本文選題：甲狀腺相關(guān)性眼病　切入點：眼眶成纖維細胞　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,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胞因子之一,可通過促使成纖維細胞增殖、向肌成纖維細胞(myofibroblast,MFB)表型轉(zhuǎn)分化及細胞外基質(zhì)(extracellular matrix,ECM)的合成,最終導(dǎo)致纖維化的形成。在此過程中,MFB持續(xù)存在或凋亡過程缺陷可導(dǎo)致纖維化進行性發(fā)展(α-SMA是MFB標志性蛋白),MFB成為控制纖維化的重要靶細胞。而TGF-β1是一種多功能的細胞因子,具有抑制炎癥反應(yīng)及抗腫瘤作用,若直接阻斷TGF-β1抗纖維化則對機體產(chǎn)生不利的影響。因此,希望在闡明TGF-β1致纖維化作用機制的同時尋找其下游信號通路,結(jié)締組織生長因子(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。本課題旨在通過體外培養(yǎng)TAO眼外肌來源的成纖維細胞(orbital fibroblasts,OF),觀察TGF-β1能否通過促進成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)的信號傳導(dǎo)通路,初步探討TGF-β1促進OF轉(zhuǎn)分化為MFB及誘導(dǎo)OF表達CTGF的信號傳導(dǎo)通路,同時觀察了CTGF對OF轉(zhuǎn)分化為MFB的作用以及CTGF自分泌調(diào)節(jié)的信號傳導(dǎo)通路,希望進一步明確TGF-β1在TAO眼外肌纖維化中的發(fā)病機制,為TAO眼外肌纖維化治療提供相應(yīng)的分子靶點。 方法 1.應(yīng)用免疫組化SP法檢測TGF-β1、CTGF、P-Smad2/Smad3、Smad4、Smad7在TAO患者眼外肌中的表達; 2.采用組織塊貼壁法原代培養(yǎng)TAO患者眼外肌來源的成纖維細胞并采用HE染色及免疫細胞化學(xué)技術(shù)進行鑒定; 3.采用Real-time PCR觀察TGF-β1誘導(dǎo)OF表達ECM主要成分TIMP-1 mRNA、COL-I mRNA、COLIII mRNA及FN mRNA水平的變化;采用細胞免疫熒光觀察TGF-β1誘導(dǎo)OF表達TIMP-1、COL-I、COL III及FN蛋白及其定位; 4.采用MTT法檢測TGF-β1誘導(dǎo)OF增殖的效應(yīng);Real-time PCR和Western-blot檢測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TGF-β1誘導(dǎo)OF合成α-SMAmRNA、CTGF mRNA及蛋白水平變化;細胞免疫熒光檢測α-SMA、CTGF蛋白表達定位及Smad3磷酸化變化;Western-blot檢測SB431542抑制劑對MAPK家族(p38、ERK、JNK)磷酸化的影響;同時采用Real-time PCR檢測CTGF誘導(dǎo)OF轉(zhuǎn)分化作用及CTGF自身調(diào)節(jié)信號傳導(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胞及內(nèi)皮細胞中存在較正常眼外肌高表達的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; 2.甲狀腺相關(guān)眼病眼外肌來源成纖維細胞原代培養(yǎng)成功并穩(wěn)定傳代,細胞呈長梭形,細胞免疫化學(xué)鑒定:波形蛋白(Vimentin)染色陽性,而角蛋白(Keratin)、結(jié)蛋白(Desmin)、S~(-1)00染色均陰性,證明為中胚層來源的成纖維細胞; 3.Realtime-PCR檢測10μg·L~(-1)TGF-β1刺激OF的時間效應(yīng):TIMP~(-1) mRNA在3 h和6 h分別為對照組的1.50倍和2.46倍(P0.01); COL-I mRNA在24 h和48 h分別為對照組的5.49、3.69倍(P0.01);COL-IIImRNA在24 h和48 h分別為對照組的2.14、1.63倍(P0.01);FN mRNA在12 h和24 h后分別為對照組的2.45、1.53倍(P0.01)。