摘要
目的研究C-Jun氨基末端激酶1(JNK1)基因表达沉默对肺成纤维细胞MRC-5增殖的影响。方法设计针对JNK1基因的小RNA分子(siRNA),以脂质体转染法将双链siRNA转入MRC-5细胞后,采用流式细胞术检测最佳转染浓度,RT-PCR检测JNK1 mRNA表达变化,Western blot检测总JNK、磷酸化JNK(P-JNK)蛋白表达,用CCK-8法检测siRNA对MRC-5细胞增殖的影响。结果 siRNA转染MRC-5细胞的最佳浓度为50nmol/L;设计的两个靶位点siRNA,均可有效降低JNK1 mRNA表达,其中siRNA1抑制效果较siRNA2明显;siRNA1降低总JNK及P-JNK蛋白表达,有效抑制MRC-5细胞的增殖。结论 体外合成的siRNA可降低MRC-5细胞中JNK1基因的表达,降低JNK磷酸化水平,明显抑制细胞增殖。
Objective To investigate the effects of lipofectamin mediated siRNA targeting C - Jun N - terminal kinase （JNK1） on the proliferation of human lung fibroblasts, so as to reveal the relationship between JNK1 and cell proliferation in ashthma. Methods siR- NA sequence against JNK1 and nonsense sense oligonueleotides were designed. After recombinant being produled,flow eytometry was used to detect the optimal concentration of siRNA to transfect. RT - PCR and Western blot were used to test knockdown effect in MRC - 5 cells. CCK - 8 was used to observe cell proliferation. Results The optimal concentration of transfection is 50nmol/L. RT - PCR and Western blot results indicated that the expression of JNK1 was significantly decreased compared with the other control groups. CCK - 8 revealed that cell proliferation rate was markedly inhibited in JNK1 RNAi group in comparison with control groups. Conclusion Recombinant lipo- fectamin expressing siRNA targeting JNK1 was constructed successfully, and cell proliferation was significantly inhibited after JNK1 knockdown in MRC -5 ,which paves the way for further research on JNK pathway involved in development of asthma.
出处
《医学研究杂志》
2013年第3期34-37,共4页
Journal of Medical Research
基金
国家自然科学基金资助项目(30973232)
关键词
JNK1
RNA干扰
细胞增殖
C - Jun N - terminal kinasel
RNA interference
Cell proliferation
