【作者】 陶灵；

【导师】 李世荣；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 增生性瘢痕是烧伤、整形乃至整个创伤修复领域亟待解决的难题之一,成纤维细胞是其发病的关键效应细胞,生长因子对成纤维细胞的增殖、分化具有重要的调控作用。结缔组织生长因子(CTGF)是新近发现的一种具有强烈促纤维化作用的生长因子,有研究证实[1],CTGF能促进体外增生性瘢痕成纤维细胞(HSF)增殖及其向肌成纤维细胞(MyoF)的转分化,但其细胞内信号转导机制尚不清楚,未见相关报道。JAK-STATs通路是近几年发现的细胞因子信息内传的非常重要的一条通路,是参与人体内部分生理(如造血)和病理反应(如部分肿瘤、类风湿性关节炎、脑损伤、支气管哮喘等)的共同通路之一,已证实与多种疾病发生发展及防治密切相关[2-5]。目前已知有多种细胞因子(如干扰素-α、β、γ,白细胞介素-2、4、6等)、生长因子(表皮生长因子、血小板衍生生长因子等)、生长激素、瘦素等通过JAK-STATs途径进行信号转导并调控部分细胞的增殖分化过程。鉴于以上原因,我们选择其作为参与CTGF调控人增生性瘢痕成纤维细胞增殖分化过程的信号通路进行筛选鉴定。方法:本研究通过原代培养人增生性瘢痕成纤维细胞,将对象分为①对照组:人增生性瘢痕成纤维细胞组;②CTGF刺激人增生性瘢痕成纤维细胞组。采用Western blot、免疫荧光化学染色、凝胶阻滞电泳(EMSA)、RT-PCR方法,分以下两大部分进行研究,第一大部分为JAK-STATs信号分子的初步筛选,包括①取0min、5 min、10 min、20 min、30 min、45 min、60 min、90 min八个时相点,采用western-blot方法检测各时相点JAK1、JAK2、JAK3、TYK2、STAT1、STAT2、STAT3、STAT4、STAT5、STAT6十类蛋白活化(磷酸化)情况,并筛选出对CTGF刺激其磷酸化程度呈反应性增高的蛋白(JAK1、STAT1);②对筛选出来的蛋白(STAT1)用免疫荧光化学染色方法观察其胞浆到胞核的核转位的时相变化并比较组间差异;③利用凝胶阻滞电泳(EMSA)方法进一步验证此蛋白(STAT1)与DNA的结合能力及持续时间。第二大部分,将对象分为四组:①STAT1 ASODN+CTGF组;②CTGF刺激组;③STAT1 ASODN转染组;④空白对照组。在上述研究基础上,对筛选出的信号分子(STAT1)应用特异性抑制剂(STAT1 ASODN)进行阻断,用MTT法测定各组间细胞增殖差异, RT-PCR观察α-SMA的表达以反映人增生性瘢痕成纤维细胞向肌成纤维细胞转分化情况,以了解STAT1对CTGF调控人增生性瘢痕成纤维细胞增殖分化过程的影响。结果:CTGF刺激人增生性瘢痕成纤维细胞后JAK-STATs通路所有蛋白(总蛋白以“t-”表示)均以非磷酸化和磷酸化(以“p-”表示)两种形式存在,在起始状态下即出现磷酸化条带,之后其磷酸化程度逐渐增强,在30min左右达峰值,再随时间延长逐渐下降呈一倒抛物线。Western blot显示胞浆及胞核中“p-JAK1/ t-JAK1”比值和“p-STAT1/ t-STAT1”比值随CTGF作用时间延长逐渐增大,至30min时达最强,之后逐渐下降。其余JAK-STATs蛋白虽均有不同程度的磷酸化,但并未随CTGF刺激出现反应性增强趋势。应用免疫荧光化学染色法检测STAT1的核转位,发现磷酸化STAT1蛋白在刺激30min左右达最强,比较组间差异发现:瘢痕成纤维细胞组(++):荧光明亮,胞浆、胞核分布基本一致;CTGF刺激瘢痕成纤维细胞组(+++-++++):荧光闪亮,呈明显的亮绿色,胞核中荧光强度增加,核聚集现象明显;应用凝胶阻滞电泳法,采用不同浓度CTGF刺激时发现检测STAT1与DNA的结合能力呈CTGF浓度依赖趋势,当CTGF 10ng/ml时达到峰值。同时,我们用10ng/ml CTGF刺激人增生性瘢痕成纤维细胞在不同时相点(0、10、20、30、45、60、90、120min)STAT1与DNA的结合能力及持续时间时发现:当CTGF刺激10min时,SIF已开始活化,45-60min达峰值,之后逐渐减弱,且较对照组明显增强。根据以上结果,初步筛选出JAK1、STAT1蛋白为参与CTGF调控人增生性瘢痕成纤维细胞增殖分化过程的信号分子。为进一步验证上述结论,我们将对象分为四组:①STAT1 ASODN+CTGF组;②CTGF刺激组;③STAT1 ASODN转染组;④空白对照组,观察STAT1对CTGF调控人增生性瘢痕成纤维细胞增殖分化过程的影响。在RT-PCR确定STAT1 ASODN已成功转入的前提下,用MTT测定转染前后细胞增殖程度变化时发现,增生性瘢痕成纤维细胞增殖程度呈②>④>①③的趋势变化,转染后细胞增殖明显受到抑制,结果具有统计学意义(P<0.05)。同时,我们采用RT-PCR比较转染前后a-SMA的差异,发现转染前后a-SMA/GAPDH条带灰度值无明显差异(P>0.05),结果不具有统计学意义。结论:1、在CTGF促进人增生性瘢痕成纤维细胞增殖与转分化的同时,JAK1和STAT1蛋白被不同程度活化,其活化程度随CTGF的刺激反应性增强,胞核中磷酸化STAT1荧光强度明显增加,核聚集现象明显;STAT1与DNA的结合能力增强。2、STAT1 ASODN转染后人增生性瘢痕成纤维细胞增殖能力明显下降,但并未完全抑制其增殖过程;3、STAT1 ASODN转染后人增生性瘢痕成纤维细胞向肌成纤维细胞的转分化能力则未见明显改变。以上研究结果提示:STAT1参与了CTGF调控人增生性瘢痕成纤维细胞增殖过程,但并非是唯一途径;而JAK1则可能作为其上游因子,通过JAK1-STAT1通路参与了CTGF促进人增生性瘢痕成纤维细胞增殖过程。这从一定程度上揭示了CTGF调控人增生性瘢痕成纤维细胞增殖过程的信号转导机制,从而为抑制瘢痕纤维化提供了新的研究方向,有助于更深入地调控CTGF的促纤维化作用,为增生性瘢痕及其它纤维化疾病的研究及防治提供新的思路。更多还原

【Abstract】 Hypertrophic scar is a quiz which needs to be solved in burn surgery.plastic surgery and all the trauma field.as the key effector cell,fibroblast is regulated by growth factor in the aspect of proliferation and differentiation.Connective tissue growth factor(CTGF) is a kind of fator which can promote fibrosis intensively and urge vitro-human hypertrophic scar fibroblast to differentiate to myofibroblast[1].But there is rare reports to its signal transduction mechanism.JAK-STATs pathway is a very important one which is found in recent years and play a part in cytokine signal transduction,and it paticipates in many physiological functions(eg. haematogenesis)and patho-functions(eg. Tumor.arthritis deformans.brain injured.bronchial asthma) [2-5].A lot of cytokines(eg. Interferon-α.β.