【摘要】：研究背景 細(xì)胞因子的胞內(nèi)功能發(fā)揮，一般需要與細(xì)胞膜受體結(jié)合后經(jīng)特定信號傳導(dǎo)通路進(jìn)行，或由受體介導(dǎo)入胞后發(fā)揮其生物學(xué)活性。Kallistatin(Kal)是一種組織激肽釋放酶結(jié)合蛋白，，也是一種絲氨酸蛋白酶抑制劑，具有多種生物學(xué)功能，如降血壓、舒張血管、抗炎、抗氧化、抗纖維化、促進(jìn)新生內(nèi)膜的形成、抑制血管生成和腫瘤的生長等。目前發(fā)現(xiàn)的Kal膜受體有肝素硫酸蛋白聚糖（HSPGs），介導(dǎo)Kal的抗新生血管生成作用；有低密度脂蛋白受體相關(guān)蛋白6（LRP6），介導(dǎo)Kal的抗腫瘤作用；還有Kruppel樣轉(zhuǎn)錄因子4（Kruppel-like factor4，KLF4），介導(dǎo)Kal的抗炎作用。這三個Kal受體的發(fā)現(xiàn)，在一定程度上解釋了Kal部分功能，但尚不足以闡釋Kal的多功能特點。我們判斷蛋白，Kal的多功能應(yīng)該是多靶點多通路作用的結(jié)果，應(yīng)該還有未被發(fā)現(xiàn)的Kal膜受體。 研究目的 分離、鑒定Kal細(xì)胞膜結(jié)合蛋白，驗證細(xì)胞膜結(jié)合蛋白在Kal抗血管生成和抗腫瘤中的作用，為揭示Kal的多功能性以及涉及的分子機(jī)制等奠定基礎(chǔ)。 研究方法 （1）制備重組人Kal（rhKal），利用Pull-Down、LC-MC/MC、免疫共沉淀、免疫熒光等技術(shù)分離、鑒定rhKal細(xì)胞膜結(jié)合蛋白。 （2）利用siRNA、抗體封閉等技術(shù)，分析rhKal膜結(jié)合蛋白在rhKal抗血管生成和抗腫瘤作用中的作用。 （3）利用流式細(xì)胞儀、Western Blotting等方法，分析細(xì)胞膜結(jié)合蛋白對介導(dǎo)rhKal入胞的作用。 （4）采用細(xì)胞培養(yǎng)及分析方法，分析rhKal與候選rhKal細(xì)胞膜結(jié)合蛋白所涉及的信號通路。 （5）利用皮下移植瘤模型，分析候選rhKal細(xì)胞膜結(jié)合蛋白介導(dǎo)rhKal的抗血管生成和抗腫瘤活性的分子機(jī)制。 研究結(jié)果 （1）首次發(fā)現(xiàn)核仁素、整合素β3、金屬基質(zhì)蛋白酶2（MMP2）、血管內(nèi)皮生長因子受體1（VEGFR1）、VEGFR2為rhKal細(xì)胞膜結(jié)合蛋白。 （2）首次發(fā)現(xiàn)核仁素是介導(dǎo)rhKal入胞的重要受體，并參與rhKal體內(nèi)外抗腫瘤新生血管形成的過程。 （3）首次發(fā)現(xiàn)核仁素介導(dǎo)rhKal抗血管生成的分子機(jī)制是：rhKal通過而下調(diào)蛋白激酶CK2的表達(dá)抑制核仁素磷酸化，從而抑制由VEGF和堿性成纖維細(xì)胞因子（bFGF）所誘導(dǎo)的血管內(nèi)皮細(xì)胞增殖。 （4）首次發(fā)現(xiàn)整合素β3可以介導(dǎo)rhKal的抗腫瘤活性。 （5）首次發(fā)現(xiàn)整合素β3介導(dǎo)rhKal抗腫瘤活性的分子機(jī)制為：rhKal抑制整合素β3、黏著斑激酶（FAK）、類固醇激素受體共活化因子（Src）的磷酸化。 研究結(jié)論 （1）核仁素是介導(dǎo)rhKal入胞的受體，并參與rhKal的體內(nèi)外抗腫瘤新生血管形成和抗腫瘤活性。 （2）整合素β3是介導(dǎo)rhKal抗腫瘤活性的重要受體。
[Abstract]:Background to study the intracellular function of cytokines, it is generally necessary to bind to cell membrane receptors and pass through specific signal transduction pathways. Kallistatin (Kal) is a tissue kallikrein releasing enzyme binding protein and a serine protease inhibitor with many biological functions, such as lowering blood pressure, relaxing blood vessels, anti-inflammation, anti-oxidation. Anti-fibrosis, promoting neointimal formation, inhibiting angiogenesis and tumor growth. The present Kal membrane receptors include heparin sulfate proteoglycan (HSPGs),) -mediated anti-angiogenesis of Kal, low-density lipoprotein receptor-associated protein 6 (LRP6), which mediates the anti-tumor effect of Kal, and Kruppel like transcription factor 4 (Kruppel-like factor4,KLF4), which mediates the anti-inflammatory effect of Kal. The discovery of these three Kal receptors, to some extent, explains the partial function of Kal, but it is not sufficient to explain the multifunctional characteristics of Kal. We