大鼠骨髓间充质干细胞和内皮祖细胞的分离培养及鉴定

背景：骨髓间充质干细胞的分离和培养扩增至今缺乏统一的方法。内皮祖细胞可以由外周血或骨髓中的单个核细胞诱导分化而成,但因获取骨髓单个核细胞的方法各异,分离效果亦有较大差异。 目的：拟在体外分离培养大鼠骨髓间充质干细胞和内皮祖细胞,并对其进行鉴定。 设计、时间及地点：细胞学体外观察,于2008-10/2009-02在安徽医科大学第一附属医院中心实验室完成。 材料：清洁级健康雄性SD大鼠2只,由安徽省动物中心提供。 方法：抽取大鼠骨髓,通过密度梯度离心法提取单个核细胞,采用差速贴壁法分离骨髓间充质干细胞。原代骨髓间充质干细胞培养48 h后,收集未贴壁细胞,加入含胎牛血清、血管内皮生长因子、成纤维细胞生长因子、胰岛素样生长因子1、重组人上皮生长因子的EGM-2完全培养液诱导骨髓间充质干细胞向内皮祖细胞分化。设立3组：分别为早期贴壁且普通培养的内皮祖细胞、2次贴壁且诱导培养的内皮祖细胞、大鼠成熟主动脉内皮细胞,采用硝酸还原酶法间接测量细胞培养液中一氧化氮的含量。 主要观察指标：骨髓间充质干细胞与内皮祖细胞体外培养情况,细胞培养液中一氧化氮的含量。 结果：原代培养的骨髓间充质干细胞呈梭形,24 h内大部分细胞贴壁,9~10 d可达90%融合,纯化扩增后骨髓间充质干细胞呈均匀一致的长梭性,传代周期为8 d左右,流式细胞仪检测第3代骨髓间充质干细胞CD34,CD45呈阴性,CD105呈阳性。2次贴壁的内皮祖细胞培养3 d内贴壁,6 d形成集落,8~10 d出现条索状结构,呈微血管样生长,2周时大部分细胞呈多角形,细胞集落相互连接,呈典型的“铺路石”样,7~10 d细胞DiI-acLDL、FITC-UEA-1双染呈阳性,流式细胞仪检测Flk-1,CD133阳性。2次贴壁且诱导培养的内皮祖细胞培养液中一氧化氮含量明显高于早期贴壁且普通培养的内皮祖细胞(P < 0.05),但低于大鼠成熟主动脉内皮细胞(P < 0.05)。 结论：差速贴壁法能有效地分离、纯化、扩增大鼠骨髓间充质干细胞,加入多种细胞生长因子诱导后具有较强地向内皮细胞方向分化的能力,且2次贴壁细胞已具有部分内皮细胞的功能。

Isolation, culture and identification of rat bone marrow mesenchymal stem cells and endothelial progenitor cells

BACKGROUND: There are no unified methods to isolate, culture and amplify bone marrow mesenchymal stem cells (BMSCs). Endothelial progenitor cells (EPCs) can be harvested by inducing peripheral blood or bone marrow mononuclear cells. However, the method of collecting bone marrow mononuclear cells is various, and the isolation efficiency is different. OBJECTIVE: To isolate and identify BMSCs and EPCs in vitro. DESIGN, TIME AND SETTING: The cytological in vitro study was performed at the Central Laboratory, First Affiliated Hospital, Anhui Medical University from October 2008 to February 2009. MATERIALS: Two clean healthy male Sprague Dawley rats were supplied by the Anhui Animal Center. METHODS: Rat bone marrow was extracted to harvest mononuclear cells by density gradient centrifugation. BMSCs were isolated by the differential adhesion. Following 48 hours of primary culture of BMSCs, non-adhered cells were collected and induced by EGM-2 complete medium supplemented with fetal bovine serum, vascular endothelial growth factor, fibroblast growth factor, insulin-like growth factor-1 and recombinant human epidermal growth factor. There were three groups: early adherence + common culture EPCs, twice adherence + induction culture EPCs, and rat mature aorta EPCs. Nitric oxide content was directly measured in the medium using nitrate reductase method. MAIN OUTCOME MEASURES: The following parameters were measured: in vitro culture of BMSCs and EPCs, and nitric oxide content in the medium. RESULTS: Primary culture of BMSCs was in a spindle-shape, within 24 hours the majority of cells were adherent, 9-10 days up to 90% cells were confluent. Purified BMSCs were amplified and uniform, long spindle, with the passage cycle for 8 days. Flow cytometry detection showed that cells were CD34-, CD45-negative and CD105-positive. Second adherent of EPCs induced by 3 days culture showed colony formation following six days, appeared cord-like structure and microvessel-like growth at 8-10 days. At 2 weeks, the majority of cells were polygonal, colony-forming unit was interconnected, showing a typical "cobblestone"-like. At 7-10 days, cells were double stained DiI-acLDL and FITC-UEA-1. Flow cytometry showed Flk-1-positive and CD133-positive. Nitric oxide content was significantly higher in the culture medium compared with the ordinary cultured cells (P < 0.05), but lower than mature aorta endothelial cells (P < 0.05). CONCLUSION: Differential adhesion can effectively isolate, purify and amplify rat BMSCs, which has strong ability to differentiate into EPCs following treatment of multiple cell growth factors. Second adherent of cells has partial function of EPCs.