摘要
目的筛选高效dsRNA/mdr1,以备研究RNA干扰逆转肝细胞癌多药耐药之用。方法首先根据siRNA设计原则,以多药耐药基因(mdr1)为靶基因,设计并选择4~5条siRNA/mdr1,经BLAST后体外转录法合成dsRNA/mdr1,用Oligofectamine试剂分别转染HepG2/mdr1,然后从mRNA、蛋白(P-gp)表达水平和细胞功能变化评价HepG2/mdr1耐药性被逆转的程度,比较各个dsRNA/mdr1的逆转效率,筛选出有效的siRNA/mdr1。结果成功合成5条dsRNA/mdr1(其中1条为阴性对照),dsRNA/mdr1-4mRNA表达(18.73±1.33)%、蛋白表达变化(79.1±1.6)%^(16.8±0.4)%与其他各组细胞比较,有显著性差异;细胞内柔红霉素(DNR)累积量也较其他组明显增加(平均荧光强度79.58,阳性率84.25%,P<0.05)。结论体外转录法结合脂质体转染适用于筛选高效siRNA,肯定了siRNA干扰序列能够有效阻抑mdr1基因编码蛋白p170的功能。
Objective To screen effective dsRNA/mdr1 for studying the reversal of muhidrug resistance of hepatocellular carcinoma （HCC） by RNA interference （RNAi）. Methods dsRNA/mdr1 targeting muhidrug resistance gene （mdr1） was designed and synthesized by in vitro transcription. HepG2/mdr1 in 6 groups was transfected with the complex of different dsRNA/mdrl （dsRNA/mdr1-1, dsRNA/mdr1-2, dsRNA/mdr1-3, dsRNA/mdr1-4, dsRNA/mdr1-5 ） using Oligofectamine. Then the cells were collected to measure the expression of mRNA/mdr1, P-glycoprotein and the accumulation of DNR. Results Five dsRNA/mdr1 were successfully synthesized including one negative. The expression of mRNA/mdr1 [ （ 18.73 ±1.33 ）% ] and P-glycoprotein [ from （79.1 ± 1.6） % before transfection down to （ 16.8 ±0.4） % ] in dsRNA/mdr1-4 was significantly lower than that of other groups （ P 〈 0.05 ）. Accumulation of DNR in dsRNA/mdr1-4 was higher than that in other groups （P 〈 0.05 ）. Conclusion In vitro transcription combined with liposome is fit to screen the effective dsRNA/mdr1, siRNA/mdr1 could suppress the expression of P-glycoprotein coded by mdrl gene.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2008年第1期35-38,共4页
Journal of Third Military Medical University
基金
国家自然科学基金(30170925)
广西科学基金(0728102)~~
关键词
肝细胞癌
多药耐药
RNA干扰
hepatocellular carcinoma
muhidrug resistance
RNA interference
