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目的 :探讨地塞米松对人α1( )原胶原基因调控序列的作用。方法 :用组织块法原代培养人皮肤成纤维细胞并进行传代培养作为本研究的模型细胞。 (1)采用 Brd U掺入的 EL ISA法 ,测定经不同浓度地塞米松处理 2 4h后成纤维细胞的增殖情况。 (2 )构建含长短不一的人α1( )原胶原基因 5′侧翼序列与氯霉素乙酰基转移酶 (CAT)报告基因的 3种重组质粒 ;以脂质体法转染人皮肤成纤维细胞 ;EL ISA法测定不同浓度地塞米松作用 2 4h后 ,转染了 3种重组质粒的成纤维细胞的 CAT表达量。结果 :(1)在加入 2 % FCS或 10 % FCS的培养条件下 ,1× 10 - 9～ 1× 10 - 4 m ol/ L 地塞米松处理 2 4h后 ,各浓度组成纤维细胞增殖值之间以及与对照组之间差异均无显著性 (P>0 .0 5 )。 (2 ) 3种重组体转染成纤维细胞 ,并经地塞米松处理 2 4h后 ,转染细胞的 CAT相对表达量测定 :地塞米松组与对照组之间、地塞米松较高浓度组 (1× 10 - 5 m ol/ L)与较低浓度组 (1× 10 - 6mol/ L)之间 ,差异均有显著性 (P<0 .0 5 )。结论 :地塞米松在 1× 10 - 9～ 1× 10 - 4 mol/ L 浓度范围内 ,对所研究的模型细胞——人皮肤成纤维细胞增殖无明显作用。地塞米松对胶原基因启动序列具有负性调控作用 ,且存在剂量依赖关系。
Objective: To investigate the effect of dexamethasone on human α1 () procollagen gene regulatory sequence. Methods: Human dermal fibroblasts were primarily cultured by tissue block method and subcultured as the model cells in this study. (1) The Brd U incorporation of EL ISA was used to determine the proliferation of fibroblasts after 24 h treatment with different concentrations of dexamethasone. (2) Construction of three recombinant plasmids containing the 5 ’flanking sequence of human α1 () procollagen gene and the chloramphenicol acetyltransferase (CAT) reporter gene with different lengths; transfecting human skin fibroblasts by liposome Cells; EL ISA method for determination of different concentrations of dexamethasone 2 4h, transfected with three kinds of recombinant plasmid fibroblasts CAT expression. Results: (1) After treated with 1 × 10 - 9 ~ 1 × 10 - 4 mol / L dexamethasone for 24 hours under the culture conditions of 2% FCS or 10% FCS, the proliferation of fibroblasts There was no significant difference between the control group and the control group (P> 0.05). (2) After transfection of fibroblasts with three recombinant plasmids and treated with dexamethasone for 24 hours, the relative expression levels of CAT in transfected cells were measured: the dexamethasone group and the control group, the dexamethasone higher concentration group (1 × 10-5 mol / L) and lower concentration group (1 × 10-6 mol / L), the difference was significant (P <0.05). Conclusion: Dexamethasone has no significant effect on the proliferation of human dermal fibroblasts in the concentration range of 1 × 10 -9 ~ 1 × 10 -4 mol / L. Dexamethasone had a negative regulatory effect on the promoter sequence of collagen genes in a dose-dependent manner.