抑制乳腺癌耐药蛋白基因逆转肝癌多药耐药的体内实验

In Vivo Experimental Study on Reversion of Multidrug-resistance of Hepatocellular Carcinoma by Suppression of Breast Cancer Resistance Protein Gene

目的探讨小片段RNA干扰抑制乳腺癌耐药蛋白(BCRP)基因及其蛋白表达在裸鼠体内逆转人肝癌组织多药耐药(MDR)的可行性。方法建立裸鼠多药耐药肝细胞癌模型,随机分为A组和B组,A组(对照)注射生理盐水40μl+Lipo-fectamine200010μl,B组注射BCRP基因RNAi质粒pSUPER-BCRP40μl+Lipofectamine200010μl,两组均行瘤内注射1次。5d后,各组均经腹腔注射阿霉素5mg/kg化疗,每5d给药1次,共4次。彩色B超测量肿瘤体积;化疗结束后1周处死裸鼠,RT-PCR及Westernblot法检测各组裸鼠肿瘤组织中BCRP基因mRNA的转录水平及其蛋白的表达水平。结果B组每次化疗后肿瘤体积均明显缩小;除第1次化疗外,其余各次化疗后肿瘤体积均小于A组;化疗结束后,B组与A组相比,肿瘤组织中BCRP基因mRNA的转录水平和BCRP蛋白的表达水平均明显降低。结论BCRP基因RNAi质粒pSUPER-BCRP可有效降低裸鼠肝癌组织BCRP基因mRNA的转录水平及其蛋白的表达水平,在一定程度上逆转MDR,为从基因水平逆转MDR提供了初步的实验依据。

Objective Toinvestigate the feasibility of reversingmultidrug-resistance（MDR）of human hepatocellular carcinoma by suppression of breast cancer resistance protein（BCRP） mRNA transcription and protein expression with small interfering RNA （siRNA）in nude mice. Methods The model nude mice with multidrug-resistant hepatocellular carcinoma were randomly divided into control（A）and test（B）groups. Forty microliter of physiological saline ＋ 10 μl of Lipofectamine 2000 was injected into the carcinomas of mice in group A, and 40 μl of plasmid pSUPER-BCRP ＋ 10 μl of Lipofectamine 2000 to those in group B. Five days later, all the mice were injected i.p. with amycin at a dosage of 5 mg / kg, every 5 d for 4 times. The sizes of carcinomas were determined by color B type ultrasound inspection. The nude mice were killed one week after completion of chemotherapy, and the BCRP mRNA transcription and protein expression levels in their carcinoma tissues were determined by RT-PCR and Western blot respectively. Results The sizes of carcinomas of mice in group B decreased significantly after each chemotherapy, and, except those after the first chemotherapy, were significantly smaller than those in group A. After completion of chemotherapy, both the BCRP mRNA transcription and protein expression levels in carcinoma tissues of mice in group B decreased significantly as compared with those in group A. Conclusion RNAi plasmid pSUPER-BCRP decreased the BCRP mRNA transcription and protein expression levels in hepatocellular carcinoma tissues of nude mice effectively and reversed MDR at a certain degree, which provided a primary experimental basis for reversion of MDR at gene level.