本文選題：甲狀腺相關(guān)性眼病　切入點(diǎn)：眼眶成纖維細(xì)胞　出處：《第二軍醫(yī)大學(xué)》2011年博士論文

【摘要】：目的 甲狀腺相關(guān)性眼病(thyroid-associated ophthalmopathy,TAO)眼外肌纖維化在病理學(xué)上具有不可逆的特點(diǎn),可導(dǎo)致患者出現(xiàn)斜視、復(fù)視,甚至壓迫性神經(jīng)病變而導(dǎo)致失明。目前發(fā)病機(jī)制不明,藥物及手術(shù)治療效果均欠佳。TGF-β1是導(dǎo)致纖維化最重要的細(xì)胞因子之一,可通過(guò)促使成纖維細(xì)胞增殖、向肌成纖維細(xì)胞(myofibroblast,MFB)表型轉(zhuǎn)分化及細(xì)胞外基質(zhì)(extracellular matrix,ECM)的合成,最終導(dǎo)致纖維化的形成。在此過(guò)程中,MFB持續(xù)存在或凋亡過(guò)程缺陷可導(dǎo)致纖維化進(jìn)行性發(fā)展(α-SMA是MFB標(biāo)志性蛋白),MFB成為控制纖維化的重要靶細(xì)胞。而TGF-β1是一種多功能的細(xì)胞因子,具有抑制炎癥反應(yīng)及抗腫瘤作用,若直接阻斷TGF-β1抗纖維化則對(duì)機(jī)體產(chǎn)生不利的影響。因此,希望在闡明TGF-β1致纖維化作用機(jī)制的同時(shí)尋找其下游信號(hào)通路,結(jié)締組織生長(zhǎng)因子(connective tissue growth factor,CTGF)屬于即可早期基因CNN家族,在TGF-β1作用下,可由成纖維細(xì)胞分泌,CTGF能被多種因子轉(zhuǎn)錄激活,其中以TGF-β1最引人注目,在TGF-β1的下游發(fā)揮重要的生物學(xué)作用。本課題旨在通過(guò)體外培養(yǎng)TAO眼外肌來(lái)源的成纖維細(xì)胞(orbital fibroblasts,OF),觀察TGF-β1能否通過(guò)促進(jìn)成OF增殖、向MFB表型轉(zhuǎn)分化及ECM的合成,最終導(dǎo)致TAO眼外肌纖維化的形成。同時(shí)選擇TGF-β1/Smads、絲裂原活化蛋白激酶(mitogen -activated protein kinase,MAPK)、磷脂酰肌醇3激酶(phosphatidylinositol 3-kinase,PI3K)信號(hào)傳導(dǎo)通路,利用特異性抑制劑(SB431542 for TGF-β1/Smads、SB203580 for P38/MAPK、PD98059 for ERK/MAPK、SP600125 for JNK/MAPK、LY294002 for PI3K)分別阻斷其相應(yīng)的信號(hào)傳導(dǎo)通路,初步探討TGF-β1促進(jìn)OF轉(zhuǎn)分化為MFB及誘導(dǎo)OF表達(dá)CTGF的信號(hào)傳導(dǎo)通路,同時(shí)觀察了CTGF對(duì)OF轉(zhuǎn)分化為MFB的作用以及CTGF自分泌調(diào)節(jié)的信號(hào)傳導(dǎo)通路,希望進(jìn)一步明確TGF-β1在TAO眼外肌纖維化中的發(fā)病機(jī)制,為T(mén)AO眼外肌纖維化治療提供相應(yīng)的分子靶點(diǎn)。 方法 1.應(yīng)用免疫組化SP法檢測(cè)TGF-β1、CTGF、P-Smad2/Smad3、Smad4、Smad7在TAO患者眼外肌中的表達(dá); 2.采用組織塊貼壁法原代培養(yǎng)TAO患者眼外肌來(lái)源的成纖維細(xì)胞并采用HE染色及免疫細(xì)胞化學(xué)技術(shù)進(jìn)行鑒定; 3.