药前酶基因修饰的骨髓间充质干细胞对黑色素瘤靶向治疗的研究

Stem Cells Modified by Mediated Prodrug Gene in Targeted Therapy of Human Melanoma

目的:利用药前酶基因修饰的骨髓间充质干细胞(BMSCs)提高恶性黑色素瘤局部化疗效果。方法:从雄性大鼠股骨、胫骨骨髓中分离、培养大鼠BMSCs并鉴定;将细胞色素氧化酶P4502E1(CYP2E1)以含增强绿色荧光蛋白(enhanced green fluorescent protein,EGFP)的重组腺病毒为载体转染BMSCs,用RT-PCR和荧光显微镜观察绿色荧光的方法来检测目的基因的表达;将人黑色素瘤细胞MV3注射到裸鼠背部皮下,建立裸鼠MV3移植瘤模型,将移植瘤模型分为4组,分别给予CYP2E1-BMSCs及达卡巴嗪,单用CYP2E1-BMSCs,单用达卡巴嗪以及生理盐水治疗,并检测各组的体内局部抗肿瘤效应。结果:成功分离培养大鼠BMSCs,形态上均一,贴壁生长,以长梭形为主,体积小,核浆比大,呈明显的同向性改变,漩涡状盘旋排列;流式细胞术检测提示高表达CD29、CD71,低表达CD34、CD45;体外经不同诱导体系培养后提示具有成骨、成脂、成软骨潜能;鉴定后的BMSCs经重组腺病毒转染后,荧光显微镜可观察到转基因靶细胞EGFP表达,RT-PCR法从mRNA水平检测到目的基因在转基因细胞中表达;荧光显微镜观察治疗后裸鼠的肿瘤及各脏器的组织切片提示CYP2E1-BMSCs能聚集在肿瘤组织及转移灶周围,CYP2E1-BMSCs联合化疗药物组局部凋亡率明显高于单纯化疗组及空白对照。结论:CYP2E1-BMSCs在体内具有协同化疗药物抗肿瘤的作用,并有向肿瘤细胞聚集的特点。

Objective： To enhance the local chemotherapy effect of human melanoma by utilizing rats bone marrow mesenchymal stem cells（BMSCs） modified by mediated prodrug gene. Methods： BMSCs were separated from ratg bone marrow of femur and tibia, cultured in vitro and identified, which were transfected by recombinant adenovirus mediate CYP2E1 gene （pAdSCMV-CYP2E1-EGFP）. RT- PCR method and fluorescence microscope were adoped to detect the expression of CYP2E1 in genetic modified BMSCs （CYP2E1-BMSCs）. The cells of MV3 were injected subcutaneously into back of nude mice to build the MV3 transplantation tumor model. Then the MV3 transplantation tumor model mice were divided into 4 groups： group 1 received treatment of CYP2E1-BMSCs combined with dacarbazine ; group 2 received treatment of CYP2E1-BMSCs alone ; group 3 dacarbazin alone ; goup 4 physi- ological saline alone（ control group）. Results： The rats＇BMSCs were successfully separated and cul- tured. BMSCs were uniform in morphology, featured by adherent growth, small size, long spindle shape, large karyoplasmic ratio, obvious same direction change, and swirling and spiral arrangment. Flow cytometry detection indicated high expression level of CD29, CD71, and low expression level of CD29, CDT1. Incubation in different induction system in vitro indicated that BMSCs had potential in osteogenesis, adipogenesis and chondrogenesis. After identification and transfection, EGFP expression in transgenosis targeted cells could be observed under fluorescence microscope and CYP2E1 target gene expression in mRNA expression level could be detected by RT-PCR. Under fluorescence microscope, after treatment, tissue slice of nude mices＇tumor and other organs showed that CYP2E1-BMSCs could gather around tumor tissue and metastatic foci, and the apoptosis rate of group 1 （ CYP2E1-BMSCs combined with dacarbazine） was significantly higher than that of group 3 （ dacarbazine alone） and con- trol group （physiological saline alone）. Conclusion： CYP2E1-BMSCs can significantly improve the potency of dacarbazine in vivo, and can gather around tumor tissue. Therefore CYP2EI-BMSCs play a role in synergistic effect of chemotherapy drugs on tumor.