本文關(guān)鍵詞：DEDD與Smad3相互作用負(fù)性調(diào)控TGF-β介導(dǎo)的生物學(xué)活性　出處：《中國(guó)協(xié)和醫(yī)科大學(xué)》2009年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： DEDD Smad3 TGF-β 相互作用 腫瘤

【摘要】： 轉(zhuǎn)化生長(zhǎng)因子β(TGF-β)是一類(lèi)對(duì)細(xì)胞增殖、分化、遷移、凋亡以及細(xì)胞外基質(zhì)形成、血管形成、免疫、發(fā)育均發(fā)揮調(diào)節(jié)作用的分泌型細(xì)胞因子。該家族調(diào)節(jié)異常可以導(dǎo)致各種疾病發(fā)生,例如腫瘤、纖維化、自身免疫性疾病以及血管性疾病等。TGF-β家族發(fā)揮其生物學(xué)功能主要通過(guò)Smads蛋白,Smads是TGF-β家族細(xì)胞內(nèi)信號(hào)傳導(dǎo)分子。目前在哺乳動(dòng)物中已經(jīng)發(fā)現(xiàn)八種Smads蛋白,根據(jù)功能不同可分為三類(lèi)：受體激活型Smads、通用介質(zhì)型Smads、抑制型Smads。TGF-β信號(hào)在整個(gè)傳遞過(guò)程中均受到不同層面的調(diào)節(jié),因此TGF-β家族具有生物功能多樣性。細(xì)胞內(nèi)多種蛋白都可與Smads相互作用,這可以促進(jìn)TGF-β信號(hào)與其他信號(hào)途徑發(fā)生“整合”。 死亡受體結(jié)構(gòu)域(Death effector domains, DEDs)是蛋白發(fā)生相互作用的區(qū)域,含有DED的蛋白可以介導(dǎo)由死亡受體觸發(fā)的程序性細(xì)胞死亡或凋亡。DED本身沒(méi)有酶活性而是作為其他DEDs的結(jié)合區(qū)域,因而促進(jìn)蛋白復(fù)合體形成。FADD、caspase8以及DEDD均屬于含有DED結(jié)構(gòu)域的蛋白。研究表明,DEDD通過(guò)活化caspase3或capase6在CD95介導(dǎo)的凋亡途徑中發(fā)揮重要作用。近來(lái)發(fā)現(xiàn)DEDD也參與細(xì)胞周期調(diào)控并抑制有絲分裂進(jìn)程。然而,關(guān)于DEDD是否通過(guò)與關(guān)鍵的蛋白相互作用參與其他信號(hào)轉(zhuǎn)導(dǎo)途徑以及DEDD本身生物學(xué)功能尚未清楚。 我們實(shí)驗(yàn)室前期以全長(zhǎng)的Smad3蛋白為“釣餌”,通過(guò)酵母雙雜交系統(tǒng)篩選出DEDD與Smad3相互作用。本次研究中,我們應(yīng)用免疫共沉淀和GST-pull down方法均證明DEDD與Smad3可以發(fā)生相互作用,這是首次報(bào)道Smad3與含有DED結(jié)構(gòu)域蛋白之間的相互作用。我們發(fā)現(xiàn)TGF-β可以下調(diào)DEDD與Smad3的結(jié)合,而且DEDD與磷酸化的Smad3結(jié)合能力減弱。雙熒光素酶報(bào)告基因?qū)嶒?yàn)證明DEDD通過(guò)其DED結(jié)構(gòu)域可以抑制TGF-β/Smad3介導(dǎo)的轉(zhuǎn)錄活性,而且Smad3可以協(xié)同增強(qiáng)DEDD對(duì)NF-κB基因的轉(zhuǎn)錄。細(xì)胞免疫熒光染色和Western blotting實(shí)驗(yàn)證明DEDD可以抑制Smad3入核作用。免疫沉淀實(shí)驗(yàn)證明DEDD可以抑制Smad3磷酸化以及Smad3與Smad4復(fù)合體形成作用。Western blotting和RT-PCR實(shí)驗(yàn)證明DEDD抑制TGF-β靶基因的轉(zhuǎn)錄和翻譯,而且TGF-β抑制DEDD蛋白表達(dá)和基因轉(zhuǎn)錄。細(xì)胞劃痕實(shí)驗(yàn)和Transwell實(shí)驗(yàn)證明DEDD抑制TGF-β介導(dǎo)的細(xì)胞遷移和侵襲作用,而且DEDD本身也具有抑制細(xì)胞遷移和侵襲作用。Annexin V-FITC細(xì)胞凋亡檢測(cè)實(shí)驗(yàn)和MTT實(shí)驗(yàn)證明DEDD本身可以誘導(dǎo)細(xì)胞凋亡和生長(zhǎng)阻滯。最后,我們應(yīng)用臨床結(jié)腸癌病人組織標(biāo)本進(jìn)行免疫組化分析得到：與正常組織相比,DEDD蛋白表達(dá)在結(jié)腸癌病人組織下降�？傊�,我們的結(jié)果表明通過(guò)DEDD與Smad3的相互作用在TGF-β信號(hào)途徑和CD95途徑之間建立嶄新的聯(lián)系,DEDD成為T(mén)GF-β信號(hào)途徑的抑制因素。
[Abstract]:Transforming growth factor 尾 (TGF- 尾) is a kind of cell proliferation, differentiation, migration, apoptosis and extracellular matrix formation, angiogenesis, immune. A secreted cytokine that plays a regulatory role in development. Abnormal regulation in this family can lead to various diseases such as tumours and fibrosis. Autoimmune diseases and vascular diseases. TGF- 尾 family play its biological function mainly through Smads protein. Smads is a signal transduction molecule of TGF- 尾 family. At present, eight Smads proteins have been found in mammals, which can be classified into three types according to their functions: receptor-activated Smads. The signal of Smads, Smads.TGF- 尾 is regulated by different levels during the whole process of transmission. Therefore, TGF- 尾 family has a variety of biological functions. Many