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背景:体外扩增法培养血管内皮祖细胞操作简便、费用低廉是实验中获取内皮祖细胞的主要方法。目的:拟从兔的骨髓中通过体外扩增法分离培养出血管内皮祖细胞,为进一步观察自体血管内皮祖细胞移植促进血管内皮的修复提供细胞学基础。设计、时间及地点:开放性实验,于2005-03/2006-02在解放军第二军医大学长征医院内科实验室完成。材料:6~8月龄新西兰大白兔8只,雌雄不拘,体质量(2.5±0.5)kg,抽取骨髓,密度离心法分离骨髓单个核细胞方法:将骨髓单个核细胞接种于密度为1×106cm-2,加入含有血管内皮细胞生长因子、碱性成纤维细胞生长因子的M199培养基中体外扩增培养7d,通过DiL标记的乙酰化低密度脂蛋白和FITC标记的凝集素BS-1双染法鉴定血管内皮祖细胞,显示红色荧光的为吞噬了乙酰化低密度脂蛋白的细胞,绿色荧光为结合BS-1的细胞,双染色为橙色荧光。免疫荧光染色及流式细胞仪检测CD133,CD34,Flk-1的表达。主要观察指标:①细胞形态观察。②血管内皮祖细胞的增殖能力。③乙酰化低密度脂蛋白和凝集素BS-1双染法鉴定血管内皮祖细胞结果。④血管内皮祖细胞免疫组织化学鉴定结果。⑤血管内皮祖细胞表面标志物流式细胞仪检测结果。结果:①细胞形态观察:新分离的骨髓单个核细胞呈圆形,培养72h后可见贴壁细胞呈集落样生长,细胞呈圆形或不规则形,核分裂相明显,至培养第7天成片生长的细胞集落相互连接,呈梭形的内皮样细胞。②血管内皮祖细胞的增殖能力:培养2~4d血管内皮祖细胞增殖较快,之后增殖速度减缓,生长曲线呈典型“S”形外观,培养第6,7天血管内皮祖细胞生长再次增快,吸光度值分别达到0.58±0.15和0.62±0.23。③在血管内皮祖细胞的胞质中,出现与乙酰化低密度脂蛋白结合的红色荧光聚集,阳性率达95%以上;与凝集素BS-1结合率几乎达100%;两者双染色率达90%以上。④血管内皮祖细胞免疫组织化学及流式细胞仪检测细胞表面标志物CD133,FlK-1,CD34均呈阳性。结论:体外扩增法成功地从兔骨髓中分离培养出具有血管内皮祖细胞特征的细胞群体。
BACKGROUND: In vitro expansion of cultured endothelial progenitor cells is simple and cost-effective. It is the main method to obtain endothelial progenitor cells. OBJECTIVE: To isolate and culture endothelial progenitor cells from bone marrow of rabbits by in vitro amplification, and to provide a cytological basis for further observation of autologous vascular endothelial progenitor cells transplantation to promote vascular endothelial repair. DESIGN, TIME AND SETTING: The open experiment was performed at the Internal Medicine Laboratory of Changzheng Hospital, Second Military Medical University, March 2005 to February 2006. MATERIALS: Eight New Zealand white rabbits aged 6-8 months were randomly divided into male and female. The body weight was (2.5 ± 0.5) kg. The bone marrow was sampled and the bone marrow mononuclear cells were separated by density centrifugation. Methods: Bone marrow mononuclear cells were inoculated on a density of 1 × 106cm -2, and cultured in M199 medium supplemented with vascular endothelial growth factor and basic fibroblast growth factor for 7 days. The cells were double-stained with DiL-labeled acetylated low-density lipoprotein and FITC-labeled lectin BS-1 Vascular endothelial progenitor cells were identified, showing red fluorescence as cells phagocytosed with acetylated low density lipoprotein, green fluorescence as BS-1 binding cells, and double staining as orange fluorescence. Immunofluorescence staining and flow cytometry were used to detect the expression of CD133, CD34 and Flk-1. MAIN OUTCOME MEASURES: ① Cell morphology observation. ② vascular endothelial progenitor cell proliferation. ③ acetylated low-density lipoprotein and lectin BS-1 double staining identification of endothelial progenitor cells results. ④ vascular endothelial progenitor cells identified by immunohistochemistry results. ⑤ vascular endothelial progenitor cell surface markers flow cytometry test results. RESULTS: ① Morphological observation: Newly isolated bone marrow mononuclear cells were round in shape. After cultured for 72h, the adherent cells showed colony-like growth with round or irregular cells with obvious mitosis. Of the cell colonies connected to each other, spindle-shaped endothelium-like cells. ② Proliferation capacity of endothelial progenitor cells: The endothelial progenitor cells proliferated rapidly 2 ~ 4 days after culture, and then the proliferation rate slowed down. The growth curve showed the typical “S” shape appearance. On the 6th and 7th day, the endothelial progenitor cells grew again The absorbency value reached 0.58 ± 0.15 and 0.62 ± 0.23 respectively. ③ In the cytoplasm of vascular endothelial progenitor cells, red fluorescent aggregation with acetylated low density lipoprotein appeared, the positive rate reached more than 95%; the binding rate to lectin BS-1 almost reached 100%; the double staining rate More than 90%. ④ The endothelial progenitor cells were detected by immunohistochemistry and flow cytometry. The cell surface markers CD133, FlK-1 and CD34 were all positive. Conclusion: The cell population with the characteristics of vascular endothelial progenitor cells was successfully isolated from rabbit bone marrow by in vitro amplification.