目的 研究細(xì)胞色素P450（Cyt P450）基因修飾的臍帶間充質(zhì)干細(xì)胞（UC-MSCs）聯(lián)合環(huán)磷酰胺（CPA）對(duì)急性B淋巴細(xì)胞白血病的治療作用。方法 通過基因工程方法設(shè)計(jì)Cyt P450 2B6（CYP2B6）基因序列，采用慢病毒載體基因轉(zhuǎn)染UC-MSCs，得到可高表達(dá)CYP2B6蛋白的CYP2B6-MSC種子細(xì)胞。采用實(shí)時(shí)熒光定量PCR（qRT-PCR）檢測(cè)CYP2B6基因的表達(dá)評(píng)估轉(zhuǎn)染效果；采用流式細(xì)胞術(shù)檢測(cè)CYP2B6細(xì)胞表面標(biāo)志物表達(dá)；采用體外誘導(dǎo)分化檢測(cè)CYP2B6細(xì)胞分化能力。將CYP2B6-MSC與Nalm-6共培養(yǎng)，CCK8法和Annexin-V FITC/PI試劑盒檢測(cè)UC-MSCs/CYP2B6-MSC聯(lián)合CPA對(duì)Nalm-6細(xì)胞的增殖和凋亡的影響。結(jié)果 qRT-PCR檢測(cè)結(jié)果顯示，CYP2B6-MSC可高表達(dá)CYP2B6蛋白，而hUC-MSC（對(duì)照組細(xì)胞）不表達(dá)CYP2B6蛋白；流式細(xì)胞術(shù)及誘導(dǎo)分化檢測(cè)發(fā)現(xiàn)，CYP2B6基因修飾后的MSC流式表型及分化能力均未有顯著變化；腫瘤殺傷實(shí)驗(yàn)發(fā)現(xiàn)，CYP2B6-MSC與Nalm-6細(xì)胞共培養(yǎng)，同時(shí)加入CPA后，與UC-MSC及未加CPA組比較，CYP2B6-MSC+CPA對(duì)Nalm-6細(xì)胞具有顯著的增殖抑制及促凋亡作用（P<0.01）。結(jié)論 CYP2B6基因修飾后的MSC聯(lián)合CPA對(duì)Nalm-6具有顯著的生長(zhǎng)抑制和促凋亡作用。

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.

R965

國(guó)家自然科學(xué)基金資助項(xiàng)目（81400078）