proteins can interact with Smads, which can promote the "integration" of TGF- 尾 signal with other signaling pathways. Death effector domain (DEDs) is a region in which proteins interact with each other. Proteins containing DED can mediate programmed cell death or apoptosis triggered by death receptors. DED itself has no enzyme activity but acts as a binding region of other DEDs. Therefore, promoting the formation of protein complex. FADDD caspase8 and DEDD belong to the protein containing DED domain. DEDD plays an important role in CD95 mediated apoptosis by activating caspase3 or capase6. Recently, it has been found that DEDD also participates in cell cycle regulation and inhibits mitosis. ... but... It is not clear whether DEDD participates in other signal transduction pathways by interacting with key proteins and the biological function of DEDD itself. In our laboratory, the full-length Smad3 protein was used as "bait", and the interaction between DEDD and Smad3 was screened by yeast two-hybrid system. We used both immunoprecipitation and GST-pull down methods to prove that DEDD and Smad3 could interact with each other. This is the first report on the interaction between Smad3 and DED domain proteins. We found that TGF- 尾 can down-regulate the binding of DEDD to Smad3. Moreover, the binding ability of DEDD to phosphorylated Smad3 was weakened. Double luciferase reporter gene experiment showed that DEDD could inhibit transcriptional activity mediated by TGF- 尾 / Smad3 through its DED domain. Sex. Moreover, Smad3 can enhance the transcription of NF- 魏 B gene by DEDD. Cellular immunofluorescence staining and Western. Blotting assay showed that DEDD could inhibit the nucleation of Smad3, and immunoprecipitation assay showed that DEDD could inhibit the phosphorylation of Smad3 and the Smad3 / Smad4 complex. Forming action. Western. Blotting and RT-PCR experiments demonstrated that DEDD inhibited the transcription and translation of TGF- 尾 target genes. Moreover, TGF- 尾 inhibited DEDD protein expression and gene transcription. Cell scratch assay and Transwell assay demonstrated that DEDD inhibited TGF- 尾 -mediated cell migration and invasion. And DEDD itself can inhibit cell migration and invasion. Annexin. V-FITC cell apoptosis assay and MTT assay proved that DEDD itself can induce apoptosis and growth arrest. Finally. We used clinical colon cancer tissue samples for immunohistochemical analysis: compared with normal tissues, the expression of DDEDD protein in colon cancer patients decreased. Our results show that a new relationship between TGF- 尾 signaling pathway and CD95 pathway is established by the interaction of DEDD and Smad3. DEDD is a factor that inhibits TGF- 尾 signaling pathway.
【學(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R363

【共引文獻(xiàn)】

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2 陶小紅;荔枝核提取物體外抗腫瘤生物活性及其機(jī)理的初步研究[D];暨南大學(xué);2009年

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本文編號(hào)：1405161