Gs)α亚基(Gs protein alphala subunit,Gsα)的表达,筛选出抑制Gsα蛋白效果最明显的特异短发夹RNA(short hairpin RNA,shRNA)表达质粒。方法:构建3条靶向gnas基因的特异与RNA质粒载体(shRNA1-3),以含非特异性shRNA编码序列的质粒载体为阴性对照(HK),用脂质体LipofectamineTMLTX&PLUS转染原代培养的大鼠心肌细胞,并设空白对照组,转染后通过半定量RT-PCR和Western blot法检测GsαmRNA和蛋白的表达情况,以内参照三磷酸甘油醛脱氢酶(GAPDH)进行标化。结果:shRNA1-3均使Gsα的mRNA和蛋白质表达明显降低,差异有统计学意义(P<0.01),且shRNA3的抑制效果最显著,阴性对照质粒组与空白对照组Gsα表达差异无统计学意义。结论:利用RNAi技术成功下调了原代培养的心肌细胞中Gsα的表达,并筛选出沉默效果最明显的shRNA真核表达质粒,为心肌细胞的转染提供了可行的方法。
OBJECTIVE: To selectively downregulate the expression of Gs alpha subunit (Gs alpha) on rat cardiomyocytes by RNA interference (RNAi), and to screen out the most obvious inhibitory effect of Gs alpha protein Of short hairpin RNA (shRNA) expression plasmid. METHODS: Three specific and RNA plasmid vectors (shRNA1-3) targeting gnas gene were constructed. The plasmid vector containing the non-specific shRNA coding sequence was used as a negative control (HK). LipofectamineTMLTX & PLUS The control group was established. After transfection, the expression of GsαmRNA and protein was detected by semi-quantitative RT-PCR and Western blot, and glycosylphosphate triphosphate dehydrogenase (GAPDH) was used for internalization. Results: shRNA1-3 mRNA and protein expression of Gsα significantly decreased, the difference was statistically significant (P <0.01), and the inhibitory effect of shRNA3 most significant, the negative control plasmid group and the blank control group Gsα expression was not statistically significant . CONCLUSION: The expression of Gsα in primary cultured cardiomyocytes was successfully down-regulated by RNAi technology and the most obvious shRNA eukaryotic expression plasmid was screened out, which provided a feasible method for transfection of cardiomyocytes.