【摘要】：目的:探討過表達(dá)膜聯(lián)蛋白A2受體(annexin A2 receptor,AX2R,AXIIR,c5orf39)對(duì)新生血管形成的作用研究。方法:根據(jù)Gene Bank的編碼序列,用primer premier 5軟件設(shè)計(jì)AX2R的PCR(polymerase chain reaction,PCR)引物,真核重組質(zhì)粒Lenti-AX2R的制備方法包括以下步驟:通過PCR合成人的AX2R基因片段;將擴(kuò)增的核苷酸序列片段與真核質(zhì)粒連接,該插入片段的酶切位點(diǎn)分別為Xbar I和Sal I,再將所述的真核重組質(zhì)粒轉(zhuǎn)化到感受態(tài)細(xì)菌中,涂板子、挑克隆、搖菌擴(kuò)增,使用堿裂解法抽提真核重組質(zhì)粒;通過DNA測(cè)序篩選出正確插入的重組質(zhì)粒克隆。將構(gòu)建好的質(zhì)粒Lenti-AX2R轉(zhuǎn)染人胚腎細(xì)胞(293T),采用Western blotting和RT-PCR方法檢測(cè)真核過表達(dá)質(zhì)粒的蛋白和mRNA(messenger ribonucleic acid,mRNA)的表達(dá)水平。將Lenti-AX2R包裝為慢病毒感染視網(wǎng)膜血管內(nèi)皮細(xì)胞(human retinal endothelial cells,HRECs)和臍靜脈內(nèi)皮細(xì)胞(human umbilical vein endothelial cells,HUVECs),以達(dá)到內(nèi)皮細(xì)胞過表達(dá)AX2R基因的目的。通過細(xì)胞增殖實(shí)驗(yàn)、細(xì)胞遷移實(shí)驗(yàn)和管養(yǎng)形成實(shí)驗(yàn)檢測(cè)過表達(dá)AX2R對(duì)血管內(nèi)皮細(xì)胞功能的影響,采用小鼠主動(dòng)脈環(huán)血管形成實(shí)驗(yàn)檢測(cè)過表達(dá)AX2R在體外對(duì)新生血管形成的影響。采用流式細(xì)胞儀檢測(cè)過表達(dá)AX2R對(duì)HRECs和HUVECs兩種細(xì)胞細(xì)胞周期的影響,并用Western blotting檢測(cè)Cyclin A1、Cyclin B1、Cyclin D1、Cyclin E1、CDK1和p-CDC2一系列細(xì)胞周期相關(guān)蛋白的表達(dá)水平;利用熒光顯微鏡觀察Lenti-EGFP-AX2R融合質(zhì)粒在HRECs和293T細(xì)胞的定位情況;采用Western blotting和RT-PCR檢測(cè)蛋白降解相關(guān)通路分子KLF2(Krüppel like transcription factor 2)、血管內(nèi)皮細(xì)胞生長因子(vascular endothelial growther factor,VEGF)、血管內(nèi)皮細(xì)胞生長因子受體2(vascular endothelial growther factor receptor 2,VEGFR2)蛋白和基因表達(dá)水平。結(jié)果:通過將構(gòu)建的過表達(dá)AX2R質(zhì)粒的測(cè)序結(jié)果與BALST比對(duì),發(fā)現(xiàn)與Gene Bank中公布的序列一致;Western blotting和RT-PCR的檢測(cè)結(jié)果顯示,同空白對(duì)照組和陰性對(duì)照組相比,AX2R的蛋白表達(dá)水平和基因表達(dá)水平均顯著升高。過表達(dá)AX2R慢病毒通過感染HRECs和HUVECs一定的時(shí)間后,同對(duì)照組相比,細(xì)胞增殖的數(shù)量、細(xì)胞向遠(yuǎn)處遷移的細(xì)胞數(shù)和細(xì)胞形成管狀結(jié)構(gòu)的數(shù)量均明顯減少;在體外培養(yǎng)預(yù)先感染過表達(dá)AX2R慢病毒的小鼠主動(dòng)脈環(huán)發(fā)現(xiàn),實(shí)驗(yàn)組小鼠主動(dòng)脈環(huán)開始長出新生血管的初始時(shí)間明顯延遲,并且萌出的新生血管不論是血管的長度、數(shù)量以及血管形態(tài)的完整度都明顯弱于其他兩組。流式細(xì)胞儀檢測(cè)結(jié)果采用Modfitsoftware軟件分析顯示在過表達(dá)AX2R組,處于G1期的細(xì)胞數(shù)不變,處于S期的細(xì)胞數(shù)增加,處于G2期的細(xì)胞數(shù)減少,細(xì)胞周期相關(guān)蛋白cyclin B1和cyclin E1的表達(dá)水平?jīng)]有變化,CDK1和p-CDC2的蛋白表達(dá)水平升高,cyclin A1和cyclin E1的蛋白表達(dá)水平降低;熒光顯微鏡觀察結(jié)果發(fā)現(xiàn)Lenti-EGFP-AX2R在HRECs和293T細(xì)胞內(nèi)的表達(dá)蛋白成團(tuán)聚集存在;Western blotting和RT-PCR檢測(cè)結(jié)果顯示HRECs和HUVECs過表達(dá)了AX2R后,KLF2的蛋白和基因表達(dá)水平升高,VEGF和VEGFR2的表達(dá)水平則是下降的。結(jié)論:成功構(gòu)建了AX2R的過表達(dá)質(zhì)粒Lenti-AX2R。Lenti-AX2R能夠抑制HRECs和HUVECs兩種內(nèi)皮細(xì)胞的遷移、增殖、管樣形成和抑制小鼠主動(dòng)脈環(huán)新生血管的形成,可能是通過抑制細(xì)胞周期和KLF2的蛋白降解相關(guān)通路來發(fā)揮作用的。
[Abstract]:Objective: To investigate the effect of Annexin A2 receptor (AX2R, AXIIR, c5orf39) on angiogenesis. The method comprises the following steps: using a PCR (polymerase chain reaction (PCR) primer of a primer premixer 5 software to design an AX2R according to the coding sequence of the GeneBank, wherein the preparation method of the true nuclear recombinant plasmid Lenti-AX2R comprises the following steps of: synthesizing a human AX2R gene fragment by PCR; connecting the amplified fragment sequence fragment with the true nuclear plasmid; The enzyme cutting sites of the inserted fragment are Xbar I and Sal I, respectively, and then the true nuclear recombinant plasmid is transformed into the recombinant bacterium, the plate is coated, the clone and the shaking bacteria are amplified, the true nuclear recombinant