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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)。(2)C组及E组表现出比D组更低的PGP mRNA水平(P<0.05)。(3)E组BCRP和LRP表达水平低于C、D组(P<0.05)。(4)C、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)。(5)C、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-n HAP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 explore the effect and molecular mechanism of P53 and Rb anti-oncogene on multidrug resistance of hepatic metastatic foci by nano-targeted therapy. Methods Liposomes with polylysine and polylysine modified hydroxyapatite nanoparticle emulsion were used as carriers to transfer the recombinant plasmids containing the wild type P53 and Rb genes into the hepatic VX2 liver metastases via hepatic artery together or separately Localized, Western blotting and in situ laser scanning confocal microscopy were used to determine the expression of P53 and Rb co-expressed proteins in metastatic lesions. The efficacy of the tumor was evaluated by testing the chemosensitivity of the tumor to mitomycin (MMC), 5-fluorouracil (5-FU) and doxorubicin (ADM). Finally, real-time RT-PCR and ECL were used to detect multidrug resistance protein (PGP), multidrug resistance-related protein 1 (MRP1), lung The difference of expression of LRP and BCRP between two groups. Results (1) The nanoplex-p53 / lipiodol group (C), the nanoplex-Rb / lipiodol group (D), the nanoplex- (p53 + Rb) group and the nanoplex- The mRNA expression of PGP, BCRP and MRP1 in the / lipiodol group (E) decreased (P <0.05). (2) Group C and group E showed lower PGP mRNA level than group D (P <0.05). (3) The expression of BCRP and LRP in group E was lower than that in group C and D (P <0.05). (4) The expression of PGP protein in group C and E was significantly lower than that in group A, while the expression of BCRP and LRP in group E was significantly lower than that in group A, but the expression of MRP1 in group C and D was lower than that in group A (P <0.05). (5) Group C, D and E showed higher sensitivity to chemotherapeutic ADM and MMC than group A; Group D and E had higher sensitivity to chemotherapy drug 5-FU than group A (P <0.05 ). Conclusion The Pll-n HAP-mediated P53 gene cooperates with the targeted Rb gene in nano-targeted therapy and enhances its sensitivity to chemotherapeutic drugs by regulating the expression of important genes in multiple drug resistance mechanisms of tumor cells. P53 gene combined with Rb gene targeting of nano-gene therapy may become multi-drug resistant liver metastases effective treatment.