纳米颗粒介导的P53联合Rb基因靶向治疗对兔VX2肝转移癌多药耐药的影响及分子机制

摘要

目的 联合P53及Rb抗癌基因,通过纳米基因靶向治疗技术,探索其对肝转移癌灶多药耐药的影响及其分子机制.方法 以超液化碘油及多聚赖氨酸修饰的羟基磷灰石纳米颗粒乳剂为载体,将含有野生型P53、Rb基因的重组表达质粒经肝动脉共同或者分别转运至兔VX2肝转移癌灶局部,采用蛋白质印迹法及原位激光扫描共焦显微镜确定P53及Rb共表达蛋白在转移灶的表达.通过检测肿瘤对化疗药物丝裂霉素(MMC)、5-氟尿嘧啶(5-FU)和阿霉素(ADM)的敏感性来评价疗效.最后应用实时反转录聚合酶链反应(real time RT-PCR)及电化学发光法(ECL)蛋白杂交技术检测多药耐药蛋白(PGP),多药耐药相关蛋白1(MRP1),肺耐药蛋白(LRP),乳腺癌耐药蛋白(BCRP)的表达差异.结果 ①与对照碘油组(A)和nanoplex/lipiodol组(B)相比,nanoplex-p53/lipiodol组(C),nanoplex-Rb/lipiodol组(D),nanoplex-(p53+Rb)/lipiodol组(E)PGP、BCRP、MRP1 mRNA表达均降低(P<0.05).②C组及E组表现出比D组更低的PGP mRNA水平(P<0.05).③E组BCRP和LRP表达水平低于C、D组(P<0.05).④C、E组PGP蛋白表达显著低于A组,仅E组BCRP和LRP蛋白表达显著低于A组,C、D、E组MRP1蛋白表达均低于A组(P<0.05).⑤C、D、E组表现出比A组更高的对化疗药ADM和MMC的敏感性;D、E组比A组更高的对化疗药5-FU的敏感性(P<0.05).结论 以Pll-nHAP为载体的P53基因协同Rb基因的纳米靶向治疗,通过调节肿瘤细胞多重耐药机制中重要基因的表达,增强了其对化疗药的敏感性.P53基因联合Rb基因的纳米基因靶向治疗有可能成为针对肝转移灶多药耐药的有效治疗方法.%Objective To investigate the effects of antioncogenes, P53 and Rb on the multidrug resistance of liver metastasis and its molecular mechanism through nano-gene targeted therapy. Methods Ultra-liquefied lipiodol and Pll-modified nHAP emulsions were used as carriers. The recombinant expressing plasmids containing wild-type P53 and Rb genes were transferred to the local lesion of the rabbit VX2 liver metastasis by hepatic arteries jointly or separately. The expression of P53 and Rb co-expression proteins in the metastases was determined by Western blotting and in situ confocal laser microscopy. The efficacy was evaluated by measuring the sensitivity of the tumor to chemotherapeutic drugs, mitomycin (MMC), 5-fluorouracil (5-FU) and doxorubicin (ADM). The differences in the ex-pression of multidrug resistance protein (PGP), multidrug resistance-associated protein 1 (MRP1), lung resistance pro-tein (LRP) and breast cancer resistance protein (BCRP) were determined by real time RT-PCR and ECL protein hy-bridization. Results ①Compared with the control group (A) and nanoplex-lipiodol group (B), the mRNA expression of PGP, BCRP, MRP1 decreased in the nanoplex-p53/lipiodol group (C), nanoplex-Rb/lipiodol group (D) and nanoplex-(p53+Rb)/lipiodol (E) group (P<0.05).②Group C and group E showed lower PGP mRNA levels than those in group D (P<0.05). ③The expression levels of BCRP and LRP in group E were lower than those in group C and D ( P<0.05). ④The PGP protein expression in groups C and E was lower than that in the group A;the BCRP and LRP protein expres-sion in group E were significantly lower than those in group A; and the MRP1 protein expression in groups C, D and E were lower than that in group A (P<0.05).⑤Groups C, D and E showed a higher sensitivity to the chemotherapeutic drugs ADM and MMC than the group A; groups D and E showed a higher sensitivity to the chemotherapeutic drug 5- FU than the group A (P<0.05). Conclusion The sensitivity of P53 gene-mediated Rb gene targeted chemotherapy with Pll-nHAP as a carrier to chemotherapeutic drugs is enhanced by regulating the expression of significant genes in multidrug resistance mechanism of tumor cells. The nano-gene targeted therapy of P53 gene and Rb gene may be an effective treatment for multidrug resistance of liver metastasis.