组织激肽释放酶结合蛋白细胞膜受体的分离鉴定与其抗肿瘤分子机制的研究
发布时间：2018-08-26 15:58
【摘要】：研究背景 细胞因子的胞内功能发挥，一般需要与细胞膜受体结合后经特定信号传导通路进行，或由受体介导入胞后发挥其生物学活性。Kallistatin(Kal)是一种组织激肽释放酶结合蛋白，，也是一种丝氨酸蛋白酶抑制剂，具有多种生物学功能，如降血压、舒张血管、抗炎、抗氧化、抗纤维化、促进新生内膜的形成、抑制血管生成和肿瘤的生长等。目前发现的Kal膜受体有肝素硫酸蛋白聚糖（HSPGs），介导Kal的抗新生血管生成作用；有低密度脂蛋白受体相关蛋白6（LRP6），介导Kal的抗肿瘤作用；还有Kruppel样转录因子4（Kruppel-like factor4，KLF4），介导Kal的抗炎作用。这三个Kal受体的发现，在一定程度上解释了Kal部分功能，但尚不足以阐释Kal的多功能特点。我们判断蛋白，Kal的多功能应该是多靶点多通路作用的结果，应该还有未被发现的Kal膜受体。 研究目的 分离、鉴定Kal细胞膜结合蛋白，验证细胞膜结合蛋白在Kal抗血管生成和抗肿瘤中的作用，为揭示Kal的多功能性以及涉及的分子机制等奠定基础。 研究方法 （1）制备重组人Kal（rhKal），利用Pull-Down、LC-MC/MC、免疫共沉淀、免疫荧光等技术分离、鉴定rhKal细胞膜结合蛋白。 （2）利用siRNA、抗体封闭等技术，分析rhKal膜结合蛋白在rhKal抗血管生成和抗肿瘤作用中的作用。 （3）利用流式细胞仪、Western Blotting等方法，分析细胞膜结合蛋白对介导rhKal入胞的作用。 （4）采用细胞培养及分析方法，分析rhKal与候选rhKal细胞膜结合蛋白所涉及的信号通路。 （5）利用皮下移植瘤模型，分析候选rhKal细胞膜结合蛋白介导rhKal的抗血管生成和抗肿瘤活性的分子机制。 研究结果 （1）首次发现核仁素、整合素β3、金属基质蛋白酶2（MMP2）、血管内皮生长因子受体1（VEGFR1）、VEGFR2为rhKal细胞膜结合蛋白。 （2）首次发现核仁素是介导rhKal入胞的重要受体，并参与rhKal体内外抗肿瘤新生血管形成的过程。 （3）首次发现核仁素介导rhKal抗血管生成的分子机制是：rhKal通过而下调蛋白激酶CK2的表达抑制核仁素磷酸化，从而抑制由VEGF和碱性成纤维细胞因子（bFGF）所诱导的血管内皮细胞增殖。 （4）首次发现整合素β3可以介导rhKal的抗肿瘤活性。 （5）首次发现整合素β3介导rhKal抗肿瘤活性的分子机制为：rhKal抑制整合素β3、黏着斑激酶（FAK）、类固醇激素受体共活化因子（Src）的磷酸化。 研究结论 （1）核仁素是介导rhKal入胞的受体，并参与rhKal的体内外抗肿瘤新生血管形成和抗肿瘤活性。 （2）整合素β3是介导rhKal抗肿瘤活性的重要受体。
[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.
【学位授予单位】：华侨大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R96
本文编号：2205407
[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.
【学位授予单位】：华侨大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R96
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相关期刊论文 前3条
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3 Masayuki Nagahashi;Subramaniam Ramachandran;Omar M Rashid;Kazuaki Takabe;;Lymphangiogenesis:A new player in cancer progression[J];World Journal of Gastroenterology;2010年32期
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