猪脐静脉血管内皮细胞体外培养研究

发布时间：2018-02-24 06:31

  本文关键词： 猪 血管内皮细胞 细胞鉴定 细胞体外培养 端粒酶 PPARγ　出处：《湖南农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的：对猪脐静脉血管内皮细胞进行体外培养并使其永生化,进而为研究过氧化物酶体增殖物激活受体PPARγ对猪胎盘及胎盘血管发生的影响提供良好的细胞模型。方法：采用胶原酶对新鲜仔猪脐静脉血管内皮细胞进行消化分离,获得原代细胞并在CO2培养箱条件下进行体外培养；显微镜观察细胞生长的形态学变化,免疫组化法进行细胞鉴定；利用ICELLigence细胞功能分析仪监测体外培养细胞的生长曲线变化,并采用流式细胞术检测细胞周期和凋亡；采用端粒酶转入的方法进行体外培养猪脐静脉血管内皮细胞的永生化处理,并用G418药物筛选阳性单克隆细胞；利用细胞功能分析仪监测激动剂和抑制剂对该细胞增殖能力的影响。结果：(1)原代细胞生长状态良好,呈小三角形、椭圆形、梭形、多边形单层生长,界限清晰,融合后呈铺路石状排列,细胞增殖能力强。多次传代后细胞增殖能力减弱,细胞活力差,凋亡细胞增多。兔抗人第八因子相关抗原(FⅧAg)抗体和兔抗人CD31多克隆抗体鉴定均呈阳性。(2)原代细胞转入端粒酶后,转染效检证明端粒酶成功转入细胞内；转染细胞药筛14d后获得单克隆细胞,并能够增殖传代,细胞状态良好。(3)当激动剂浓度为5μMol/ml时,可以促进PPARγ蛋白的表达,加速原代细胞和单克隆细胞的生长增殖,增强细胞活力；当抑制剂浓度为20μMol/ml时,明显抑制PPARγ蛋白的表达,从而抑制细胞的生长增殖,降低细胞活力。结论：本研究由猪脐静脉血管分离获得的原代培养细胞,经体外培养观察和细胞特征性标志物检测,符合血管内皮细胞的基本特征；原代培养细胞经端粒酶转染和药筛处理获得转染阳性单克隆细胞,并表现出良好的生长增殖能力；使用PPARγ激动剂或抑制剂药物干预,原代培养细胞和转染的单克隆细胞均表现出类似的促进或抑制细胞生长的反应。
[Abstract]:Objective: to culture porcine umbilical vein endothelial cells in vitro and immortalize them. So as to provide a good cell model to study the effect of peroxisome proliferator activated receptor (PPAR 纬) on placenta and placental angiogenesis. Methods: fresh umbilical vein endothelial cells were digested and separated by collagenase. Primary cells were obtained and cultured in vitro in CO2 incubator; morphological changes of cell growth were observed under microscope and identified by immunohistochemical method; growth curves of cultured cells were monitored by ICELLigence cell function analyzer. Cell cycle and apoptosis were detected by flow cytometry, immortalization of porcine umbilical vein endothelial cells in vitro was treated by telomerase transfer, and positive monoclonal cells were screened by G418. The effects of agonists and inhibitors on the proliferation of the cells were monitored by cell function analyzer. Results: the primary cells were in good growth state, small triangle, ellipse, fusiform, polygonal monolayer growth, and the boundary was clear. After fusion, the cells were arranged in paving stone, the ability of cell proliferation was strong. After repeated passages, the ability of cell proliferation was weakened, and the activity of cells was poor. The number of apoptotic cells was increased. Both rabbit anti-human factor 8th antigen F 鈪，

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