Wang Ji-shi, Hu Xiao-yan, Zhang Wei, Yuan Jun, Yang Yuan, Fang Qin
Department of Hematology, First Affiliated Hospital of Guiyang Medical College, Guiyang 550004, China.
Zhonghua Yi Xue Za Zhi. 2010 Aug 10;90(30):2130-5.
To construct the human cytochrome P-450 CYP2E1 (CYP2E1) recombinant adenovirus vector and detect its anti-tumor effects in combination with chemotherapeutic drugs so as to provide rationales for gene directed enzyme prodrug therapy (GDEPT).
CYP2E1 cDNA was cloned from human liver and a recombinant adenovirus vector constructed at a titer of 1 × 10(12) pfu/ml. Human bone marrow-derived mesenchymal stem cells (BMSCs) were separated, cultured, purified and detected. The tropism of BMSCs for cancer cells was detected by Transwell technique. These recombinant vectors were transferred into BMSCs and A375 cells and the expressions of EGFP and CYP2E1 detected by fluorescence microscope, RT-PCR and Western blot respectively. Inverted microscope, MTT and Annexin V-FITC/PI were employed to detect the anti-tumor effect of CYP2E1 recombinant adenovirus vectors in combination with chemotherapeutic prodrug dacarbazine (DTIC).
The recombinant adenovirus vectors pAd5CMV-NpA-CYP2E1 and pAd5CMV-NpA-EGFP were constructed successfully. BMSCs were successfully separated and they could migrate in vitro through a polycarbonate filter toward K562 and A375 cells in the lower chamber. Fluorescence microscope was used to detect the expression of EGFP while both RT-PCR and Western blot revealed a high expression of CYP2E1 in gene-transfected group cells. The dead cell counts of co-culture group of gene-transfected BMSCs and wild type A375 cells were significantly higher than those of the control under inverted microscope. The results of MTT showed that the growth inhibition of gene-transfected group cells was increased with DTIC concentration in a concentration dependent manner. IC(50) of group BMSCs-CYP2E1 + A375 was (0.17 ± 0.13) mmol/L, and that of the control group was (0.65 ± 0.20) mmol/L (P < 0.01). The DTIC concentration at which BMSCs were relatively safe might be selected at 0.05 mmol/L. Annexin V-FITC/PI confirmed that the apoptosis rate of BMSCs-CYP2E1 + A375 group was significantly higher than that of the control group after a 48-hour treatment of 0.05 mmol/L DTIC (26.8 ± 2.0 vs 8.7 ± 1.3, P < 0.01).
With an in vitro tropism for cancer cells, BMSCs transferred with CYP2E1 recombinant adenovirus vectors have anti-tumor effects probably synergistically with chemotherapeutic drugs.
构建人细胞色素P-450 CYP2E1（CYP2E1）重组腺病毒载体，并检测其与化疗药物联合应用时的抗肿瘤作用，为基因导向酶前药疗法（GDEPT）提供理论依据。
从人肝脏中克隆CYP2E1 cDNA，构建重组腺病毒载体，滴度为1×10(12) pfu/ml。分离、培养、纯化并检测人骨髓间充质干细胞（BMSCs）。采用Transwell技术检测BMSCs对癌细胞的趋向性。将这些重组载体分别转入BMSCs和A375细胞，通过荧光显微镜、RT-PCR和Western blot分别检测EGFP和CYP2E1的表达。采用倒置显微镜、MTT法和Annexin V-FITC/PI法检测CYP2E1重组腺病毒载体与化疗前药达卡巴嗪（DTIC）联合应用时的抗肿瘤作用。
成功构建重组腺病毒载体pAd5CMV-NpA-CYP2E1和pAd5CMV-NpA-EGFP。成功分离出BMSCs，其可在体外通过聚碳酸酯滤膜向下方小室中的K562和A375细胞迁移。用荧光显微镜检测EGFP的表达，RT-PCR和Western blot均显示基因转染组细胞中CYP2E1高表达。倒置显微镜下，基因转染的BMSCs与野生型A375细胞共培养组的死细胞数明显高于对照组。MTT结果显示，基因转染组细胞的生长抑制率随DTIC浓度的增加呈浓度依赖性升高。BMSCs-CYP2E1 + A375组的IC(50)为(0.17±0.13) mmol/L，对照组为(0.65±0.20) mmol/L（P<0.01）。BMSCs相对安全的DTIC浓度可能为0.05 mmol/L。Annexin V-FITC/PI证实，0.05 mmol/L DTIC处理48小时后，BMSCs-CYP2E1 + A375组的凋亡率明显高于对照组（26.8±2.0对8.7±1.3，P<0.01）。
转染CYP2E1重组腺病毒载体的BMSCs对癌细胞具有体外趋向性，可能与化疗药物协同发挥抗肿瘤作用。