不同濃度TGF-β1刺激OF 24 h后劑量效應(yīng):與空白對照組相比,TIMP~(-1)mRNA在5μg·L~(-1)和10μg·L~(-1)時分別為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);細胞免疫熒光染色顯示TIMP~(-1)、COL-I、COL III及FN蛋白的表達位于OF細胞漿; 4.MTT法檢測顯示TGF-β1(1～10μg·L~(-1)TGF-β1)可促進OF增殖,各濃度與對照組相比(P0.05); Realtime-PCR和Western-blot檢測顯示在一定的濃度范圍內(nèi),TGF-β1均以時間和劑量依賴的方式誘導(dǎo)OF表達α-SMA、CTGF mRNA,各濃度與空白對照組相比,(P0.05)其中CTGF mRNA在12h達到高峰,α-SMA mRNA在24h達到高峰,SB431542顯著抑制劑了α-SMA mRNA的表達(與對照相比,P0.05),同時PD98059、SB203580也抑制劑了α-SMA mRNA的表達(與空白對照相比P0.05);SB431542和SP600125抑制了CTGFmRNA的表達(與對照相比,P0.05);Western-blot檢測出相對應(yīng)的結(jié)果;同時Western-blot檢測TGF-β1誘導(dǎo)OF,可使Smad3磷酸化改變,在30min時達到頂點;SB431542抑制劑對MAPK家族磷酸化沒有影響。同時Realtime-PCR檢測一定的濃度范圍內(nèi)(0.5～50μg·L~(-1))CTGF可以試劑和劑量依賴的方式誘導(dǎo)OF轉(zhuǎn)分化表達α-SMA mRNA,(與對照組相比P0.05)SB431542和SP600125抑制了CTGF自分泌的表達(與對照相比,P0.05),細胞免疫熒光檢測出10μg·L~(-1)TGF-β1誘導(dǎo)OF表達α-SMA和CTGF蛋白,定位于OF細胞漿;細胞免疫組化觀察到OF表達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,而Smad7則相反,表達定位于成纖維細胞,少量表達在內(nèi)皮細胞,TGF-β1/Smad通路可能參與了TAO眼外肌纖維化的發(fā)病過程; 2.組織塊貼壁法培養(yǎng)的細胞符合甲狀腺相關(guān)眼病眼外肌成纖維細胞的形態(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以劑量和時間依賴的方式誘導(dǎo)OF表達細胞外基質(zhì)主要成分TIMP~(-1)、COL-I、COL III及FN mRNA;細胞免疫熒光染色顯示TGF-β1可誘導(dǎo)OF表達TIMP~(-1)、COL-I、COL III及FN蛋白,蛋白位于OF細胞漿;TGF-β1可通過促進ECM的合成參與TAO眼外肌纖維化的發(fā)病過程。 4. TGF-β1對OF增殖有增加的趨勢,但P0.05,無統(tǒng)計學(xué)意義;TGF-β1可誘導(dǎo)OF向MFB表型轉(zhuǎn)分化及表達CTGF,CTGF的表達高峰早于α-SMA;CTGF可誘導(dǎo)OF向MFB表型轉(zhuǎn)分化;細胞免疫熒光可檢測到α-SMA及CTGF蛋白的表達并定位于OF細胞漿;TGF-β1誘導(dǎo)OF向MFB表型轉(zhuǎn)分化表達α-SMA通TGF-β1/Smad及P38/MAPK和ERK/MAPK信號傳導(dǎo)通路;TGF-β1誘導(dǎo)OF表達CTGF通TGF-β1/Smad及JNK/MAPK信號傳導(dǎo)通路;CTGF自分泌調(diào)節(jié)通過JNK/MAPK信號傳導(dǎo)通路;TGF-β1可引起Smad3磷酸化改變,SB431542對MAPK信號通路磷酸化水平?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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