【作者】 陆燕；

【导师】 魏锐利；

【作者基本信息】 第二军医大学 ， 眼科学， 2011， 博士

【摘要】 目的甲状腺相关性眼病（thyroid-associated ophthalmopathy,TAO）眼外肌纤维化在病理学上具有不可逆的特点,可导致患者出现斜视、复视,甚至压迫性神经病变而导致失明。目前发病机制不明,药物及手术治疗效果均欠佳。TGF-β1是导致纤维化最重要的细胞因子之一,可通过促使成纤维细胞增殖、向肌成纤维细胞（myofibroblast,MFB）表型转分化及细胞外基质（extracellular matrix,ECM）的合成,最终导致纤维化的形成。在此过程中,MFB持续存在或凋亡过程缺陷可导致纤维化进行性发展（α-SMA是MFB标志性蛋白）,MFB成为控制纤维化的重要靶细胞。而TGF-β1是一种多功能的细胞因子,具有抑制炎症反应及抗肿瘤作用,若直接阻断TGF-β1抗纤维化则对机体产生不利的影响。因此,希望在阐明TGF-β1致纤维化作用机制的同时寻找其下游信号通路,结缔组织生长因子（connective tissue growth factor,CTGF）属于即可早期基因CNN家族,在TGF-β1作用下,可由成纤维细胞分泌,CTGF能被多种因子转录激活,其中以TGF-β1最引人注目,在TGF-β1的下游发挥重要的生物学作用。本课题旨在通过体外培养TAO眼外肌来源的成纤维细胞（orbital fibroblasts,OF）,观察TGF-β1能否通过促进成OF增殖、向MFB表型转分化及ECM的合成,最终导致TAO眼外肌纤维化的形成。同时选择TGF-β1/Smads、丝裂原活化蛋白激酶（mitogen -activated protein kinase,MAPK）、磷脂酰肌醇3激酶（phosphatidylinositol 3-kinase,PI3K）信号传导通路,利用特异性抑制剂（SB431542 for TGF-β1/Smads、SB203580 for P38/MAPK、PD98059 for ERK/MAPK、SP600125 for JNK/MAPK、LY294002 for PI3K）分别阻断其相应的信号传导通路,初步探讨TGF-β1促进OF转分化为MFB及诱导OF表达CTGF的信号传导通路,同时观察了CTGF对OF转分化为MFB的作用以及CTGF自分泌调节的信号传导通路,希望进一步明确TGF-β1在TAO眼外肌纤维化中的发病机制,为TAO眼外肌纤维化治疗提供相应的分子靶点。方法1.应用免疫组化SP法检测TGF-β1、CTGF、P-Smad2/Smad3、Smad4、Smad7在TAO患者眼外肌中的表达;2.采用组织块贴壁法原代培养TAO患者眼外肌来源的成纤维细胞并采用HE染色及免疫细胞化学技术进行鉴定;3.采用Real-time PCR观察TGF-β1诱导OF表达ECM主要成分TIMP-1 mRNA、COL-I mRNA、COLIII mRNA及FN mRNA水平的变化;采用细胞免疫荧光观察TGF-β1诱导OF表达TIMP-1、COL-I、COL III及FN蛋白及其定位;4.采用MTT法检测TGF-β1诱导OF增殖的效应;Real-time PCR和Western-blot检测TGF-β1诱导OF合成α-SMAmRNA、CTGF mRNA及蛋白水平变化;预先添加各种信号通路抑制剂（SB431542 for TGF-β1/Smads、SB203580 for P38/MAPK、PD98059 for ERK/MAPK、SP600125 for JNK/MAPK、LY294002 for PI3K）后,采用Real-time PCR和Western-blot检测TGF-β1诱导OF合成α-SMAmRNA、CTGF mRNA及蛋白水平变化;细胞免疫荧光检测α-SMA、CTGF蛋白表达定位及Smad3磷酸化变化;Western-blot检测SB431542抑制剂对MAPK家族（p38、ERK、JNK）磷酸化的影响;同时采用Real-time PCR检测CTGF诱导OF转分化作用及CTGF自身调节信号传导通路。结果1.免疫组化结果显示TAO眼外肌组织成纤维细胞及内皮细胞中存在较正常眼外肌高表达的TGF-β1、CTGF、P-Smad2/Smad3、Smad4,但Smad7在TAO纤维化眼外肌组中较正常眼外肌低表达;2.甲状腺相关眼病眼外肌来源成纤维细胞原代培养成功并稳定传代,细胞呈长梭形,细胞免疫化学鉴定:波形蛋白（Vimentin）染色阳性,而角蛋白（Keratin）、结蛋白（Desmin）、S^-100染色均阴性,证明为中胚层来源的成纤维细胞;3.Realtime-PCR检测10μg·L^-1TGF-β1刺激OF的时间效应:TIMP^-1 mRNA在3 h和6 h分别为对照组的1.50倍和2.46倍（P<0.01）; COL-I mRNA在24 h和48 h分别为对照组的5.49、3.69倍（P<0.01）;COL-IIImRNA在24 h和48 h分别为对照组的2.14、1.63倍（P<0.01）;FN mRNA在12 h和24 h后分别为对照组的2.45、1.53倍（P<0.01）。