摘要
目的 :建立稳定转染维生素 D受体 (VDR)反义 c DNA的人骨肉瘤细胞系。方法 :构建 VDR反义 c DNA的真核表达载体 ,用 L ipofectamine法转染人骨肉瘤细胞系 HOS- 86 0 3,经 G418筛选后获得稳定转染细胞株 ,并用免疫组化技术检测内源性 VDR蛋白的表达情况 ;采用瞬时转染报告基因技术从靶基因水平检测 VDRas3细胞内 VDR的转录激活功能。结果 :筛选出 6株抗 G418细胞克隆 (VDRas1～ 6 ) ,其中 VDRas3细胞内源性 VDR蛋白的表达量低于对照细胞。激素作用 72 h后 ,对照细胞的报告基因氯霉素乙酰转移酶 (CAT)转录活性增加约 3.5倍 ,而 VDRas3细胞 CAT的转录基本不受激素诱导。结论 :获得了稳定转染 VDR反义 c DNA的细胞系 ,为今后进一步研究 1,2 5 (OH) 2 D3及其类似物调节细胞增殖分化的分子机制提供了一个良好的细胞模型。
Objective: To establish a human osteosarcoma cell line which stably transfected human vitamin D receptor(VDR) antisense cDNA. Methods: The eukaryotic expression vector harboring VDR antisense cDNA was constructed, and was transfected into the human osteosarcoma cell line HOS 8603 by lipofectamine method. The stable transfectants were screened by G418 and the expression of endogenous VDR were further detected at protein level by immunohistochemical analysis. The transactivation function mediated by VDR of the VDRas3 cells was detected at reporter gene level by transient transfection method. Results: Six subclones (VDRas1 6) were isolated, and the level of endogenous VDR expression in the VDRas3 cells was decreased significantly compared with that in the control cells. The transcriptional activity of the reporter gene CAT in the control cells was increased by 3.5 fold when treated with 1×10 -6 mol/L 1,25(OH) 2 D 3 for 72 h, but the transcription of CAT in the VDRas3 cells could not be induced by 1,25(OH) 2 D 3. Conclusion:A cell line stably expressing VDR antisense cDNA is established for the further study of the molecular mechanisms of 1,25 (OH) 2 D 3 effect and its analogues on proliferation and differentiation of the human osteosarcoma cell line. [
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2001年第3期242-244,共3页
Academic Journal of Second Military Medical University
基金
国家自然科学基金!资助项目 (3990 0 0 5 6 )