plasmid is extracted by using the alkaline lysis method, and the correct inserted recombinant plasmid clone is screened by DNA sequencing. The constructed plasmid, Lenti-AX2R, was transfected into human embryonic kidney cells (293T). Western blotting and RT-PCR were used to detect the expression level of protein and mRNA (mRNA) of the eukaryotic expression plasmid. Lenti-AX2R is packaged as a lentivirus-infected retinal vascular endothelial cell (HRCs) and human umbilical vein endothelial cells (HUVECs) to achieve the purpose of endothelial cell overexpression of the AX2R gene. The effect of AX2R on vascular endothelial cell function was detected by cell proliferation assay, cell migration experiment and tube culture experiment. CyclinD1, Cyclin B1, Cyclin D1, Cyclin E1, CDK1 and p-CDC2 were detected by Western blotting using flow cytometry. The localization of Lenti-EGFP-AX2R fusion plasmid in HRCs and 293T cells was observed by fluorescence microscopy. Western blotting and RT-PCR were used to detect protein degradation related pathway molecules KLF2 (vascular endothelial growth factor, VEGF). Vascular endothelial growth factor receptor 2 (VEGFR2) protein and gene expression level. Results: Compared with the control group and the negative control group, the expression level of AX2R and the level of gene expression were significantly higher than that of the control group and the negative control group. Compared with the control group, the number of cell proliferation, the number of cells migrating to the distance and the number of cells forming the tubular structure were significantly decreased compared with the control group after the expression AX2R lentivirus was infected with HRCs and HUVECs for a certain time. In vitro culture of mouse aortic rings that had been previously infected with the expression of the AX2R lentivirus showed that the initial time of the newborn blood vessel was significantly delayed by the aortic rings of the experimental group and that the newly-infected new blood vessels were of the length of the blood vessel, The number and the completeness of the vessel morphology were significantly weaker in the other two groups. The results of flow cytometry showed that the number of cells in G1 phase was unchanged, the number of cells in G1 phase was increased, the number of cells in G2 phase was decreased, and the expression levels of cyclin B1 and cyclin E1 were not changed in G2 phase. The expression levels of CDK1 and p-CDC2 increased, and the protein expression levels of CYP1A1 and CD2E1 were decreased. The results of fluorescence microscopy showed that the expression of Lenti-EGFP-AX2R in HRCs and 293T cells was clustered. Western blotting and RT-PCR showed that HRCs and HUVECs had been overexpressing AX2R. The level of protein and gene expression of KLF2 increased, and the expression level of VEGF and VEGFR2 decreased. Conclusion: The overexpression plasmid Lenti-AX2R, Lenti-AX2R, which successfully constructed the AX2R, can inhibit the migration, proliferation, tube-like formation and inhibition of the neovascularization in the aortic rings of mice, and may play a role in inhibiting the cell cycle and the protein degradation-related pathway of KLF2.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R774

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