体外扩增法分离培养兔骨髓来源的血管内皮祖细胞

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。

In vitro isolation and culture of rabbit bone marrow-derived vascular endothelial progenitor cells

BACKGROUND: The in vitro amplification is a primary method for harvesting endothelial progenitor cells (EPCs) due to its simple operation and low cost.OBJECTIVE: To isolate EPCs from rabbit bone marrow to further observe the effects of autologous EPCs on promoting vascular endothelial repair.DESIGN, TIME AND SETTING: An open experiment was performed at the laboratory of Department of Internal Medicine, Changzheng Hospital, Second Military Medical University of Chinese PLA between March 2005 and February 2006. MATERIALS: Eight New Zealand rabbits of either gender, aged 6-8 months, weighing (2.5:L-0.5) kg, were included in this study. Rabbit bone marrow was taken for isolation of bone marrow mononuclear cells by density centrifugation. METHODS: Bone marrow-derived mononuclear cells were inoculated at 1×106/cm2 and cultured for 7 days in M199 medium containing vascular endothelial growth factors and basic fibroblast growth factors. EPCs were identified by Dil-labeled acetylated low-density lipoprotein (Dil- Ac-LDL) and FITC-labeled lectin BS-1 staining. Cells that phagocytized Ac-LDL displayed red fluorescence, cells that combined with lectin BS-1 showed green fluorescence, and cells that were labeled with both exhibited orange fluorescence. Expression levels of CD133, CD134, and Flk-lwere detected using immunofluorescent staining and through the use of flow cytometer.MAIN OUTCOME MEASURES: ① Cellular morphology observation. ② Proliferative capacity of EPCs.③EPCs identified by Dil- Ac-LDL and FITC-labeled lectin BS-1. ④ lmmanohistocbemical identification of EPCs. ⑤Flow cytometry identification of EPC surface marker.RESULTS: ① Cellular morphological observation: the newly isolated bone marrow-derived mononuclear cells exhibited a round appearance. Following 72-hour culture, adherent cells grew in colony cluster, presenting with round or irregular appearance, and nuclear division was obvious. By day 7, flaky cell colonies mutually connected together, presenting with shuttle-shaped endothelioid cells.② Proliferative capability of EPCs: in the 2-4 days of culture, EPCs proliferated fast, and the proliferation slowed down thereafter, exhibiting a typical "S" -shaped appearance. By days 6 and 7, EPC proliferation accelerated again, with the absorbance values of 0.58±0.15 and 0.62±0.23, respectively. ③ Over 95% of EPC cytoplasm exhibited red fluorescence after stained with Ac-LDL, appropriately 100% of cytoplasm exhibited green fluorescence after stained with FITC-labeled lectin BS-1, and over 90% of cytoplasm exhibited orange fluorescence after double staining. ④ Immonohistochemistry and flow cytometry results revealed positive expression of EPC surface markers CD133, FIK-1, and CD34.CONCLUSION: Cell population with EPC characteristics can be successfully isolated from rabbit bone marrow by in vitro amplification.