STAT1在结缔组织生长因子刺激人增生性瘢痕成纤维细胞增殖分化中的作用

Function of STAT1 in proliferation and differentiation of human hypertrophic scar fibroblast induced by connective tissue growth factor

目的基于既往研究，验证信号转导和转录活化因子1（STAT1）是否参与结缔组织生长因子（CTGF）刺激人增生性瘢痕成纤维细胞增殖分化过程。方法取6例增生性瘢痕患者的手术切除组织，培养成纤维细胞。应用凝胶阻滞电泳（EMSA）方法测定不同浓度（0、5、7．5、10、15ng／m1）CTGF刺激45min和10ng／mlCTGF刺激不同时间（0、10、20、30、45、60、90、120min）对瘢痕成纤维细胞STAT1与DNA结合能力的影响。将瘢痕成纤维细胞分为CTGF刺激组、STAT1反义寡脱氧核苷酸（ASODN）转染组、STAT1 ASODN＋CTGF组和空白对照组，用噻唑盐（MTT）法测定各组细胞培养1、2、3d的增殖活性，RT-PCR测定α-平滑肌肌动蛋白（α-SMA）mRNA表达以反映瘢痕成纤维细胞向肌成纤维细胞分化活性。结果瘢痕成纤维细胞中STAT1与DNA的结合活性随CTGF浓度而增强，当CTGF浓度为10ng／ml时达峰值；10ng／ml的CTGF刺激45～60min达峰值。MTT法检测显示CTGF刺激组培养1—3d增殖活性明显均高于空白对照组（均P〈0．05），而STAT1 ASODN转染组和STAT1 ASODN＋CTGF组均明显低于空白对照组和CTGF刺激组（均P〈0．05）。RT—PCR检测结果显示，CTGF刺激组、STAT1 ASODN转染组、STAT1 ASODN＋CTGF组和空白对照组瘢痕成纤维细胞向肌成纤维细胞分化活性分别为0．78±0．08、0．38±0．09、0．76±0．10和0．d0±0．12，STAT1 ASODN转染组和空白组均明显低于CTGF刺激组和STAT1 ASODN＋CTGF组（均P〈0．05）。结论STAT1 ASODN可明显抑制瘢痕成纤维细胞的增殖活性，并阻断CTGF对细胞增殖的刺激作用，提示STAT1参与CTGF调控人增生性瘢痕成纤维细胞增殖过程。

Objective To validate whether STAT1 participated in the process of CTGF-indueed proliferation and differentiation of human hypertrophic scar fibroblast （hHSF） on the basis of previous researches. Methods hHSF was co-cultivated with 6 patients＇ hypertrophic scar specimens. Eleetrophoretic mobility shift assay （EMSA） was used to verify binding ability between DNA and STAT1 with the stimulus of different concentrations of CTGF（0,5, 7. 5, 10, 15 ng/ml） at 45th min and the stimulus of 10 ng/ml CTGF at different phase point（0, 10, 20, 30, 45, 60, 90, and 120 min）. We divided cells into CTGF group, STAT1 ASODN group, STAT1 ASODN ＋ CTGF group and control group. And MTT was used to detect the proliferation of hHSF on days1, 2 and 3, and RT-PCR to detect α-smouth muscle actin mRNA to follow the differentiation. Results EMSA showed that the binding ability between STAT1 and DNA depended on the concentration of CTGF and peaked with the stimulation of 10 ng/ml CTGF. And at the same time, it peaked at 45 - 60 min with 10 ng/ml CTGF. MTT showed that cellular proliferation of CTGF group was much higher than that of control group （ all P 〈 0. 05 ）. And those of STAT1 ASODN group and STAT1 ASODN ＋ CTGF group were much lower than those of control group and CTGF group （ all P 〈 0.05 ）. RT-PCR showed that differentiation activation from fibroblast to myofibroblast of CTGF group, STAT1 ASODN group, STAT1 ASODN ＋ CTGF group and control group were 0. 78 ± 0. 08, 0. 38 ± 0. 09, 0. 76 ± 0. 10, and 0.40 ± 0. 12, respectively. Differentiation activation of STAT1 ASODN group and control group were much lower than those of CTGF group and STAT1 ASODN ＋ CTGF group （ all P 〈 0. 05 ） Conclusions STAT1 ASODN is important in the proliferating process of hHSF and it blocks the stimulation of CTGF on hHSF proliferation. The above result revealed that STAT1 participates in the process of hHSF proliferation induced by connective tissue growth factor.