细胞色素P450基因修饰的脐带间充质干细胞联合环磷酰胺对急性B淋巴细胞白血病的治疗作用

目的 研究细胞色素P450（Cyt P450）基因修饰的脐带间充质干细胞（UC-MSCs）联合环磷酰胺（CPA）对急性B淋巴细胞白血病的治疗作用。方法 通过基因工程方法设计Cyt P450 2B6（CYP2B6）基因序列,采用慢病毒载体基因转染UC-MSCs,得到可高表达CYP2B6蛋白的CYP2B6-MSC种子细胞。采用实时荧光定量PCR（qRT-PCR）检测CYP2B6基因的表达评估转染效果;采用流式细胞术检测CYP2B6细胞表面标志物表达;采用体外诱导分化检测CYP2B6细胞分化能力。将CYP2B6-MSC与Nalm-6共培养,CCK8法和Annexin-V FITC/PI试剂盒检测UC-MSCs/CYP2B6-MSC联合CPA对Nalm-6细胞的增殖和凋亡的影响。结果 qRT-PCR检测结果显示,CYP2B6-MSC可高表达CYP2B6蛋白,而hUC-MSC（对照组细胞）不表达CYP2B6蛋白;流式细胞术及诱导分化检测发现,CYP2B6基因修饰后的MSC流式表型及分化能力均未有显著变化;肿瘤杀伤实验发现,CYP2B6-MSC与Nalm-6细胞共培养,同时加入CPA后,与UC-MSC及未加CPA组比较,CYP2B6-MSC+CPA对Nalm-6细胞具有显著的增殖抑制及促凋亡作用（P<0.01）。结论 CYP2B6基因修饰后的MSC联合CPA对Nalm-6具有显著的生长抑制和促凋亡作用。

Therapy effect for B acute lymphocytic leukemia using umbilical cord derived mesenchymal stem cells transferred with cytochrome P450 combined with cyclophosphamide

Objective To transfect Cytochrome P450 2B6 (CYP2B6) into umbilical cord mesenchymal stem cells (UC-MSCs) with lentivirus vector. The anti-tumor effect of B acute Lymphocytic Leukemia with UC-MSCs-CYP2B6 cooperated with Cyclophosphamide (CPA) was measured to provide laboratory database for gene directed enzyme prodrug therapy (GDEPT) which used BMSCs as vehicles. Methods Cyp2502b6 (CYP2B6) gene sequence was designed by gene engineering method, and the CYP2B6-MSC seed cells with high expression of CYP2B6 protein were obtained by transfection of UC MSCs with lentivirus vector gene. The expression of CYP2B6 gene was detected by real-time fluorescent quantitative PCR (qRT-PCR) to evaluate the transfection effect; the expression of CYP2B6 cell surface markers was detected by flow cytometry; the differentiation ability of CYP2B6 cell was detected by inducing differentiation in vitro. The effects of UC-MSCs/CYP2B6-MSC combined with CPA on the proliferation and apoptosis of Nalm-6 cells were detected by CCK8 and annexin-V FITC/PI kit. Results The results of qRT-PCR showed that CYP2B6-MSC could highly express CYP2B6 protein, while hUC-MSC (control group cells) did not express CYP2B6 protein. Flow cytometry and induced differentiation detection showed that the flow phenotype and differentiation ability of CYP2B6 gene modified MSC had no significant change. Compared with UC-MSC and without CPA, CYP2B6-MSC + CPA significantly inhibited the proliferation and promoted the apoptosis of Nalm-6 cells (P < 0.01). Conclusion These data suggest that MSCs expressing CYP2B6 with CPA could represent a promising treatment for B acute Lymphocytic Leukemia to test in future clinical trials.