细胞色素氧化酶P450 3A4基因联合环磷酰胺的体外抗肿瘤效应  

The anti-tumor effect of Cytochrome oxidase P450 3A4 combined with Cyclophosphamide in vitro

作　　者：张巍[1] 王季石[1] 杨远[1] 方琴[2] 

机构地区：[1]贵阳医学院第一附属医院血液科,贵阳550004 [2]贵阳医学院第一附属医院药剂科,贵阳550004

出　　处：《复旦学报（医学版）》2009年第4期384-388,共5页Fudan University Journal of Medical Sciences

基　　金：国家自然科学基金项目(30760276)

摘　　要：目的克隆细胞色素氧化酶P4503A4(CYP3A4)基因,构建真核表达质粒并导入K562细胞进行表达,研究CYP3A4联合环磷酰胺(CPA)的体外抗肿瘤效应。方法利用RT-PCR从人肝细胞中克隆CYP3A4基因,构建重组真核表达载体。脂质体法导入K562细胞,RT-PCR和Western blot检测CYP3A4基因在K562细胞中的表达并筛选稳定转染的细胞克隆。MTT法检测CYP3A4联合CPA对肿瘤细胞的抑制作用。结果成功克隆CYP3A4基因并构建了真核表达质粒pcDNA3.1/myc-HisA(+)-CYP3A4。RT-PCR和Western blot分别显示转基因组的CYP3A4 mRNA和蛋白质表达水平均显著高于转空载体组和对照组。MTT示转基因组IC50值为1.09016mmol/L,明显低于转空载体组和对照组。酶诱导剂和抑制剂分别能够减低和增高转基因组IC50。结论CYP3A4体外具有联合CPA的抗肿瘤作用,这种作用能够被CYP3A4相应的诱导剂和抑制剂增强或减低。Objective To clone CYP3A4 gene and to construct CYP3A4 recombinant mammalian expression vectors which are transferred into K562 cells for expression, and detect the anti-tumor effect of CYP3A4 combined with Cyelophospharnide （CPA） in vitro. Methods Mammalian expression vector containing CYP3A4 gene cloned from human hepatocytes by RT-PCR were constructed and transferred into K562 cells via liposome. The expression of CYP3A4 was detected by RT-PCR and Western blot respectively. MTT detected the anti tumor effect of CYP3A4 recombinant mammalian expression vectors combined with CPA. Results We cloned CYP3A4 gene and constructed the recombinant mammalian expression vectors pcDNA3, 1/myc-His A （ ＋ ）-CYP3A4 successfully. Both RT-PCR and Western blot showed significantly higher mRNA and protein expression of CYP3A4 in genetransfected group than in empty vector-transfeeted controls and in empty cells controls. The IC50, values were could recom could mmol/L in gene-transfected group, which were significantly lower than in other two groups and reduced and increased by the revulsant and inhibitor respectively. Conclusions CYP3A4 ant mammalian expression vectors had anti tumor effect cooperating with CPA, and the effect inereased and reduced by the revulsant and inhibitor of CYP3A4 respectively.

关 键 词：细胞色素氧化酶P450 3A4 环磷酰胺 基因介导的酶前药治疗 肿瘤治疗 基因转染 

分 类 号：Q786[生物学—分子生物学]