不同浓度TGF-β1刺激OF 24 h后剂量效应:与空白对照组相比,TIMP^-1mRNA在5μg·L^-1和10μg·L^-1时分别为1.79、1.46倍（P<0.01）; COL-I mRNA在1μg·L^-1和10μg·L^-1分别1.94、3.29倍（p<0.01）; COL- III mRNA在5μg·L^-1和10μg·L^-1分别1.52、3.28倍（P<0.01）;FN mRNA在1μg·L^-1和10μg·L^-1分别为1.30、2.45倍（P<0.01）;细胞免疫荧光染色显示TIMP^-1、COL-I、COL III及FN蛋白的表达位于OF细胞浆;4.MTT法检测显示TGF-β1(1～10μg·L^-1TGF-β1)可促进OF增殖,各浓度与对照组相比（P>0.05）; Realtime-PCR和Western-blot检测显示在一定的浓度范围内,TGF-β1均以时间和剂量依赖的方式诱导OF表达α-SMA、CTGF mRNA,各浓度与空白对照组相比,（P<0.05）其中CTGF mRNA在12h达到高峰,α-SMA mRNA在24h达到高峰,SB431542显著抑制剂了α-SMA mRNA的表达（与对照相比,P<0.05）,同时PD98059、SB203580也抑制剂了α-SMA mRNA的表达（与空白对照相比P<0.05）;SB431542和SP600125抑制了CTGFmRNA的表达（与对照相比,P<0.05）;Western-blot检测出相对应的结果;同时Western-blot检测TGF-β1诱导OF,可使Smad3磷酸化改变,在30min时达到顶点;SB431542抑制剂对MAPK家族磷酸化没有影响。同时Realtime-PCR检测一定的浓度范围内(0.5～50μg·L^-1)CTGF可以试剂和剂量依赖的方式诱导OF转分化表达α-SMA mRNA,（与对照组相比P<0.05）SB431542和SP600125抑制了CTGF自分泌的表达（与对照相比,P<0.05）,细胞免疫荧光检测出10μg·L^-1TGF-β1诱导OF表达α-SMA和CTGF蛋白,定位于OF细胞浆;细胞免疫组化观察到OF表达α-SMA。结论1.TGF-β1、CTGF、P-Smad2/Smad3、Smad4在TAO患者眼外肌组织中较正常组织中高表达,而Smad7则相反,表达定位于成纤维细胞,少量表达在内皮细胞,TGF-β1/Smad通路可能参与了TAO眼外肌纤维化的发病过程;2.组织块贴壁法培养的细胞符合甲状腺相关眼病眼外肌成纤维细胞的形态学和免疫学特征,可作为研究甲状腺相关性眼病眼外肌纤维化的体外模型;3. Realtime-PCR显示在一定的浓度范围内,TGF-β1以剂量和时间依赖的方式诱导OF表达细胞外基质主要成分TIMP^-1、COL-I、COL III及FN mRNA;细胞免疫荧光染色显示TGF-β1可诱导OF表达TIMP^-1、COL-I、COL III及FN蛋白,蛋白位于OF细胞浆;TGF-β1可通过促进ECM的合成参与TAO眼外肌纤维化的发病过程。4. TGF-β1对OF增殖有增加的趋势,但P>0.05,无统计学意义;TGF-β1可诱导OF向MFB表型转分化及表达CTGF,CTGF的表达高峰早于α-SMA;CTGF可诱导OF向MFB表型转分化;细胞免疫荧光可检测到α-SMA及CTGF蛋白的表达并定位于OF细胞浆;TGF-β1诱导OF向MFB表型转分化表达α-SMA通TGF-β1/Smad及P38/MAPK和ERK/MAPK信号传导通路;TGF-β1诱导OF表达CTGF通TGF-β1/Smad及JNK/MAPK信号传导通路;CTGF自分泌调节通过JNK/MAPK信号传导通路;TGF-β1可引起Smad3磷酸化改变,SB431542对MAPK信号通路磷酸化水平没有影响。更多还原

【Abstract】 ObjectiveExtraocular muscle fibrosis of thyroid-associated ophthalmopathy （TAO） is inreversible in pathology,the patients complain of strabisums, diplopia and even lose their sight because of compressed optic neuropathy.Transforming growth factor-β1 （TGF-β1）is one of the most important profibrotic cytokines,it can lead to proliferation of orbital fibroblasts（OF）, induce OF transdifferentiation to myofibroblast（MFB）and stimulate OF to express components of extracellular matrix（ECM）. Studies have confirmed that TGF-β1 play an important role in the pathogenesis of Extraocular muscle fibrosis of TAO and the abnormally expressive TGF-β1, （connective tissue growth factor, CTGF）and P-Smad2/Smad3 in OF of TAO.However,the molecule mechanism and signal ransduction pathways are uncertain.This research was designed to study the expression ofα-smooth muscle actin（α-SMA）,CTGF and TGF-β1/Smad pathway and key enzymes of mitogen- activated protein kinase（MAPK） and phosphatidylinositol 3-kinase（PI3K）signal transduction pathways in orbital fibroblasts after TGF-β1stimulation and the correlation betweenα-SMA,CTGF and the above signal transduction pathways,to investigation the expression changes and significance of TGF-β1 in OF