体外分离培养大鼠骨髓内皮祖细胞及其生物学特性鉴定

Isolation,culture and biological characterization of rat bone marrow-derived endothelial progenitor cells

背景:现阶段急需建立一种稳定的体外分离培养内皮祖细胞的方法,骨髓中内皮祖细胞的含量丰富,增殖能力强,而且容易获得,操作简单。目的:拟在体外分离培养大鼠骨髓内皮祖细胞,并对其生物学特性进行鉴定。设计、时间及地点:细胞学体外观察,于2008-08/12在重庆医科大学附属第一医院中心实验室完成。材料:清洁级2周龄SD大鼠20只,由重庆医科大学实验动物中心提供。方法:无菌条件下分离大鼠股骨和胫骨,用预冷至4℃的0.01mol/L磷酸盐缓冲液冲洗骨髓腔,以密度梯度离心法收集单个核细胞层,含体积分数为20%胎牛血清的M199培养液重悬细胞,制成单细胞悬液后进行细胞计数,按1×109L-1浓度接种到预先用纤维连接蛋白包被的培养瓶中,37℃、体积分数为5%的CO2饱和湿度恒温培养。主要观察指标:倒置显微镜观察细胞形态变化,免疫细胞化学检测内皮祖细胞表面特异性抗原CD133、内皮祖细胞表面抗原CD34和血管内皮生长因子受体KDR的表达,并进行流式细胞仪定量分析和细胞生长曲线分析。结果:新分离获得的骨髓单个核细胞为小圆形,培养4d后贴壁细胞呈"集落"样生长,中间为圆形贴壁细胞,外周梭形细胞较多,传代后梭形细胞首尾相连,呈"毛细血管"样排列。培养第4,7,10,14天贴壁细胞CD34,KDR,CD133均呈阳性表达,CD133阳性细胞第10天时开始减少。流式细胞仪检测结果显示,CD133/KDR双阳性细胞率逐渐升高,10d时达峰值,随后下降。KDR阳性细胞率随培养时间的延长而逐渐升高。原代细胞生长曲线显示细胞在第3～6天处于指数生长期,贴壁细胞大量增殖,呈"集落"样生长。结论:采用密度梯度离心法可成功从SD大鼠骨髓中分离获得内皮祖细胞,加入M199培养液后能够贴壁增殖,并可以分化为内皮细胞。

BACKGROUND： A steady method that can isolate endothelial progenitor cells （EPCs） need to be explored. Studies show that the EPCs riches in bone marrow with strong reproductive activity, which can be easily obtained. OBJECTIVE： To evaluate the isolating culture and biological characterization of EPCs derived from rat marrow. DESIGN, TIME AND SETTING： The in vitro cytology experiment was pedormed at the central laboratory of the First Affiliated Hospital of Chongqing Medical University from August to December 2008. MATERIALS： Twenty SD rats, with two-week-old, were obtained from Experimental Animal Center of Chongqing Medical University. METHODS： femurs and tibias of rats were isolated under sterile conditions, and then the bone marrow cavity was washed by 4℃ 0.01 mol/L phosphate buffer （PBS）. Mononuclear cells were collected from marrow suspension by density gradient centrifugation, followed by cultured with M199 culture medium containing 20% fetal bovine serum to obtained cell suspension. The cells were vaccinated into culture flask （coated with fibronectin） with the density of 1×10^9/L, and incubated at 37℃ in 5% CO2 atmosphere. MAIN OUTCOME MEASURES： Morphologic change of cells was observed by an invert microscope, the expression of CD133, CD34 as well as KDR was detected by immunocytochemistry, and the flow cytometry was used to analyze the growth curve. RESULTS： The adherent cells formed clusters that were found to be characterized by density packed round cells surrounded by spindle-likes cells. Adherent cells were identified positive for CD133, CD34 and KDR by immunocytochemistry on days 4, 7, 10 and 14. CD133-positive cells reduced after 10 days. Flow cytometry analysis indicated that the number of CD133/KDR double positive cells gradually increased to the maximum at day 10 and then declined. KDR-positive cells gradually increased up with the time. EPCs growth curve showed that cells proliferated fast on days 3 6. CONCLUSION： EPCs can be isolated and cultured by density gradient centrifugation from marrow suspension of SD rats. These cells exhibit the characterization of endothelial cells, and can differentiate into endothelial cells.