雌激素膜受体所介导的PI（3）K传导通路激活在内皮祖细胞增殖和迁移中的作用

发布时间：2018-06-21 20:59

本文选题：雌激素 + 内皮祖细胞　；参考：《复旦大学》2005年博士论文

【摘要】：第一部分外周血中内皮祖细胞的分离和培养目的:研究外周血中内皮祖细胞分离、培养及和鉴定的方法。方法:从年轻志愿者的外周血中用密度梯度离心方法分离出单个核细胞,在添加VEGF的M199培养液中培养,每隔3～4d换一次液。培养5d后,消化贴壁细胞,用DiI—ac-LDL及FITC-UEA-1对其进行免疫荧光分析。结果:培养3～4d单核细胞开始贴壁,14d左右开始出现索条状结构,免疫荧光鉴定显示90%的贴壁细胞呈双荧光染色阳性。结论:外周血中富含内皮祖细胞,在一定的培养条件下,可分化为内皮样细胞。第二部分雌激素对内皮祖细胞增殖和迁移作用的影响目的研究雌激素对内皮祖细胞增殖及迁移的影响及细胞培养时间和雌激素浓度对其影响,并研究雌激素作用下细胞周期变化和促细胞增殖和迁移的细胞表面抗原VE-cadherin,Flt-1和KDR mRNA和蛋白水平的变化。方法从年轻志愿者的外周血分离出单个核细胞,在内皮细胞培养条件下培养,诱导,分化出内皮祖细胞。取培养7d的EPCs,并分别加入不同浓度(0,10~(-6)mol/L,10~(-7)mol/L,10~(-8)mol/L,10~(-9)mol/L或10~(-10)mol/L)的17β—雌二醇作用24h后,进行细胞增殖实验分析(MTS)和细胞迁移实验,观察不同浓度雌激素对内皮祖细胞增殖及迁移能力的影响。分别取4×10~5培养5d,7d,9d,13d,20d的内皮祖细胞,加入10~(-8)mol/L雌激素作用24小时,消化贴壁细胞并在显微镜下计数。分别取培养5d,7d,9d,13d的内皮祖细胞,加入10~(-8)mol/L的雌激素作用24h后检测内皮祖细胞在细胞周期各时相的分布,进一步研究雌激素对细胞增殖的影响。分别取培养5d,7d,9d,13d的内皮祖细胞,加入10~(-8)mol/L的雌激素作用24h后,分别用Real-time和流式细胞技术检测内皮细胞表面特异性抗原Flt-1,KDR和VE-cadherin mRNA和蛋白水平表达的变化。结果增殖实验MTS分析表明雌激素能显著增强内皮祖细胞的增殖活性,雌激素浓度为10~(-8)mol/L—10~(-9)mol/L时,增殖活性最强。手工计数内皮祖细胞显示雌激素作用后内皮祖细胞数目显著增多,8-10d的内皮祖细胞增殖活性达到高峰。迁移实验也表明雌激素能够促进内皮祖细胞的迁移,雌激素浓度
[Abstract]:Part I isolation and culture of endothelial progenitor cells from peripheral blood objective: to study the methods of isolation, culture and identification of endothelial progenitor cells in peripheral blood. Methods: mononuclear cells were isolated from the peripheral blood of young volunteers by density gradient centrifugation. The mononuclear cells were cultured in M199 medium supplemented with VEGF. After 5 days of culture, adherent cells were digested and immunofluorescence analysis was performed with DiI-ac-LDL and FITC-UEA-1. Results: after 3 days of culture, mononuclear cells began to adhere to the wall for 14 days. The immunofluorescence analysis showed that 90% of the adherent cells were double fluorescent staining positive. Conclusion: the peripheral blood is rich in endothelial progenitor cells and can differentiate into endothelial like cells under certain culture conditions. Part II effects of estrogen on proliferation and migration of endothelial progenitor cells objective to study the effects of estrogen on proliferation and migration of endothelial progenitor cells The effect of cell culture time and estrogen concentration on it, The changes of cell cycle and VE-cadherin1 and KDR mRNA and protein levels were studied. Methods Mononuclear cells were isolated from the peripheral blood of young volunteers, cultured in endothelial cells, induced and differentiated into endothelial progenitor cells. EPCs were cultured for 7 days and were treated with 17 尾 -estradiol (17 尾 -estradiol / L) at different concentrations of 10 ~ (-6) mol / L ~ (-10) ~ (-7) mol / L ~ (10) ~ (-8) mol / L ~ (-1) ~ (-10) mol / L) for 24 hours. Cell proliferation assay was performed to analyze the effects of different concentrations of estrogen on the proliferation and migration of endothelial progenitor cells (EPCs). Endothelial progenitor cells (EPCs) cultured at 4 脳 10 ~ 5 for 5 d ~ 7 d ~ 9 d ~ (13) d ~ (-1) for 20 d were treated with 10 ~ (-8) mol / L estrogen for 24 h, then the adherent cells were digested and counted under microscope. Endothelial progenitor cells (EPCs) were cultured for 5 d, 7 d, 9 d and 13 d, respectively. The distribution of endothelial progenitor cells (EPCs) in each phase of cell cycle was detected after 24 hours of treatment with 10 ~ 8mol / L estrogen, and the effect of estrogen on cell proliferation was further studied. Endothelial progenitor cells (EPCs) were cultured for 5 d, 7 d, 9 d and 13 d, respectively. The expression of VEGF mRNA and VE-cadherin mRNA and protein were detected by Real-time and flow cytometry, respectively. Results the results of MTS analysis showed that estrogen could significantly enhance the proliferation of endothelial progenitor cells. When the concentration of estrogen was 10 ~ (-8) mol / L ~ (-9) mol / L, the proliferative activity was the highest. Manual counting of endothelial progenitor cells showed that the number of endothelial progenitor cells increased significantly after estrogen treatment, and the proliferation activity of endothelial progenitor cells reached the peak after 8-10 days. The migration experiment also showed that estrogen could promote the migration of endothelial progenitor cells and the concentration of estrogen.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R329.2

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