人VEGF165基因转染人外周血内皮祖细胞的实验研究

发布时间：2018-04-10 15:16

本文选题：内皮祖细胞 + 血管内皮细胞生长因子　；参考：《安徽医科大学》2010年硕士论文

【摘要】： 目的：随着人口老龄化和饮食结构改变,下肢缺血性疾病已成为老年人的常见病和多发病,是截肢致残的主要原因,已成为血管外科研究的一个难点和热点。促进组织血管新生研究已发展成为一种治疗缺血性疾病的新方法。治疗性血管新生可通过三种方法得以实现：生长因子蛋白治疗,细胞移植以及基因转染。血管内皮祖细胞和血管内皮生长因子在血管形成和促血管新生的过程中起着重要作用,本研究拟将基因治疗和干细胞治疗方法相结合,探讨人血管内皮细胞生长因子165(hVEGF165)基因转染人外周血内皮祖细胞(EPCs)及对EPCs增殖和分泌VEGF和NO能力的影响。方法：取经过rhG-CSF动员的健康成人的外周血,采用密度梯度离心法提取单个核细胞,接种在hFN包被的培养板上,M199培养液(含FBS和VEGF)培养,培养至第7d,对贴壁细胞进行EPCs的鉴定：(1)倒置显微镜下观察贴壁细胞形态；(2)激光共聚焦显微镜观察经DiI-acLDL和FITC-UEA-I双染色的贴壁细胞；然后对pcDNA3. 1-hVEGF165质粒进行扩增,抽提,并利用双酶切法及测序进行鉴定,实验分为三组：脂质体介导pcDNA3.1-hVEGF165质粒转染组、pcDNA3.1空质粒转染组和空白对照组。通过ELISA法和硝酸还原酶法分别测定各组上清液中VEGF和一氧化氮(NO)的含量；噻唑蓝(MTT)检测hVEGF165基因修饰对EPCs增殖的影响。结果：FITC-UEA-I和DiI-ac-LDL双染色阳性细胞为正在分化的EPCs,发黄色荧光；脂质体介导pcDNA3.1-hVEGF165质粒转染组EPCs培养基上清液中VEGF和NO表达量明显高于其他两组(P0.01)；VEGF质粒转染对EPCs的增殖无明显影响。结论：人外周血EPCs可以成功转染hVEGF165基因,同时能表达一定浓度的VEGF蛋白,并可促进NO的分泌,而对EPCs的活性无明显影响。为进一步研究VEGF165基因和EPCs结合治疗缺血性疾病提供实验依据。
[Abstract]:Objective: with the aging of population and the change of dietary structure, ischemic disease of lower extremity has become a common disease and frequent disease in the elderly, which is the main cause of amputation and disability, and has become a difficult and hot spot in vascular surgery.Promoting tissue angiogenesis has developed into a new method for the treatment of ischemic diseases.Therapeutic angiogenesis can be achieved in three ways: growth factor protein therapy, cell transplantation, and gene transfection.Vascular endothelial progenitor cells (VECs) and vascular endothelial growth factors (VEGF) play an important role in angiogenesis and angiogenesis.To investigate the effect of human vascular endothelial growth factor 165 (hVEGF165) gene transfection on the proliferation and secretion of VEGF and no in human peripheral blood endothelial progenitor cells (EPCs).Methods: the peripheral blood of healthy adults mobilized by rhG-CSF was extracted by density gradient centrifugation. The mononuclear cells were cultured in M199 medium (including FBS and VEGF) on the hFN coated culture plate.On the 7th day of culture, the adherent cells were identified by EPCs. The adherent cells were observed under inverted microscope. The adherent cells were observed by confocal laser microscopy (DiI-acLDL and FITC-UEA-I) and then pcDNA3.The 1-hVEGF165 plasmid was amplified, extracted and identified by double enzyme digestion and sequencing. The experiment was divided into three groups: liposome-mediated pcDNA3.1-hVEGF165 plasmid transfection group (pcDNA3.1 empty plasmid transfection group) and blank control group (control group).The contents of VEGF and nitric oxide (no) in supernatant of each group were determined by ELISA method and nitrate reductase method respectively, and the effect of hVEGF165 gene modification on EPCs proliferation was detected.Results the expression of VEGF and no in the supernatant of EPCs medium in the liposome-mediated pcDNA3.1-hVEGF165 plasmid transfection group was significantly higher than that in the other two groups. The expression of VEGF and no in the supernatant of EPCs medium in the liposome-mediated pcDNA3.1-hVEGF165 plasmid transfection group was significantly higher than that in the other two groups.Conclusion: human peripheral blood EPCs can successfully transfect the hVEGF165 gene, express a certain concentration of VEGF protein, promote the secretion of no, but have no effect on the activity of EPCs.To provide experimental basis for further study on the combination of VEGF165 gene and EPCs in the treatment of ischemic diseases.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346

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