【摘要】：目的 研究核糖核酸酶抑制因子（RI）蛋白與血管生成素（ANG）及其突變體蛋白的相互作用。探討兩者相互作用介導(dǎo)和調(diào)節(jié)膀胱癌細(xì)胞T24細(xì)胞生長(zhǎng)的影響及其分子機(jī)制。 方法 1.首先以pCMV-3×flag-ANG為模板，PCR法擴(kuò)增獲得ANG的突變體pCMV-3×flag-ANGH37A。以pcDNA3.1-RI為模板，PCR法擴(kuò)增RI編碼區(qū)序列，將其克隆至pGEX-4T-1原核表達(dá)載體中，構(gòu)建原核表達(dá)質(zhì)粒pGEX-4T-RI。再分別以pCMV-3×flag-ANG、pCMV-3×flag-ANGH37A和pcDNA3.1-RI為模板，PCR法擴(kuò)增其編碼區(qū)序列，構(gòu)建真核表達(dá)質(zhì)粒pEYFP-ANG、pEYFP-ANGH37A和pECFP-RI。 2.在T24和HEK293細(xì)胞內(nèi)進(jìn)行RI與ANG及其突變體的免疫共沉淀實(shí)驗(yàn)與熒光共振能量轉(zhuǎn)移實(shí)驗(yàn)。研究RI與ANG及其突變體在細(xì)胞內(nèi)的相互作用。在細(xì)胞外應(yīng)用GST pull-down方法研究RI與ANG及其突變體在體外的相互作用。在T24細(xì)胞和293細(xì)胞內(nèi)進(jìn)行RI與ANG及其突變體的免疫熒光共定位實(shí)驗(yàn)。此外，熒光共振能量轉(zhuǎn)移（FRET）觀察和結(jié)果也顯示RI與ANG在細(xì)胞內(nèi)的相互作用。 3.將pCMV-3×flag-ANG及pCMV-3×flag-ANGH37A轉(zhuǎn)染T24細(xì)胞和293細(xì)胞，檢測(cè)RI與ANG相互作用對(duì)T24細(xì)胞的影響及介導(dǎo)PI3K/AKT/mTOR信號(hào)通路蛋白的影響。 4.構(gòu)建裸鼠移植瘤模型，檢測(cè)ANG蛋白及其突變體對(duì)于移植瘤生長(zhǎng)，腫瘤新生血管以及自發(fā)轉(zhuǎn)移等的影響。 結(jié)果 1.重組真核表達(dá)質(zhì)粒pCMV-3×flag-ANGH37A、pEYFP-ANG、pEYFP-ANGH37A和pECFP-RI構(gòu)建正確。重組原核表達(dá)質(zhì)粒pGEX-4T-RI構(gòu)建正確。 2.實(shí)驗(yàn)結(jié)果顯示在細(xì)胞內(nèi)和細(xì)胞外，RI與ANG及其突變體均存在相互作用。 3.通過(guò)pCMV-3×flag-ANG及pCMV-3×flag-ANGH37A轉(zhuǎn)染T24細(xì)胞后，PI3K/AKT/mTOR通路的一些關(guān)鍵靶蛋白均呈現(xiàn)顯著的升高，，表明ANG可以通過(guò)此通路促進(jìn)T24細(xì)胞的生長(zhǎng)。 4.移植瘤動(dòng)物模型的實(shí)驗(yàn)結(jié)果表明，ANG蛋白及其突變體的過(guò)表達(dá)對(duì)于膀胱癌腫瘤的生長(zhǎng)具有促進(jìn)作用，并且其腫瘤組織中新生血管相比正常的腫瘤細(xì)胞組顯著增加。 結(jié)論 綜合以上研究成果，我們得出了以下的一些結(jié)論：內(nèi)源性及外源性的核糖核酸酶抑制因子（RI）蛋白與血管生成素（ANG）及其突變體蛋白均存在相互作用。并且在此基礎(chǔ)上，RI與ANG蛋白的相互作用，介導(dǎo)和調(diào)節(jié)膀胱癌細(xì)胞T24的PI3K/AKT/mTOR信號(hào)通路，進(jìn)一步的影響了膀胱癌的生長(zhǎng)及腫瘤新生血管的生成。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.14

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