摘要
目的:研究短发夹RNA(short hairpin RNA,shRNA)对人胰腺癌细胞PANC-1中突变型K-ras基因表达的抑制作用.方法:构建特异性针对胰腺癌PANC-1细胞突变型K-ras基因的重组质粒pGensil-1-P1,阴性对照质粒pGensil-1-HK并采用脂质体介导的方法转染胰腺癌细胞.应用逆转录聚合酶链反应(RT-PCR)和蛋白免疫印迹(Westernblot)检测K-ras基因mRNA和蛋白的表达,CCK-8方法测量细胞生长曲线.并建立人胰腺癌细胞PANC-1裸鼠皮下移植瘤模型,分别注射Pgenesil-1-P1、Pgenesil-1-HK质粒,动态观察并处死裸鼠计算肿瘤体积.结果:测序证实质粒表达载体构建成功,短发夹RNA(shRNA)使胰腺癌细胞PANC-1突变型K-ras基因的mRNA较未治疗组、阴性对照组相比明显减少(P=0.01);未治疗组、阴性对照组及治疗组的K-ras蛋白表达率分别为100%,99.0%±0.73%,39.9%±2.1%,前两组K-ras蛋白表达差异无统计学意义,治疗组较未治疗组K-ras蛋白表达下调了约60.1%;细胞生长受到明显抑制(P=0.02);经重组质粒治疗14d后,裸鼠皮下移植瘤生长缓慢,相较于对照组瘤体明显缩小(P=0.03).结论:shRNA对特异性转染组的胰腺癌细胞的突变K-ras基因表达有抑制作用,使细胞以及裸鼠移植瘤的生长受到抑制.
AIM： To study the specific inhibitory effect of short hairpin RNA （shRNA） on the mutant K-ras gene expression in PANC-1 cells.
METHODS： Plasmid pGensil-1 recombinant plasmids targeting mutant K-ras gene was established. After transfection into PANC-1 cells,the expression of K-ras gene was detected using RT-PCR, Western blot techniques, and the effect of cell proliferation was determined by CCK-8 test. And a tumor model of nude mouse was constructed through rejecting the recombination plasmids to observe the volume of tumor.
RESULTS： The recombinant plasmid encoding mutant-specific shRNA for K-ras inhibited the expression of mutant K-ras significantly （P = 0.01）. Rates of K-ras protein expression in nontreatment group, negative control group and treatment group were 100%, 99.0% ± 0.73%, 39.9% ± 2.1%, respectively. No significant difference in K-ras protein expression was observed between non-treatment group and negative control group. A 60.1% reduction in K-ras protein expression from the treatment group to nontreatment group indicated that cell growth was significantly inhibited （P = 0.02）; and after fourteen day＇s recombinant plasmid treatment, the volume of tumor grew slowly in nude mice and was significantly decreased compared with the control group （P = 0.03）.
CONCLUSION： The specific shRNA inhibits the K-ras expression efficiently, and the growth of cells and nude mice is inhibited.
出处
《世界华人消化杂志》
CAS
北大核心
2008年第35期3941-3945,共5页
World Chinese Journal of Digestology
