系统性硬化病成纤维细胞单克隆Ⅰ型前胶原基因转录特性的研究

Transcriptional characteristics of type I procollagen gene of fibroblast clones from patients with systemic sclerosis

目的探讨系统性硬化病（ss）患者皮损成纤维细胞高胶原合成克隆的发生机制。方法通过原位杂交法检测成纤维细胞克隆Ⅰ型前胶原理1链（COL1A1）mRNA表达，并将成纤维细胞克隆依COL1A1mRNA水平分为高、中、低组。构建5个含有人COL1A1基因不同长度转录调控序列的重组质粒，应用瞬时转染、荧光素酶报告基因技术检测5个COL1A1基因转录调控序列在不同组别成纤维细胞克隆中的启动活性。结果SS组和正常对照组不同成纤维细胞克隆COL1A1mRNA水平都具有明显差异，ss组高表达COL1A1mRNA的成纤维细胞克隆所占的比例（50．O％）高于正常对照组（12．5％），SS组成纤维细胞克隆的平均COL1A1mRNA水平显著高于正常对照组（t=3．690，P〈0．01）。5个COL1A1上游转录调控序列中-174～＋42bp在SS组和正常对照组不同成纤维细胞克隆均具有最强启动活性。COL1A1转录调控序列的活性与成纤维细胞克隆的COL1A1mRNA水平呈正相关。结论ss高胶原合成的成纤维细胞克隆的COL1A1基因在转录水平即被激活。

Objective To study the mechanisms underlying high collagen synthesis of systemic sclerosis skin fibroblasts. Methods The mRNA expression levels of procollagen alpha 1 type 1 （COL1A1） were detected by in situ hybridization to select heterogeneic fibroblast clones, and monoclonal fibroblasts were divided into three groups according to their mRNA levels of COL1A1. Meanwhile, five deletion constructs containing portions of human procollagen gene promoter were ligated into plasmids and used to transfect the three groups of fibroblast clones. The transcription of COL1A1 in these fibroblasts was assessed by transient transfection and luciferase assay. Results Fibroblast clones from both patients and normal controls showed a heterogeneity in the mRNA expression of COL1A1. Patients with systemic sclerosis had a larger proportion of fibroblasts expressing high level of COL1AlmRNA than did normal controls （50% vs 12.5%）. A significant increase was also observed in the mean mRNA levels of COL1A1 in fibroblast clones from patients with systemic sclerosis compared with those from normal controls （6.64 ± 2.48 vs 3.69 ± 2.01, t = 3.690, P 〈 0.01 ）. Maximal transcriptional activity was observed with a construct containing COL1A1 promoter from -174 bp to ＋42 bp in all fibroblast clones. The activities of COL1A1 promoter positively correlated with the mRNA levels of this gene. Conclusions COL1A1 gene might be activated at the transcriptional level in systemic sclerosis fibroblast clones with a high rate of collagen synthesis.