【摘要】 目的:构建靶向于卵巢癌耐药细胞株中高表达的H-ras癌基因的短发夹状RNA重组质粒,研究它对卵巢癌耐药细胞株SKOV3/ADM内源H-ras基因表达的抑制作用以及H-ras基因与卵巢癌发生和耐药的关系。方法:运用基因克隆技术构建靶向于H-ras基因的重组质粒pshRNA/H-ras并转染至卵巢癌耐药细胞株SKOV3/ADM,通过W est-ern blot、RT-PCR检测H-ras蛋白的表达及mRNA转录水平的变化;MTT法检测它对SK-OV3/ADM细胞增殖的抑制作用,AO/EB实验检测pshRNA/H-ras对SKOV3/ADM细胞凋亡的影响。结果:构建的两重组质粒pshRNA/H-ras1、2均能明显抑制H-ras基因的表达。蛋白质印迹结果表明,转染重组质粒pshRNA/H-ras1、2的SKOV3/ADM细胞靶蛋白的抑制率分别为86.87%和92.21%,RT-PCR检测到H-ras基因mRNA转录水平下降;pshR-NA/H-ras1、2明显抑制SKOV3/ADM细胞的增殖,MTT检测转染48h后的抑制率分别为62.4%和47.9%,AO/EB实验观测到转染24h和48h的细胞凋亡率分别为20.3%和35.6%。结论:H-ras基因在耐药的卵巢癌细胞增殖与凋亡过程中发挥作用,重组质粒pshRNA/H-ras可有效地抑制SKOV3/ADM细胞内源H-ras基因的表达和mRNA的转录,并抑制细胞的增殖和促进凋亡的发生,为化疗耐受的肿瘤细胞提供了新的基因治疗手段。更多还原

【Abstract】 Objective:To construct the small hairpin RNA recombinant plasmids targeting H-ras gene(pshRNA/H-ras) which expresses highly in ovarian cancer resistance strain SKOV3/ADM to silence endogenetic H-ras gene expression and investigate the role of H-ras gene in the development of resistant ovarian cancer.Methods:The pshRNA/H-ras1,2 were constructed by gene clone technology.The expression level of H-ras protein and mRNA were detected by Western blot and RT-PCR respectively after transfected the pshRNA/H-ras in SKOV3/ADM.The influence on proliferation and apoptosis were investigated by MTT and AO/EB.Results:The expression against H-ras proteins were inhibited 86.87% and 92.21% by pshRNA/H-ras1、2 respectively.The transcription level of H-ras mRNA was decreased obviously.The cell proliferation were inhibited 62.4% and 47.9% after transfected pshRNA/H-ras1,2 by MTT.The apoptosis were observed in AO/EB experiments and the apoptosis rate were 20.3% and 35.6% respectively after transfected 24h and 48h.Conclusions:These results demonstrate that H-ras plays an important role in proliferation of resistant ovarian cancer and the short hairpin RNA of H-ras can efficiently suppress H-ras expression and enhance the apoptosis in SKOV3/ADM,which provides a noval method for chemotherapy resistant tumors.更多还原