γ, interleukin-2.4.6) .growth factors(eg. epidermal growth factor.platelet-derived growth factor) .growth hormone.leptin regulate proliferation and differentiation of many cells through JAK-STATs.Respecting the aboved reasons,we carry out the work to study the function of JAK-STATs pathway in proliferation and differentiation of human hypertrophic scar fibroblast induced by connective tissue growth factor.Methods: cultivate hHSF with primary cultivation,then, divide the cells into two groups:①control group:hHSF②CTGF-stimulated group:hHSF with CTGF.The 1st part included:①Western-blot was used to detect proteins’activation including JAK1.JAK2.JAK3.TYK2.STAT1.STAT2.STAT3.STAT4.STAT5.STAT6 at 0min.5 min.10 min.20 min.30 min.45 min.60 min.90 min .②Immunofluorescence(IF)was used to verify nuclear translocation of the boltered protein.③Electrophoretic mobility shift assay(EMSA) was used to verify binding ability with DNA of the boltered protein.In the 2nd part,we divided cells into four groups:①STAT1 ASODN+CTGF group;②CTGF group;③STAT1 ASODN group;④control group.And then,we used MTT to detect the proliferation of hHSF and western-blot to detectα-SMA to learn the differentiation .Results: In the premise that proliferation and differentiation of CTGF-stimulated group was much higher than that of control group(p<0.05),results of western-blot showed all the JAK-STATs proteins(“t-”) exists in two styles:non-phosphorylation and phosphorylation(“p-”).The phosphorylated protein appears at the beginnning,and then increased gradually,peaked at 30min,and fell-off afer 30mins.Among all the proteins ,only the ratio of“p-JAK1/ t-JAK1”and“p-STAT1/ t-STAT1”increased following CTGF stimulation. IF results showed phosphorylated STAT1 peaked at 30min.We compared the two groups and found:①control group(++):bright,flavo-green;②CTGF group(+++-++++): blink,obviouse bright green. EMSA told us that the binding ability of STAT1 and DNA depended on the concentration of CTGF,and peaked with the stimulation of 10ng/ml CTGF.And then,we used 10 ng/ml CTGF to stimulate hHSF at 0.10.20.30.45.60.90.120min and found that SIF was activated at 10min,peaked at 45-60min,fell-off gradully after 60min.The binding ability of CTGF group was much higher than that of control group.So JAK1.STAT1 was selected in preliminary screening.And then ,we used STAT1 ASODN to block the STAT1.In the premise of sucessful transfection of STAT1 ASODN by RT-PCR,we found that the proliferation of hHSF was inhibited obviously with MTT afer ASODN transfection(P<0.05),but expression ofα-SMA changed little before and after the blockage of STAT1 ASODN(P>0.05). Conclusions:1. At the same time of CTGF-stimulated proliferation and differentiation of hHSF,JAK1and STAT1was activated to some extent,and increased following CTGF stimulation.And fluorescence intensity of phosphorylated STAT1 and binding ability of STAT1and DNA increased obviously following the activation of CTGF;2. The proliferation of hHSF was inhibited obviously but not all with MTT afer ASODN transfection(P<0.05);3. Expression ofα-SMA changed little before and after the blockage of STAT1 ASODN(P>0.05).STAT1 is important in the process of CTGF-induced proliferation of hHSF,but it’s not the only pathway to control the process.JAK1 may be the upstream element of STAT1 and may paticipate in the process of CTGF-induced proliferation of hHSF.The results above revealed the signal transduction mechanism of CTGF-induced proliferation of hHSF ,may afford new direction to inhibit scar fibrosis and contraction.更多还原