TGF-β1在TAO眼外肌纤维化中的作用及相关信号传导通路研究

发布时间：2018-03-30 04:07

  本文选题：甲状腺相关性眼病　切入点：眼眶成纤维细胞　出处：《第二军医大学》2011年博士论文

【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~(-1)00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~(-1)TGF-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(P0.01); COL-I mRNA在24 h和48 h分别为对照组的5.49、3.69倍(P0.01);COL-IIImRNA在24 h和48 h分别为对照组的2.14、1.63倍(P0.01);FN mRNA在12 h和24 h后分别为对照组的2.45、1.53倍(P0.01)。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~(-1)mRNA在5μg·L~(-1)和10μg·L~(-1)时分别为1.79、1.46倍(P0.01); COL-I mRNA在1μg·L~(-1)和10μg·L~(-1)分别1.94、3.29倍(p0.01); COL- III mRNA在5μg·L~(-1)和10μg·L~(-1)分别1.52、3.28倍(P0.01);FN mRNA在1μg·L~(-1)和10μg·L~(-1)分别为1.30、2.45倍(P0.01);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~(-1)TGF-β1)可促进OF增殖,各浓度与对照组相比(P0.05); 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,(P0.05)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,P0.05),同时PD98059、SB203580也抑制剂了α-SMA mRNA的表达(与空白对照相比P0.05);SB431542和SP600125抑制了CTGFmRNA的表达(与对照相比,P0.05);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P0.05)SB431542和SP600125抑制了CTGF自分泌的表达(与对照相比,P0.05),细胞免疫荧光检测出10μg·L~(-1)TGF-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P0.05,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。
[Abstract]:objective
Thyroid associated ophthalmopathy (thyroid-associated ophthalmopathy, TAO) fibrosis of the extraocular muscles is not reversible in pathology, can lead to patients with strabismus, diplopia, and compressive neuropathy can lead to blindness. The pathogenesis is unknown, the effect of drug treatment and surgery were poor.TGF- beta 1 which is one of the most important cytokines of fibrosis but, by promoting the proliferation of fibroblasts and myofibroblasts (myofibroblast, MFB) transdifferentiation and extracellular matrix (extracellular matrix, ECM) synthesis, eventually leading to fibrosis. In this process, the persistence of MFB or apoptosis defects can lead to fibrosis progression (alpha -SMA is MFB mark protein), MFB has become an important target cell control fibrosis. TGF- beta 1 is a multifunctional cytokine that can inhibit the inflammation and anti-tumor effect, if directly Blockade of TGF- beta 1 anti fibrosis on the body to produce adverse effects. Therefore, I hope to clarify the TGF- beta 1 also induced fibrosis mechanism for downstream signaling pathways, connective tissue growth factor (connective tissue, growth factor, CTGF) belongs to the early gene CNN family, in beta 1 TGF- function, which is produced by a fiber cells, CTGF can be a variety of transcription factor activation, which TGF- beta 1 most notably, play an important role in the downstream of TGF- beta 1. The aims of the in vitro cultured TAO fibroblasts of extraocular muscle origin (orbital fibroblasts, OF), to observe whether through TGF- beta 1 promotes the proliferation of OF the synthesis of MFB to transdifferentiation and ECM, eventually led to the formation of TAO fibrosis of extraocular muscle. At the same time choose TGF- beta 1/Smads, mitogen activated protein kinase (mitogen -activated protein kinase, MAPK), phosphatidylinositol 3 kinase (Phosp Hatidylinositol 3-kinase, PI3K) signal transduction pathway by specific inhibitors (SB431542 for TGF- SB203580 for beta 1/Smads, P38/MAPK, PD98059 for ERK/MAPK, SP600125 for JNK/MAPK, LY294002 for PI3K) were blocked by the corresponding signal transduction pathway, a preliminary study of the TGF- beta 1 OF to promote OF induced differentiation of MFB and CTGF signal transduction pathway at the same time, to observe the expression of CTGF signal transduction pathway in the transdifferentiation of OF MFB and the role of regulating the secretion of CTGF, to further clarify the TGF- beta 1 in TAO fibrosis of extraocular muscle in the pathogenesis, provide molecular targets for the corresponding TAO extraocular muscle fibrosis treatment.
Method
1. the expression of TGF- beta 1, CTGF, P-Smad2/Smad3, Smad4, Smad7 in the extraocular muscles of TAO patients was detected by immunohistochemical SP method.
2. the fibroblasts derived from the extraocular muscles of TAO patients were cultured with tissue block wall method and identified by HE staining and immunocytochemical technology.
3., Real-time PCR was used to observe TGF- beta 1 induced OF expression, TIMP-1, mRNA, COL-I mRNA, COLIII mRNA and mRNA level in OF expression. Cell immunofluorescence assay was used to observe the expression of TGF- and its localization.
4. MTT method was used to detect TGF- beta 1 induced proliferation of OF Real-time and Western-blot PCR effect; detection of TGF- beta 1 induced synthesis of OF alpha -SMAmRNA, mRNA and protein level of CTGF in advance; add a variety of signaling pathway inhibitors (SB431542 for TGF- SB203580 for beta 1/Smads, P38/MAPK, PD98059 for ERK/MAPK, SP600125 for JNK/MAPK, LY294002 for PI3K) after using Real-time, PCR and Western-blot detection of TGF- beta 1 induced synthesis of OF alpha -SMAmRNA, mRNA and protein level of CTGF cells; immunofluorescence detection of alpha -SMA, CTGF protein expression and phosphorylation of Smad3 changes; Western-blot detection of SB431542 inhibitors on MAPK family (p38, ERK, JNK) phosphorylation; at the same time using Real-time PCR detection of CTGF induced OF differentiation and CTGF self regulating signal transduction pathway.
Result
1. immunohistochemical results showed that there was a higher expression of TGF- beta 1, CTGF, P-Smad2/Smad3 and Smad4 in extraocular muscle tissue fibroblasts and endothelial cells than in normal extraocular muscles in TAO, but Smad7 was lower in the TAO fibrosis external ocular muscle group than in the normal extraocular muscles.
2. thyroid associated ophthalmopathy derived fibroblasts cultured and passaged successfully, fusiform cells, immunocytochemistry: vimentin (Vimentin) staining and cytokeratin (Keratin), desmin (Desmin), S~ (-1) 00 was negative, that of fibroblasts for mesoderm;
3.Realtime-PCR妫，

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