采用Real-time PCR觀察TGF-β1誘導(dǎo)OF表達(dá)ECM主要成分TIMP-1 mRNA、COL-I mRNA、COLIII mRNA及FN mRNA水平的變化;采用細(xì)胞免疫熒光觀察TGF-β1誘導(dǎo)OF表達(dá)TIMP-1、COL-I、COL III及FN蛋白及其定位; 4.采用MTT法檢測(cè)TGF-β1誘導(dǎo)OF增殖的效應(yīng);Real-time PCR和Western-blot檢測(cè)TGF-β1誘導(dǎo)OF合成α-SMAmRNA、CTGF mRNA及蛋白水平變化;預(yù)先添加各種信號(hào)通路抑制劑(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檢測(cè)TGF-β1誘導(dǎo)OF合成α-SMAmRNA、CTGF mRNA及蛋白水平變化;細(xì)胞免疫熒光檢測(cè)α-SMA、CTGF蛋白表達(dá)定位及Smad3磷酸化變化;Western-blot檢測(cè)SB431542抑制劑對(duì)MAPK家族(p38、ERK、JNK)磷酸化的影響;同時(shí)采用Real-time PCR檢測(cè)CTGF誘導(dǎo)OF轉(zhuǎn)分化作用及CTGF自身調(diào)節(jié)信號(hào)傳導(dǎo)通路。 結(jié)果 1.免疫組化結(jié)果顯示TAO眼外肌組織成纖維細(xì)胞及內(nèi)皮細(xì)胞中存在較正常眼外肌高表達(dá)的TGF-β1、CTGF、P-Smad2/Smad3、Smad4,但Smad7在TAO纖維化眼外肌組中較正常眼外肌低表達(dá); 2.甲狀腺相關(guān)眼病眼外肌來(lái)源成纖維細(xì)胞原代培養(yǎng)成功并穩(wěn)定傳代,細(xì)胞呈長(zhǎng)梭形,細(xì)胞免疫化學(xué)鑒定:波形蛋白(Vimentin)染色陽(yáng)性,而角蛋白(Keratin)、結(jié)蛋白(Desmin)、S~(-1)00染色均陰性,證明為中胚層來(lái)源的成纖維細(xì)胞; 3.Realtime-PCR檢測(cè)10μg·L~(-1)TGF-β1刺激OF的時(shí)間效應(yīng):TIMP~(-1) mRNA在3 h和6 h分別為對(duì)照組的1.50倍和2.46倍(P0.01); COL-I mRNA在24 h和48 h分別為對(duì)照組的5.49、3.69倍(P0.01);COL-IIImRNA在24 h和48 h分別為對(duì)照組的2.14、1.63倍(P0.01);FN mRNA在12 h和24 h后分別為對(duì)照組的2.45、1.53倍(P0.01)。不同濃度TGF-β1刺激OF 24 h后劑量效應(yīng):與空白對(duì)照組相比,TIMP~(-1)mRNA在5μg·L~(-1)和10μg·L~(-1)時(shí)分別為1.79、1.46倍(P0.01); COL-I mRNA在1μg·L~(-1)和10μg·L~(-1)分別1.94、3.29倍(p0.01); COL- III mRNA在5μg·L~(-1)和10μg·L~(-1)分別1.52、3.28倍(P0.01);FN mRNA在1μg·L~(-1)和10μg·L~(-1)分別為1.30、2.45倍(P0.01);細(xì)胞免疫熒光染色顯示TIMP~(-1)、COL-I、COL III及FN蛋白的表達(dá)位于OF細(xì)胞漿; 4.MTT法檢測(cè)顯示TGF-β1(1～10μg·L~(-1)TGF-β1)可促進(jìn)OF增殖,各濃度與對(duì)照組相比(P0.05); Realtime-PCR和Western-blot檢測(cè)顯示在一定的濃度范圍內(nèi),TGF-β1均以時(shí)間和劑量依賴(lài)的方式誘導(dǎo)OF表達(dá)α-SMA、CTGF mRNA,各濃度與空白對(duì)照組相比,(P0.05)其中CTGF mRNA在12h達(dá)到高峰,α-SMA mRNA在24h達(dá)到高峰,SB431542顯著抑制劑了α-SMA