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目的 :探索器官纤维化形成中调控 型胶原基因高水平转录的启动片段。方法 :从人α1( )胶原基因转录起始点上游 - 2 .5 kb～ + 42 bp的片段中 ,取长度不等的片段作为启动子与含氯霉素乙酰基转移酶 (CAT)报告基因的质粒组成 6个重组体 ,脂质体法转染上述重组体至正常人原代培养皮肤成纤维细胞 ,CAT- EL ISA测定细胞 CAT表达水平以比较各重组体的启动子活性。结果 :- 2 483～ + 42 bp,- 2 6 8～ + 42 bp序列具有强启动 CAT表达活性 ,而 - 10 5～ + 42 bp片段启动CAT活性最低。结论 :人α1( )胶原基因 - 2 483～ + 42 bp,- 2 6 8～ + 42 bp片段有高启动活性 ,是进一步研究纤维化相关DNA结合蛋白的重要调控靶序列。
Objective: To explore the promoter fragment of high-level transcription of regulatory collagen gene in the formation of organ fibrosis. Methods: The fragments of unequal length from 2.5 kb to + 42 bp upstream of the transcription initiation site of human α1 (superscript) collagen gene were used as promoters and the promoter of chloramphenicol acetyltransferase (CAT) reporter gene The plasmids were composed of six recombinants. The above recombinant plasmids were transfected into primary cultured human dermal fibroblasts by lipofectamine. The CAT expression levels were determined by CAT-ELISA to compare the promoter activity of each recombinant. Results: - 2 483 ~ + 42 bp, - 2 68 ~ + 42 bp sequence had strong promoter CAT activity, while -10 5 ~ + 42 bp promoter CAT activity was the lowest. CONCLUSION: Human α1 (-) collagen gene - 2 483 ~ + 42 bp and - 2 68 ~ + 42 bp have high promoter activity, which is an important regulatory target for further study of fibrosis related DNA binding proteins.