Tao Ling, Liu Jianyi, Li Zhe, Dai Xia, Li Shirong
Department of Plastic and Reconstructive Surgery, Southwestern Hospital, Third Military Medical University, Chongqing 400038, P.R. China.
Mol Med Rep. 2010 Nov-Dec;3(6):941-5. doi: 10.3892/mmr.2010.349. Epub 2010 Aug 26.
The aim of this study was to investigate whether the JAK-STAT pathway participates in the processes of proliferation and differentiation induced by connective tissue growth factor (CTGF) in human hypertrophic scar fibroblasts (hHSF). hHSF were grown as primary cultures, then treated with or without CTGF. Western blotting was used to detect JAK1, JAK2, JAK3, TYK2, STAT1, STAT2, STAT3, STAT4, STAT5 and STAT6 protein expression in the hHSF at various time points after stimulation with CTGF. Immunofluorescence and the electrophoretic mobility shift assay (EMSA) were used to identify the interacting signalling molecules and to investigate their activation, respectively. After the signalling molecules of interest were selected, a specific inhibitor (STAT1 ASODN) was used to block the JAK-STAT pathway. The MTT assay was used to detect the proliferation of hHSF, and differentiation was assessed by evaluating changes in α-SMA expression by RT-PCR. Based on the results of Western blotting, immunofluorescence and EMSA, proliferation and differentiation were much higher in hHSF treated with CTGF (p<0.05). After blocking the pathway with STAT1 ASODN, hHSF proliferation was markedly, though not entirely, inhibited, while α-SMA expression was not significantly altered. JAK1 and STAT1 are therefore likely to participate in the proliferation and differentiation of hHSF induced by CTGF. However, though the STAT1 signalling pathway plays a key role in the CTGF-induced proliferation of hHSF, it is not the only pathway controlling this process. JAK1 may be an upstream element of STAT1, and may also participate in the CTGF-induced proliferation of hHSF. These results elucidate the signal transduction mechanism of CTGF-induced hHSF proliferation, and may aid in the development of a novel method for the inhibition of scar fibrosis and contraction.
本研究旨在探讨JAK-STAT信号通路是否参与结缔组织生长因子（CTGF）诱导人增生性瘢痕成纤维细胞（hHSF）增殖和分化的过程。将hHSF进行原代培养，然后分别用CTGF处理或不处理。采用蛋白质免疫印迹法检测CTGF刺激后不同时间点hHSF中JAK1、JAK2、JAK3、TYK2、STAT1、STAT2、STAT3、STAT4、STAT5和STAT6蛋白的表达。分别采用免疫荧光法和电泳迁移率变动分析（EMSA）鉴定相互作用的信号分子并研究其激活情况。在选定感兴趣的信号分子后，使用特异性抑制剂（STAT1反义寡脱氧核苷酸）阻断JAK-STAT信号通路。采用MTT法检测hHSF的增殖情况，并通过逆转录聚合酶链反应（RT-PCR）评估α-SMA表达的变化来评价分化情况。基于蛋白质免疫印迹、免疫荧光和EMSA的结果，CTGF处理的hHSF增殖和分化水平更高（p<0.05）。用STAT1反义寡脱氧核苷酸阻断该信号通路后，hHSF的增殖受到明显但不完全的抑制，而α-SMA的表达没有明显改变。因此，JAK1和STAT1可能参与CTGF诱导的hHSF增殖和分化。然而，尽管STAT1信号通路在CTGF诱导的hHSF增殖中起关键作用，但它不是控制这一过程的唯一通路。JAK1可能是STAT1的上游元件，也可能参与CTGF诱导的hHSF增殖。这些结果阐明了CTGF诱导hHSF增殖的信号转导机制，可能有助于开发一种抑制瘢痕纤维化和挛缩的新方法。
