过表达膜联蛋白A2受体对新生血管形成的作用研究

发布时间：2018-10-19 09:46

【摘要】：目的:探讨过表达膜联蛋白A2受体(annexin A2 receptor,AX2R,AXIIR,c5orf39)对新生血管形成的作用研究。方法:根据Gene Bank的编码序列,用primer premier 5软件设计AX2R的PCR(polymerase chain reaction,PCR)引物,真核重组质粒Lenti-AX2R的制备方法包括以下步骤:通过PCR合成人的AX2R基因片段;将扩增的核苷酸序列片段与真核质粒连接,该插入片段的酶切位点分别为Xbar I和Sal I,再将所述的真核重组质粒转化到感受态细菌中,涂板子、挑克隆、摇菌扩增,使用碱裂解法抽提真核重组质粒;通过DNA测序筛选出正确插入的重组质粒克隆。将构建好的质粒Lenti-AX2R转染人胚肾细胞(293T),采用Western blotting和RT-PCR方法检测真核过表达质粒的蛋白和mRNA(messenger ribonucleic acid,mRNA)的表达水平。将Lenti-AX2R包装为慢病毒感染视网膜血管内皮细胞(human retinal endothelial cells,HRECs)和脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs),以达到内皮细胞过表达AX2R基因的目的。通过细胞增殖实验、细胞迁移实验和管养形成实验检测过表达AX2R对血管内皮细胞功能的影响,采用小鼠主动脉环血管形成实验检测过表达AX2R在体外对新生血管形成的影响。采用流式细胞仪检测过表达AX2R对HRECs和HUVECs两种细胞细胞周期的影响,并用Western blotting检测Cyclin A1、Cyclin B1、Cyclin D1、Cyclin E1、CDK1和p-CDC2一系列细胞周期相关蛋白的表达水平;利用荧光显微镜观察Lenti-EGFP-AX2R融合质粒在HRECs和293T细胞的定位情况;采用Western blotting和RT-PCR检测蛋白降解相关通路分子KLF2(Krüppel like transcription factor 2)、血管内皮细胞生长因子(vascular endothelial growther factor,VEGF)、血管内皮细胞生长因子受体2(vascular endothelial growther factor receptor 2,VEGFR2)蛋白和基因表达水平。结果:通过将构建的过表达AX2R质粒的测序结果与BALST比对,发现与Gene Bank中公布的序列一致;Western blotting和RT-PCR的检测结果显示,同空白对照组和阴性对照组相比,AX2R的蛋白表达水平和基因表达水平均显著升高。过表达AX2R慢病毒通过感染HRECs和HUVECs一定的时间后,同对照组相比,细胞增殖的数量、细胞向远处迁移的细胞数和细胞形成管状结构的数量均明显减少;在体外培养预先感染过表达AX2R慢病毒的小鼠主动脉环发现,实验组小鼠主动脉环开始长出新生血管的初始时间明显延迟,并且萌出的新生血管不论是血管的长度、数量以及血管形态的完整度都明显弱于其他两组。流式细胞仪检测结果采用Modfitsoftware软件分析显示在过表达AX2R组,处于G1期的细胞数不变,处于S期的细胞数增加,处于G2期的细胞数减少,细胞周期相关蛋白cyclin B1和cyclin E1的表达水平没有变化,CDK1和p-CDC2的蛋白表达水平升高,cyclin A1和cyclin E1的蛋白表达水平降低;荧光显微镜观察结果发现Lenti-EGFP-AX2R在HRECs和293T细胞内的表达蛋白成团聚集存在;Western blotting和RT-PCR检测结果显示HRECs和HUVECs过表达了AX2R后,KLF2的蛋白和基因表达水平升高,VEGF和VEGFR2的表达水平则是下降的。结论:成功构建了AX2R的过表达质粒Lenti-AX2R。Lenti-AX2R能够抑制HRECs和HUVECs两种内皮细胞的迁移、增殖、管样形成和抑制小鼠主动脉环新生血管的形成,可能是通过抑制细胞周期和KLF2的蛋白降解相关通路来发挥作用的。
[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.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R774

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