P15逆转肝癌多药耐药的机制研究

MDR-reversing effect of P15 to multidrug-resistant hepatocellular carcinoma cells

目的:探讨P15逆转肝癌多药耐药(MDR)的可能机制.方法:①采用顺铂(CCDP)诱导法,建立肝癌HepG2细胞动态MDR HepG2/CDDP细胞模型;②在动态MDR HepG2/CDDP细胞模型中,RT-PCR检测P15mRNA的表达,Western Blot检测P15蛋白的表达;③建立稳定转染P15正义表达载体的HepG2/CDDP-P15细胞系及转染pcDNA3.1(+)空载体的HepG2/CDDP-PC对照细胞系;④流式细胞仪检测转染细胞HepG2/CDDP-P15,HepG2/CDDP-PC和亲本HepG2/CDDP的细胞周期及阿霉素(ADR)的蓄积和潴留;⑤Western Blot检测耐药经典分子MRP-1和P-糖蛋白(P-gp)的表达.结果:HepG2/CDDP动态耐药细胞模型建立成功;P15的mRNA表达水平随着HepG2/CDDP耐药细胞株耐药表型的增强逐渐下降;上调p15基因的表达可显著提高HepG2/CDDP耐药细胞株细胞内ADR的蓄积和潴留,促进G1期细胞阻滞,抑制肿瘤细胞增殖,下调MRP-1,P-gp蛋白的表达.结论:P15与肝癌细胞MDR关系密切,其表达水平随着肝癌耐药细胞株耐药表型的增强而逐渐下降;上调P15的表达,可有效逆转肝癌HepG2/CDDP耐药细胞株的耐药表型.

AIM： To investigate the P15 expression level in HepG2/CDDP kinetic multidrug-resistant（MDR） models and its possible molecular mechanism in multidrng-resistant hepatoeellular carcinoma cells. METHODS： Anti-cancer drug induced method was used to construct the HepG2/CDDP kinetic MDR models and RT-PCR and Western blot were used to detect the P15 expression in HepG2/CDDP kinetic MDR models. P15 over-expressed HepG2/CDDP-P15 cell line and its empty vector control HepG2/ CDDP-PC cell line were constructed. Flow cytometry（FCM） was used to study the cell cycle distribution, adriamycin（ADR） accumulation and detention in HepGE/CDDP-P15 cells and its control cell lines, and classic MDR molecules-P-gp and MRP-1 were examined by Western blot. RESULTS： The HepG2/CDDP kinetic MDR model was successfully established. The mRNA level of P15 decreased with the increasing drug resistance of hepatocellular carcinoma cells. Up-regulation of expressions of P15 in HepG2/ CDDP cells inhibited the cell proliferative activity, decreased the ability of the drug transports, increased ADR accumulation and retention. CONCLUSION： Up-regulating P15 reverses the MDR phenotype of hepatocellular carcinoma cells by down-regulating the P-gp and MRP-1 expressions.