estimate that the multifunction of Kal is the result of multitarget and multipathway, and there should be an undiscovered Kal membrane receptor. Objective to isolate and identify the membrane binding proteins of Kal, and to verify the role of membrane binding proteins in the anti-angiogenesis and anti-tumor of Kal, so as to lay a foundation for the study of the multifunctional and related molecular mechanisms of Kal. Methods (1) Recombinant human Kal (rhKal), was prepared and identified by Pull-Down,LC-MC/MC, immunoprecipitation and immunofluorescence techniques. (2) rhKal cell membrane binding protein was identified by siRNA, antibody blocking technique. The role of rhKal membrane binding protein in anti-angiogenesis and anti-tumor effects of rhKal was analyzed. (3) flow cytometry and Western Blotting were used. The effects of cell membrane binding proteins on the induction of rhKal into cells were analyzed. (4) the signal pathways involved in rhKal and candidate rhKal cell membrane binding proteins were analyzed by cell culture and analysis. (5) the model of subcutaneous transplanted tumor was used. To analyze the molecular mechanism of antiangiogenesis and antitumor activity of rhKal mediated by candidate rhKal cell membrane binding protein. Results (1) nucleolus, integrin 尾 3, metalloproteinase 2 (MMP2), vascular endothelial growth factor receptor 1 (VEGFR1) VEGFR2 were first found to be rhKal cell membrane binding proteins. In vitro and in vivo, rhKal participated in the process of anti-angiogenesis. (3) the molecular mechanism of anti-angiogenesis of rhKal mediated by nucleolus was the inhibition of nucleolar phosphorylation by down-regulating the expression of protein kinase CK2. Thus inhibiting the proliferation of vascular endothelial cells induced by VEGF and basic fibroblast factor (bFGF). (4) it is the first time that integrin 尾 3 can mediate the antitumor activity of rhKal. (5) integrin 尾 3 mediated by integrin 尾 3 is first found. The molecular mechanism of the antitumor activity of rhKal is the inhibition of integrin 尾 3 and phosphorylation of (FAK), steroid hormone receptor coactivator (Src) by 1: rhKal. Conclusion (1) nucleolus is the receptor that mediates the entry of rhKal, and participates in the anti-angiogenesis and anti-tumor activity of rhKal in vitro and in vivo. (2) integrin 尾 3 is an important receptor in mediating the antitumor activity of rhKal.
【學(xué)位授予單位】：華僑大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R96

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相關(guān)期刊論文 前3條

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