摘要
目的构建MRP2基因的真核表达载体pGenSil-1-MRP2-siRNA,并观察其转染人结肠癌耐长春新碱细胞株HCT-8/V前后MRP2基因和ABCC2蛋白表达变化以及对化疗药物敏感性变化。方法设计MRP2特异性siRNA的序列,克隆至pGenSil-1质粒中,测序鉴定后,用脂质体将重组质粒转染至HCT-8/V细胞中,用定量RT-PCR和Western blot检测MRP2的表达,MTT实验测定pGenSil-1-MRP2-siRNA和长春新碱对HCT-8/V细胞增殖抑制作用。结果在结肠癌耐药细胞HCT-8/V中,RNAi明显抑制了MRP2 mRNA及蛋白的表达,在相同浓度化疗药物的作用下,RNA干扰组细胞凋亡比例显著高于对照组(P<0.01),表明细胞耐药性显著下降,对药物敏感性显著增高。结论成功构建了MRP2的siR-NA真核表达载体,并且对结肠癌耐药细胞MRP2的表达有明显抑制作用,取得良好的逆转耐药效果。
Objective To construct the RNAi eukaryotic vector of inhibitory member of MRP family （MRP2） gene and after the vector transfection into HCT-8/V cell line to observe its interfering effect on MRP2 expression and the changed sensibility to vinblastine. Methods The specific siRNA sequence was designed according to the MRP2 sequence. The sequence was cloned into pGenSil-1 and then sequenced. The recombinant plasmid was transfected into HCT-8/V cells by liposome. Then MRP2 expression in the transfected cells was analyzed by FQ-PCR and Western blotting. MrrF test was used to measure the inhibitory effects of pGenSil- 1-MRP2-siRNA on the proliferation of HCT-8/V cells treated with vinblastine. Results The overexpression of MRP2 was suppressed efficiently by the introduction of small interfering RNA that caused sequence-specific gene silenee. The apoptotie rate of HCT-8/V eells transfeeted with pGenSil-1-MRP2-siRNA inereased signifieanfly when the eells were exposed to vinblastine as eompared with that of nontransfeeted eells （P 〈0.01 ）, indieating the drug resistanee of the transfeeted eells deereased. Conclusion The RNAi eukaryotie veetor pGen- Sil-1-MRP2-siRNA is eonstrueted sueeessfully. The RNAi we eonstrueted inhibits MRP2 expression effeetively and reverses the drug resistanee of HCT-8/V eells.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2008年第12期1179-1182,共4页
Journal of Third Military Medical University
