间充质干细胞介导'酶-前药基因'CYP1A2靶向抗肿瘤效应研究

摘要

目的 将人细胞色素P450 1A2(CYP1A2)基因转染骨髓间充质干细胞(BMSC),检测转基因BMSC协同化疗前药达卡巴嗪(DTIC)对人恶性淋巴瘤细胞株Raji细胞的靶向促凋亡作用,为以间充质干细胞为载体的"基因介导的酶-前药双靶向抗肿瘤策略"提供体外实验依据.方法 从人肝细胞中克隆CYP1A2基因,构建真核表达载体,分离、鉴定并培养BMSC,脂质体法将CYP1A2基因导入BMSC和Raji细胞中,RT-PCR和Westem blot法检测CYP1A2基因的表达,Transwell法检测BMSC的肿瘤靶向性,MTT法检测转基因Raji细胞对DTIC敏感性的改变,Annexin V/PI染色标记法检测转基因BMSC联合DTIC诱导Raji细胞凋亡的作用.结果 成功克隆CYP1A2基因并将构建的真核表达载体转染BMSC和Raji细胞,流式细胞术检测体外诱导分化结果符合BMSC特性,RT-PCR和Westernblot检测到转基因细胞中CYP1A2表达,Transwell迁移实验证实BMSC有肿瘤趋向性,MTT法检测显示DTIC呈剂量依赖性抑制转CYP1A2基因的Raji细胞生长,而转CYP1A2基因的BMSC细胞对DTIC相对耐受(IC50值分别为1.67 mmol/L和7.53 mmol/L,P＜0.01).Armexin V/PI染色法显示CYP1A2能够使细胞在体外代谢DTIC,产生细胞毒效应使肿瘤细胞凋亡,而且具有旁观者效应.结论 CYP1A2可使细胞在体外代谢DTIC产生细胞毒效应.以BMSC为载体的CYP1A2-DTIC酶-前药系统可发挥双靶向抗肿瘤作用,在体外诱导人恶性淋巴瘤细胞凋亡.%Objective To explore bone marrow-derived mesenchymal stem cells(BMSC)mediated gene directed enzyme prodrug targeting anti-tumor therapy(GDEPT).Methods CYP1A2 gene was cloned from human hepatocytes by RT-PCR,and the eukaryote expression vector was constructed and transferred into Raji cells and human BMSCs via liposome.The targeted anti-tumor effect of BMSC-CYP1A2 cooperated with dacarbazine(DTIC)was measured.RT-PCR and Western blot were used to detect the expression of CYP1A2.Migration assay Was detected with Transwell Plates.MTT Was used to evaluate the growth inhibitory effect.Cell apeptosis Was determined with Annexin V-FITC/PI staining by flow cytometry(FCM).Results The results of FCM and differentiation induction were in line with the characteresties of BMSC.The expression of CYP1A2 was confirmed by RT-PCR and Western blot.Growth inhibition of Raji-CYP1A2 cells was increased with DTIC concentration in a dose-dependent manner(IC50 was 1.67 mmol/L).However,BMSC was less sensitive to the cytotoxic effects of DTIC(IC50 of 9.26 mmol/L and 7.53 mmol/L for BMSC-pcDNA3.1and BMSC-CYP1A2 cells,respectively)than Raji cells did(IC50 of 5.62 mmo1/L and 1.67 mmol/L for Raji-pcDNA3.1 and Raji-CYP1A2 cells,respectively).BMSC migrated toward Raji cells in Transwell plate.BMSC-CYP1A2 cells mediated a bystander killing effect for CYP1A2-negative Raji cells when they were cocultured with BMSC-CYP1A2 cells.Conclusion DTIC can be catalyzed by CYP1A2 in vitro.BMSC-based enzyme prodmg system of CYP1A2 and DTIC can induce lymphoma cell apoptosis targetedly via bystanderkilling effect.