大鼠外周血内皮祖细胞培养及鉴定的实验研究

发布时间：2018-06-04 03:53

本文选题：单个核细胞 + 内皮祖细胞　；参考：《郑州大学》2010年硕士论文

【摘要】：研究背景和目的内皮祖细胞(endothelial progenitor cells, EPCs)是一种多能干细胞,它能自我更新并且增殖分化为成熟的血管内皮细胞。它不但参与了胚胎发育早期的血管形成,在成年体内与血管新生及血管形成关系也最为密切。此外,EPCs还可以分泌多种促进新生血管生长的细胞因子,例如表皮生长因子(epidermal growth factor, EGF),成纤维细胞生长因子4,9(fibroblast growth factor-4,9, FGF-4,9),血管内皮生长因子(vascular endothelial growth factor, VEGF)等等。近年来,EPCs在促进受损血管的修复方面,如心脑血管疾病、糖尿病性视网膜病变及创伤愈合等多种血管类疾病的治疗中展现出极为广泛的应用前景。建立获取外周血EPCs的方法,将会为其作为种子细胞广泛应用于血管组织工程奠定基础。方法本实验采用密度梯度离心法从大鼠外周血分离单个核细胞,按不同培养条件分成4组：1组：未铺纤维连接蛋白,使用M199基本培养基(含M199培养基,10%胎牛血清)；2组：未铺纤维连接蛋白,使用M199诱导培养基(含M199培养基,10%胎牛血清,血管内皮生长因子10ng/mL,碱性成纤维细胞生长因子bFGF4ng/mL);3组：预铺纤维连接蛋白(fibronectin,FN),使用M199基本培养基；4组：预铺纤维连接蛋白,使用M199诱导培养基。分别对其进行体外诱导培养,比较其体外生长情况的不同,应用统计学软件比较细胞培养第7天细胞数量、集落数有无统计学差异,绘制各组生长曲线,并进行免疫组化染色及免疫荧光的鉴定。结果大鼠外周血来源的单个核细胞在体外呈现贴壁生长,最初培养3到4天内细胞生长较慢,约4天后出现对数生长期,10天后细胞基本长满培养瓶底。培养第7天各组细胞数及细胞集落数提示:在相同培养条件下(分别将第1组与第3组、第2组与第4组相比较),预铺纤维连接蛋白可以促进EPCs的贴壁增殖(t=4.43,P0.05；t=3.70,P0.05)。而在同样未铺或预铺纤维连接蛋白的情况下(分别将1组与2组、3组与4组比较),在培养液中加入生长因子可以促使单个核细胞更好地向EPCs分化,说明生长因子对EPCs原代靶细胞有明显的促增殖作用(t=—13.22,P0.01；t=—10.96,P0.01)。培养所得细胞表面CD34、Flk-1和CD133在不同时段以不同比率呈阳性表达。结论从大鼠外周血中经密度梯度离心法分离单个核细胞,进行诱导培养,可以获得EPCs。纤维连接蛋白有利于EPCs的贴壁生长和增殖。血管内皮生长因子及成纤维细胞生长因子促进其增殖分化。EPCs的体外成功培养将为其应用于血管组织工程提供足够数量的种子细胞来源,并为外周血干细胞移植治疗多种疾病提供新的思路。
[Abstract]:Background and purpose of the study Endothelial progenitor cells (progenitor cells, EPCs) are pluripotent stem cells, which can self-renew and proliferate and differentiate into mature vascular endothelial cells. It is not only involved in angiogenesis in early embryonic development, but also closely related to angiogenesis and angiogenesis in adults. In addition, EPCs can also secrete a variety of cytokines that promote angiogenesis, such as epidermal growth factor, fibroblast growth factor-4, FGF-4, vascular endothelial growth factor endothelial growth factor, VEGF) and so on. In recent years, EPCs have been widely used in the treatment of vascular diseases such as cardiovascular and cerebrovascular diseases, diabetic retinopathy and wound healing. The method of obtaining EPCs from peripheral blood will lay a foundation for its wide application as seed cells in vascular tissue engineering. Method Mononuclear cells were isolated from the peripheral blood of rats by density gradient centrifugation. The mononuclear cells were divided into 4 groups according to different culture conditions: group 1: uncoated fibronectin, Using M199 basic medium (containing M199 medium / 10% fetal bovine serum): group 2: uncoated fibronectin, using M199 induction medium (M199 medium containing 10% fetal bovine serum), Vascular endothelial growth factor (10 ng / mL), basic fibroblast growth factor (bFGF4ng / mLL) group 3: precoated fibronectin FNN group, M199 basic medium: precoated fibronectin group 4: precoated fibronectin group, induced by M199 medium. The cell number and colony number were compared by statistical software on the 7th day of cell culture, and the growth curves of each group were drawn. Immunohistochemical staining and immunofluorescence were performed. Result The mononuclear cells from peripheral blood of rats showed adherent growth in vitro. The growth of mononuclear cells was slower in the first 3 to 4 days, and the logarithmic growth phase appeared after 4 days. After 10 days of logarithmic growth, the cells grew to the bottom of the culture flask. The cell number and colony number of each group on the 7th day of culture indicated that under the same culture conditions (group 1 and group 3, group 2 and group 4, respectively), the precoated fibronectin could promote the proliferation of adherent cells of EPCs. In the same condition without or without fibronectin (group 1 and group 2, group 3 and group 4, respectively), the addition of growth factor in the culture medium could promote the better differentiation of mononuclear cells into EPCs. The results indicated that the growth factor had obvious effect on the proliferation of EPCs primary target cells. CD34 flk-1 and CD133 were expressed at different rates on the surface of cultured cells. Conclusion Mononuclear cells were isolated from rat peripheral blood by density gradient centrifugation. Fibronectin is beneficial to the adherent growth and proliferation of EPCs. The successful culture of vascular endothelial growth factor and fibroblast growth factor to promote proliferation and differentiation of EPCs in vitro will provide a sufficient number of seed cell sources for its application in vascular tissue engineering. It also provides a new idea for peripheral blood stem cell transplantation in the treatment of various diseases.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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