摘要
目的:研究人维生素D受体(human vitaminDreceptor,hVDR)基因翻译起始密码子Fok Ⅰ突变介导CYP3A4诱导表达的调控能力与野生型比较有无统计学差异。方法:从肝脏组织标本中提取总RNA,RT-PCR法扩增获得野生型hVDR基因cDNA序列,并用定点诱变的方法获得含Fok Ⅰ突变的hVDRcDNA序列,构建野生型和突变型的真核表达载体;同时构建含CYP3A4近端ER6元件的荧光素酶报告基因载体。将hVDR真核表达载体、CYP3A4报告基因载体和内对照载体共同瞬时转染COS-7细胞,转染24 h后用1、10、100 nmol/L的1,25(OH)2D3孵育48 h,最后裂解细胞分析荧光素酶活性。结果:Fok Ⅰ突变型和野生型hVDR真核表达载体以及CYP3A4荧光素酶报告载体均构建成功。瞬转后胞内荧光素酶活性检测显示,各浓度1,25(OH)2D3孵育后hVDR基因Fok Ⅰ突变型组与野生型组均可激活CYP3A4的转录;但各维生素D3(vitamin D3,VD3)浓度下,突变型组与野生型组的荧光素酶比活性值没有统计学差异。结论:与VD3结合后,hVDRFokI突变型与野生型均能激活CYP3A4的诱导表达,但与野生型比较。
AIM： To study whether there is significant difference in the inductive effects on CYP3A4 between human vitamin D Receptor （hVDR） wild type and start codon Fok I mutant. METHODS： Total RNA was extracted from liver samples, cDNA of hVDR wild type was acquired by RT-PCR, and cDNA with hVDR Fok I mutant was acquired using site-directed mutagenesis. Eukaryotic expression vectors of both wild type and mutant of hVDR were constructed according to cDNA. Meanwhile, luciferase reporter gene vector was constructed with proximal ER6 element of CYP3A4. COS-7 cells＇ with hVDR eukaryotic expression vectors, CYP3A4 luciferase reporter gene vector and the internal control vector were transiently transfected for 24 h and then were incubated with 1 nmol/L, 10 nmol/L and 100 nmol/L 1,25（OH）2D3 for 48 h. Finally the cells were lysed and the luciferase activity was detected with cell lysis. RESULTS： hVDR eukaryotic expression vectors of wild type and Fok I mutant, and CYP3A4 luciferase reporter gene vector were successfully construtted. The results of luciferase activity showed that CYP3A4 transcription could be activated by hVDR eukaryotic expression vectors of wild type and Fok I mutant when incubated with 1 nmol/L, 10 nmol/L and 100 nmol/L 1,25 （ OH）2D3, respectively. However, there was no significant difference between the luciferease activities of hVDR wild type and mutant. CONCLUSION： Both hVDR wild type and Fok I mutant have inductive effect on CYP3A4 when incubated with vitamin D3 （VD3）. But Fok I mutant could not significandy change the regulational capacity of wild type VDR on CYP3A4.
出处
《中国临床药理学与治疗学》
CAS
CSCD
2009年第6期664-669,共6页
Chinese Journal of Clinical Pharmacology and Therapeutics
基金
中国博士后基金一等资助项目(20070410314)
中南大学博士后基金资助项目
