RI与ANG的相互作用对膀胱癌细胞T24的影响及分子机制研究
发布时间：2018-12-31 19:11
【摘要】：目的 研究核糖核酸酶抑制因子（RI）蛋白与血管生成素（ANG）及其突变体蛋白的相互作用。探讨两者相互作用介导和调节膀胱癌细胞T24细胞生长的影响及其分子机制。 方法 1.首先以pCMV-3×flag-ANG为模板，PCR法扩增获得ANG的突变体pCMV-3×flag-ANGH37A。以pcDNA3.1-RI为模板，PCR法扩增RI编码区序列，将其克隆至pGEX-4T-1原核表达载体中，构建原核表达质粒pGEX-4T-RI。再分别以pCMV-3×flag-ANG、pCMV-3×flag-ANGH37A和pcDNA3.1-RI为模板，PCR法扩增其编码区序列，构建真核表达质粒pEYFP-ANG、pEYFP-ANGH37A和pECFP-RI。 2.在T24和HEK293细胞内进行RI与ANG及其突变体的免疫共沉淀实验与荧光共振能量转移实验。研究RI与ANG及其突变体在细胞内的相互作用。在细胞外应用GST pull-down方法研究RI与ANG及其突变体在体外的相互作用。在T24细胞和293细胞内进行RI与ANG及其突变体的免疫荧光共定位实验。此外，荧光共振能量转移（FRET）观察和结果也显示RI与ANG在细胞内的相互作用。 3.将pCMV-3×flag-ANG及pCMV-3×flag-ANGH37A转染T24细胞和293细胞，检测RI与ANG相互作用对T24细胞的影响及介导PI3K/AKT/mTOR信号通路蛋白的影响。 4.构建裸鼠移植瘤模型，检测ANG蛋白及其突变体对于移植瘤生长，肿瘤新生血管以及自发转移等的影响。 结果 1.重组真核表达质粒pCMV-3×flag-ANGH37A、pEYFP-ANG、pEYFP-ANGH37A和pECFP-RI构建正确。重组原核表达质粒pGEX-4T-RI构建正确。 2.实验结果显示在细胞内和细胞外，RI与ANG及其突变体均存在相互作用。 3.通过pCMV-3×flag-ANG及pCMV-3×flag-ANGH37A转染T24细胞后，PI3K/AKT/mTOR通路的一些关键靶蛋白均呈现显著的升高，，表明ANG可以通过此通路促进T24细胞的生长。 4.移植瘤动物模型的实验结果表明，ANG蛋白及其突变体的过表达对于膀胱癌肿瘤的生长具有促进作用，并且其肿瘤组织中新生血管相比正常的肿瘤细胞组显著增加。 结论 综合以上研究成果，我们得出了以下的一些结论：内源性及外源性的核糖核酸酶抑制因子（RI）蛋白与血管生成素（ANG）及其突变体蛋白均存在相互作用。并且在此基础上，RI与ANG蛋白的相互作用，介导和调节膀胱癌细胞T24的PI3K/AKT/mTOR信号通路，进一步的影响了膀胱癌的生长及肿瘤新生血管的生成。
[Abstract]:Objective to study the interaction of ribonuclease inhibitor (RI) protein with angiopoietin (ANG) and its mutant protein. To investigate the effects of the two interactions on the growth of bladder cancer cell line T24 and its molecular mechanism. Method 1. Firstly, the mutant pCMV-3 脳 flag-ANGH37A. of ANG was amplified by PCR using pCMV-3 脳 flag-ANG as template. Using pcDNA3.1-RI as template, RI coding region was amplified by PCR and cloned into pGEX-4T-1 prokaryotic expression vector to construct prokaryotic expression plasmid pGEX-4T-RI.. Using pCMV-3 脳 flag-ANG,pCMV-3 脳 flag-ANGH37A and pcDNA3.1-RI as templates, the coding region was amplified by PCR, and the eukaryotic expression plasmids pEYFP-ANG,pEYFP-ANGH37A and pECFP-RI. were constructed. 2. The immunocoprecipitation and fluorescence resonance energy transfer tests of RI and ANG and their mutants were carried out in T24 and HEK293 cells. To study the intracellular interaction of RI with ANG and its mutants. The interaction of RI with ANG and its mutants in vitro was studied by GST pull-down method. Immunofluorescence co-localization of RI and ANG and their mutants were performed in T 24 and 293 cells. In addition, fluorescence resonance energy transfer (FRET) observations and results also showed the interaction between RI and ANG in cells. 3. PCMV-3 脳 flag-ANG and pCMV-3 脳 flag-ANGH37A were transfected into T24 cells and 293 cells. The effect of RI and ANG interaction on T24 cells and the effect of PI3K/AKT/mTOR signaling pathway proteins were detected. 4. To investigate the effects of ANG protein and its mutants on tumor growth, tumor angiogenesis and spontaneous metastasis in nude mice. Result 1. The recombinant eukaryotic expression plasmids pCMV-3 脳 flag-ANGH37A,pEYFP-ANG,pEYFP-ANGH37A and pECFP-RI were constructed correctly. The recombinant prokaryotic expression plasmid pGEX-4T-RI was constructed correctly. 2. The results showed that RI interacted with ANG and its mutants both in and out of cells. 3. After transfection of T24 cells by pCMV-3 脳 flag-ANG and pCMV-3 脳 flag-ANGH37A, some key target proteins of PI3K/AKT/mTOR pathway increased significantly, indicating that ANG can promote the growth of T24 cells through this pathway. 