血管内皮生长因子和碱性成纤维细胞生长因子对大鼠外周血内皮祖细胞培养的影响  

Impact of vascular endothelial growth factor and basic fibroblast growth factor on culture of rat peripheral blood progenitor cells

作　　者：张建江[1] 郑莉萍[1] 王华[1] 

机构地区：[1]郑州大学第一附属医院儿内科,450052

出　　处：《中华肾脏病杂志》2010年第12期915-919,共5页Chinese Journal of Nephrology

基　　金：河南省医学科技攻关项目（200803038）

摘　　要：目的 探讨不同培养条件对大鼠外周血来源内皮祖细胞（EPC）生长情况的影响.方法 密度梯度离心法获得大鼠外周血单个核细胞,根据培养基中是否添加血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）及培养板是否预铺纤连蛋白（FN）分组培养.观察记录细胞生长情况并进行统计分析,以免疫组化和免疫荧光法进行鉴定.结果 大鼠外周血来源的单个核细胞在体外呈现贴壁生长,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,预铺FN可以促进EPC的贴壁增殖（t=4.43,P＜0.05;t=3.70,P＜0.05）.在同样预铺或未铺FN的情况下,在培养液中加入生长因子可促使单个核细胞更好地向EPC分化（t=-10.96,P＜0.01;t=-13.22,P＜0.01）.免疫组化及免疫荧光结果 显示,细胞培养第4、7、10天,细胞表面CD34、CDl33表达不断增强[（35.7±4.2）%、（60.1±3.8）%、（81.8±6.4）%;（3.2±0.9）%、（18.4±7.3）%、（32±3.8）%],第14天下降[（32.1±5.4）%,（1.9±2.7）%];而Flk-1表达在第4、7、10、14天均不断增强[（31.2±3.5）%、（40.6±5.3）%、（71.2±8.4）%、（81.5±4.1）%].结论 FN有利于内皮祖细胞的贴壁生长和增殖.VEGF及bFGF促进其增殖分化.内皮祖细胞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.Objective To observe the impact of different culture conditions on the growth of the endothelial progenitor cells（EPCs） from rat peripheral blood. Methods Mononuclear cells obtained from rat peripheral blood were isolated by using density gradient centrifugation. According to the culture medium added with vascular endothelial growth factor （VEGF）, basic fibroblast growth factor （bFGF） and the dishes were precoated with fibronectin or not, the mononuclear cells were divided into different groups and cultured under different conditions in vitro to compare the differences of the growth conditions. The different growth conditions were recorded and the result was calculated by statistics software. At last, the cells were identified by immunohistochemistry and immunofluorescence. Results The mononuclear cells from rat peripheral blood grew in the manner of keeping close to the wall in vitro. The cells and cell colonies cultured for 7 days hinted：under the same culture conditions, precoating FN was beneficial to the adherent proliferation of EPCs （t=4.43, P〈0.05; t=3.70, P〈0.05）. Excluding the impact of fibronectin, growth factors could promote mononuclear cells differentiation to EPCs, indicating that growth factors enhanced proliferation of EPCs （t =-13.22, P〈0.01; t =-10.96, P〈0.01）. Immunohistochemistry and immunofluorescence showed that, at day 4, 7, 10 of cells cultured, CD34 and CD133 expressions were increased gradually [（35.7±4.2）%, （60.1±3.8）%, （81.8±6.4）%; （3.2±0.9）%, （18.4±7.3）%,（32±3.8）%, respectively]; at day 14, both were decreased [（32.1±5.4）%, （1.9±2.7）%]; but Flk-1was increased at day 4, 7,10, 14 [（31.2±3.5）%, （40.6±5.3）%, （71.2±8.4）%, （81.5±4.1）%].Conclusions Fibronectin is conducive to adhesion and proliferation of EPCs. VEGF and bFGF play an important role in the differentiation of EPCs. The success culture of EPCs in vitro will provide a sufficient number of seed cells for its application in vascular tissue en

关 键 词：血管内皮生长因子A 成纤维细胞生长因子2 干细胞 纤连蛋白类 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]