Yang Yuan, Wang Ji-Shi, Zhang Wei, Yuan Jun, Yang Ming, Fang Qin
Department of Hematology, The First Affiliated Hospital of Gui Yang Medical College, Guiyang 550004, China.
Zhonghua Xue Ye Xue Za Zhi. 2009 Oct;30(10):667-71.
To explore bone marrow-derived mesenchymal stem cells (BMSC) mediated gene directed enzyme prodrug targeting anti-tumor therapy (GDEPT).
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 apoptosis was determined with Annexin V-FITC/PI staining by flow cytometry(FCM).
The results of FCM and differentiation induction were in line with the characteristics 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 (IC(50) was 1.67 mmol/L). However, BMSC was less sensitive to the cytotoxic effects of DTIC (IC(50) of 9.26 mmol/L and 7.53 mmol/L for BMSC-pcDNA3.1 and BMSC-CYP1A2 cells, respectively) than Raji cells did (IC(50) of 5.62 mmol/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 co-cultured with BMSC-CYP1A2 cells.
DTIC can be catalyzed by CYP1A2 in vitro. BMSC-based enzyme prodrug system of CYP1A2 and DTIC can induce lymphoma cell apoptosis targetedly via bystander killing effect.
探讨骨髓间充质干细胞（BMSC）介导的基因导向酶解前药靶向抗肿瘤治疗（GDEPT）。
通过RT-PCR从人肝细胞中克隆CYP1A2基因，构建真核表达载体，并通过脂质体将其转入Raji细胞和人BMSC。检测BMSC-CYP1A2与达卡巴嗪（DTIC）协同作用的靶向抗肿瘤效果。采用RT-PCR和Western blot检测CYP1A2的表达。用Transwell板检测迁移实验。用MTT评估生长抑制作用。通过流式细胞术（FCM）用Annexin V-FITC/PI染色法测定细胞凋亡。
FCM和分化诱导结果符合BMSC的特征。RT-PCR和Western blot证实了CYP1A2的表达。Raji-CYP1A2细胞的生长抑制随DTIC浓度的增加呈剂量依赖性增加（IC(50)为1.67 mmol/L）。然而，BMSC对DTIC的细胞毒性作用比Raji细胞更不敏感（BMSC-pcDNA3.1和BMSC-CYP1A2细胞的IC(50)分别为9.26 mmol/L和7.53 mmol/L，而Raji-pcDNA3.1和Raji-CYP1A2细胞的IC(50)分别为5.62 mmol/L和1.67 mmol/L）。在Transwell板中BMSC向Raji细胞迁移。当BMSC-CYP1A2细胞与CYP1A2阴性的Raji细胞共培养时，BMSC-CYP1A2细胞对其介导了旁观者杀伤效应。
DTIC在体外可被CYP1A2催化。基于BMSC的CYP1A2和DTIC酶解前药系统可通过旁观者杀伤效应靶向诱导淋巴瘤细胞凋亡。