4. The results of the animal model showed that the overexpression of ANG protein and its mutants promoted the growth of bladder cancer, and the neovascularization in the tumor tissue was significantly higher than that in the normal tumor cell group. Conclusion based on the above results, we draw some conclusions as follows: the endogenous and exogenous ribonuclease inhibitor (RI) protein interacts with angiopoietin (ANG) and its mutant protein. On this basis, the interaction of RI and ANG protein mediates and regulates the PI3K/AKT/mTOR signaling pathway of bladder cancer cell T24, which further affects the growth of bladder cancer and tumor angiogenesis.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.14
[Abstract]:Objective to study the interaction of ribonuclease inhibitor (RI) protein with angiopoietin (ANG) and its mutant protein. To investigate the effects of the two interactions on the growth of bladder cancer cell line T24 and its molecular mechanism. Method 1. Firstly, the mutant pCMV-3 脳 flag-ANGH37A. of ANG was amplified by PCR using pCMV-3 脳 flag-ANG as template. Using pcDNA3.1-RI as template, RI coding region was amplified by PCR and cloned into pGEX-4T-1 prokaryotic expression vector to construct prokaryotic expression plasmid pGEX-4T-RI.. Using pCMV-3 脳 flag-ANG,pCMV-3 脳 flag-ANGH37A and pcDNA3.1-RI as templates, the coding region was amplified by PCR, and the eukaryotic expression plasmids pEYFP-ANG,pEYFP-ANGH37A and pECFP-RI. were constructed. 2. The immunocoprecipitation and fluorescence resonance energy transfer tests of RI and ANG and their mutants were carried out in T24 and HEK293 cells. To study the intracellular interaction of RI with ANG and its mutants. The interaction of RI with ANG and its mutants in vitro was studied by GST pull-down method. Immunofluorescence co-localization of RI and ANG and their mutants were performed in T 24 and 293 cells. In addition, fluorescence resonance energy transfer (FRET) observations and results also showed the interaction between RI and ANG in cells. 3. PCMV-3 脳 flag-ANG and pCMV-3 脳 flag-ANGH37A were transfected into T24 cells and 293 cells. The effect of RI and ANG interaction on T24 cells and the effect of PI3K/AKT/mTOR signaling pathway proteins were detected. 4. To investigate the effects of ANG protein and its mutants on tumor growth, tumor angiogenesis and spontaneous metastasis in nude mice. Result 1. The recombinant eukaryotic expression plasmids pCMV-3 脳 flag-ANGH37A,pEYFP-ANG,pEYFP-ANGH37A and pECFP-RI were constructed correctly. The recombinant prokaryotic expression plasmid pGEX-4T-RI was constructed correctly. 2. The results showed that RI interacted with ANG and its mutants both in and out of cells. 3. After transfection of T24 cells by pCMV-3 脳 flag-ANG and pCMV-3 脳 flag-ANGH37A, some key target proteins of PI3K/AKT/mTOR pathway increased significantly, indicating that ANG can promote the growth of T24 cells through this pathway. 4. The results of the animal model showed that the overexpression of ANG protein and its mutants promoted the growth of bladder cancer, and the neovascularization in the tumor tissue was significantly higher than that in the normal tumor cell group. Conclusion based on the above results, we draw some conclusions as follows: the endogenous and exogenous ribonuclease inhibitor (RI) protein interacts with angiopoietin (ANG) and its mutant protein. On this basis, the interaction of RI and ANG protein mediates and regulates the PI3K/AKT/mTOR signaling pathway of bladder cancer cell T24, which further affects the growth of bladder cancer and tumor angiogenesis.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.14
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