and to explore its downstream signal transduction pathways and their interrelationship in above process.Methods1. To detect the expression of TGF-β1、CTGF、P-Smad2/ Smad3、Smad4、Smad7 with immunohistochemical staining in human extraocular muscle tissues of TAO patients.2. Extraocular muscle fibroblasts were primary cultured by tissue explant culture technique and identified by morphological and immunocytochemistry analysis.3. OF were treated with 10μg·L^-1 TGF-β1 at different time points （0 hours,3 hours,6 hours,12 hours,24 hours and 48 hours） and different concentrations(1μg·L^-1,5μg·L^-1,10μg·L^-1 and 20μg·L^-1) for 24 hours, and real-time quantitative RT-PCR was performed to observe the effects of TGF-β1 on the expression of TIMP^-1, COL-I, COL- III and FN mRNA.Cell immunofluorescence was used to detect expression of TIMP^-1, COL-I, COL- III and FN protein.4. MTT was used to assess the proliferation of OF by TGF-β1,real -time PCR and Western blot were used to assess the signal transduction pathways involved in TGF-β1inducedα-SMA and CTGFgene expression in OF cells,various inhibitors（SB431542 for TGF-β1/Smad, PD98059 for ERKl/2 ,SB203580 for p38 ,SP600125 for JNK ,LY294002 for PI3K/AKT,） specific to signal mediators were employed.Activation of Smad3（phosphorylation） was assessed by Western blot using antibodies against the active（phospho）forms . Real -time PCR was used to assess the signal transduction pathways involved in CTGF inducedα-SMA and CTGF gene expression in OF. Cell immunofluorescence was used to detect expression ofα-SMA and CTGF protein.Results1. The positive staining of TGF-β1、CTGF、P-Smad2/Smad3 and Smad4 mainly localized in orbital fibroblasts of TAO and were higher than those in control group,while Smad7 was lower than that in control group.2.The Extraocular muscle fibroblasts were cultured and passaged successfully.The cells showed long spindle appearance. their Vimentin staining were positive,but their Desmin staining, Keratin staining and S^-100 staining were all negative by immunocytochemistry analysis,indicating that the cells were mesoderm derived fibroblasts.3. Time effect of OF treated with 10μg·L^-1 TGF-β1:TIMP^-1 mRNA was 1.5 folds to that of control group at 3 hours and 2.46 folds at 6 hours （both p<0.01）;COL-I mRNA was 5.49 folds to that of control group at 24 hours and 3.69 folds at 48 hours,（both p<0.01）;COL- III mRNA was 2.14 folds to that of control group at 24 hours and 1.63 folds at 48 hours（both p<0.01）;FN mRNA was 2.45 folds to that of control group at 