CAJ | 학술논문

目的：建立稳定转染维生素D受体(VDR)反义cDNA的人骨肉瘤细胞系。方法：构建VDR反义cDNA的真核表达载体，用Lipofectamine法转染人骨肉瘤细胞系HOS-8603，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72h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cDNA的细胞系，为今后进一步研究1,25(OH)2D3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。
목적：건립은정전염유생소D수체(VDR)반의cDNA적인골육류세포계。방법：구건VDR반의cDNA적진핵표체재체，용Lipofectamine법전염인골육류세포계HOS-8603，경G418사선후획득은정전염세포주，병용면역조화기술검측내원성VDR단백적표체정황；채용순시전염보고기인기술종파기인수평검측VDRas3세포내VDR적전록격활공능。결과：사선출6주항G418세포극륭(VDRas1～6)，기중VDRas3세포내원성VDR단백적표체량저우대조세포。격소작용72h후，대조세포적보고기인록매소을선전이매(CAT)전록활성증가약3.5배，이VDRas3세포CAT적전록기본불수격소유도。결론：획득료은정전염VDR반의cDNA적세포계，위금후진일보연구1,25(OH)2D3급기유사물조절세포증식분화적분자궤제제공료일개량호적세포모형。
Objective: To establish a human osteosarcoma cell line whichstably 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 D3 for 72 h, but the transcription of CAT in the VDRas3 cells could not be induced by 1,25(OH)2 D3. Conclusion:A cell line stably expressing VDR antisense cDNA is established for the further study of the molecular mechanisms of 1,25-(OH)2 D3 effect and its analogues on proliferation and differentiation of the human osteosarcoma cell line.