mRNA的表達(dá)(與對(duì)照相比,P0.05),同時(shí)PD98059、SB203580也抑制劑了α-SMA mRNA的表達(dá)(與空白對(duì)照相比P0.05);SB431542和SP600125抑制了CTGFmRNA的表達(dá)(與對(duì)照相比,P0.05);Western-blot檢測(cè)出相對(duì)應(yīng)的結(jié)果;同時(shí)Western-blot檢測(cè)TGF-β1誘導(dǎo)OF,可使Smad3磷酸化改變,在30min時(shí)達(dá)到頂點(diǎn);SB431542抑制劑對(duì)MAPK家族磷酸化沒(méi)有影響。同時(shí)Realtime-PCR檢測(cè)一定的濃度范圍內(nèi)(0.5～50μg·L~(-1))CTGF可以試劑和劑量依賴(lài)的方式誘導(dǎo)OF轉(zhuǎn)分化表達(dá)α-SMA mRNA,(與對(duì)照組相比P0.05)SB431542和SP600125抑制了CTGF自分泌的表達(dá)(與對(duì)照相比,P0.05),細(xì)胞免疫熒光檢測(cè)出10μg·L~(-1)TGF-β1誘導(dǎo)OF表達(dá)α-SMA和CTGF蛋白,定位于OF細(xì)胞漿;細(xì)胞免疫組化觀察到OF表達(dá)α-SMA。 結(jié)論 1.TGF-β1、CTGF、P-Smad2/Smad3、Smad4在TAO患者眼外肌組織中較正常組織中高表達(dá),而Smad7則相反,表達(dá)定位于成纖維細(xì)胞,少量表達(dá)在內(nèi)皮細(xì)胞,TGF-β1/Smad通路可能參與了TAO眼外肌纖維化的發(fā)病過(guò)程; 2.組織塊貼壁法培養(yǎng)的細(xì)胞符合甲狀腺相關(guān)眼病眼外肌成纖維細(xì)胞的形態(tài)學(xué)和免疫學(xué)特征,可作為研究甲狀腺相關(guān)性眼病眼外肌纖維化的體外模型; 3. Realtime-PCR顯示在一定的濃度范圍內(nèi),TGF-β1以劑量和時(shí)間依賴(lài)的方式誘導(dǎo)OF表達(dá)細(xì)胞外基質(zhì)主要成分TIMP~(-1)、COL-I、COL III及FN mRNA;細(xì)胞免疫熒光染色顯示TGF-β1可誘導(dǎo)OF表達(dá)TIMP~(-1)、COL-I、COL III及FN蛋白,蛋白位于OF細(xì)胞漿;TGF-β1可通過(guò)促進(jìn)ECM的合成參與TAO眼外肌纖維化的發(fā)病過(guò)程。 4. TGF-β1對(duì)OF增殖有增加的趨勢(shì),但P0.05,無(wú)統(tǒng)計(jì)學(xué)意義;TGF-β1可誘導(dǎo)OF向MFB表型轉(zhuǎn)分化及表達(dá)CTGF,CTGF的表達(dá)高峰早于α-SMA;CTGF可誘導(dǎo)OF向MFB表型轉(zhuǎn)分化;細(xì)胞免疫熒光可檢測(cè)到α-SMA及CTGF蛋白的表達(dá)并定位于OF細(xì)胞漿;TGF-β1誘導(dǎo)OF向MFB表型轉(zhuǎn)分化表達(dá)α-SMA通TGF-β1/Smad及P38/MAPK和ERK/MAPK信號(hào)傳導(dǎo)通路;TGF-β1誘導(dǎo)OF表達(dá)CTGF通TGF-β1/Smad及JNK/MAPK信號(hào)傳導(dǎo)通路;CTGF自分泌調(diào)節(jié)通過(guò)JNK/MAPK信號(hào)傳導(dǎo)通路;TGF-β1可引起Smad3磷酸化改變,SB431542對(duì)MAPK信號(hào)通路磷酸化水平?jīng)]有影響。
[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媯，

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