12 hours and 1.53 folds at 24 hours（both p<0.01 ）;Dose effects of OF treated with different concentrations TGF-β1 for 24 hours :TIMP^-1 mRNA was 1.79 folds to that of control group at 5μg·L^-1 and 1.46 folds at 10μg·L^-1 （ both p<0.01）;COL-I mRNA was 1.94 folds to that of control group at 1μg·L^-1 and 3.29 folds at 10μg·L^-1 （both p<0.01）;COL- III mRNA was 1.52 folds to that of control group at 5μg·L^-1 and 3.28 folds at 10μg·L^-1（both p<0.01）;FN mRNA was 1.3 folds to that of control group at 1μg·L^-1 and 2.45 folds at 10μg·L^-1, both p<0.01). Cell immunofluorescence showed that OF expression of TIMP^-1, COL-I, COL- III and FN protein by TGF-β1.4. Real -time PCR and Western blot showed that bothα-SMA ,CTGF mRNA and protein expression were higher than those in control group（p<0.05）. SB431542, PD98059 and SB203580 partly attenuated TGF-β1stimulated expression ofα-SMA（p<0.05） ,while the JNKinhibitor SP600125 or the PI3K inhibitor LY294002 had no such impact（（p>0.05））.SB431542 and SP60125 partly attenuated TGF-β1stimulated expression of CTGF（p<0.05）,while the P38inhibitor SB203580 or the PI3K inhibitor LY294002 or ERK inhibitor PD98059 had no such impact（p>0.05）.TGF-β1 induced Smad3 phosphorylation ,SB431542 effectively inhibited the phosphorylation of Smad3 stimulated by TGF-β1but not that of the components of the MAPK pathways.Real -time PCR showed that CTGF can stimulate the expression ofα-SMA in OF in a time and dose-dependent manner, and SP600125 significantly attenuated CTGFstimulated expression of CTGF in OF（p<0.05）.Conclusion1. TGF-β1/Smad pathway may play an important role in the pathogensis of extraocular muscle fibrosis of TAO.2. The cells cultured by tissue explant culture technique are consistent with typical extraocular muscle fibroblasts characteristics in morphology and immunocytochemical observation.The extraocular muscle fibroblasts are used as cell model in vitro for further research on the mechanism of extraocular muscle fibrosis of thyroid -associated ophthalmopathy.3.TGF-β1 can stimulate the expression of TIMP-1 mRNA,COL-I mRNA, COL- III mRNA and FN mRNA in OF in a time and dose-dependent manner, it may paly an important role in the pathogenesis of extraocular muscle fibrosis with TAO.4. SB431542 significantly attenuated TGF-β1 stimulated expression of CTGF andα-SMA,While MAPK partly attenuated TGF-β1 stimulated expression of CTGF andα-SMA. CTGF can stimulate the expression ofα-SMA in OF,and SP600125 significantly attenuated CTGF stimulated expression of CTGF in OF.更多还原