BACKGROUND:Currently, there is no uniform, standardized approach to isolate, purify and proliferate bone marrow mesenchymal stem cells. Chlormethylbenzamido-1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (CM-Dil) is a stable, reliable, high marking and simple marker. OBJECTIVE:To develop the methods for isolation, culture and identification of rat bone marrow mesenchymal stem cells in vitro. METHODS:Two male Sprague-Dawley rats, weighing 50-100 g were taken to col ect the bilateral femur and tibia bone marrow under sterile conditions, and then, primary bone marrow mesenchymal stem cells were isolated and cultured using bone marrow adherent separation and density gradient centrifugation. cells were amplified and purified through timely and repeated passage, and labeled at the third generation with fluorescent dyes CM-Dil in vitro as a source of donor cells. RESULTS AND CONCLUSION:Bone marrow mesenchymal stem cells were cultured successful y in vitro using bone marrow adherent separation and density gradient centrifugation separation methods, but the former was superior to the latter in the number of cultured cells significantly, while the two methods were not different significantly in terms of cellviability and proliferation. Flow cytometry results showed that the positive rates of cultured cells were 17.5%for CD34, 97.9%for CD44, and 91%for CD90. CM-Dil can label bone marrow mesenchymal stem cells successful y, which is a stable, reliable, high marking and simple marker.%背景：目前，对骨髓间充质干细胞的分离、纯化和扩增还没有统一的、标准化的方法。CM-DiI作为荧光标记物稳定、可靠、标记率高、标记简便。 目的：建立SD大鼠骨髓间充质干细胞体外分离培养及标记的方法。 方法：取2只体质量50-100 g雄性SD大鼠，无菌条件下采集双侧股骨、胫骨骨髓，用全骨髓贴壁分离法和密度梯度离心法培养出原代骨髓间充质干细胞，通过及时、反复传代对细胞进行扩增纯化，在体外用荧光活性染料CM-DiI标记第3代骨髓间充质干细胞后作为供体细胞来源。 结果与结论：用全骨髓贴壁分离法和密度梯度离心法两种方法均能成功体外分离培养骨髓间充质干细胞，经流式细胞仪分析，培养出的细胞CD34阳性率为17.5%，CD44阳性率为97.9%、CD90阳性率为91%，与骨髓间充质干细胞表面抗原一致。但培养出的细胞数量全骨髓贴壁分离法明显多于密度梯度离心法，两种方法培养骨髓间充质干细胞的细胞活力和增殖能力无明显差异。CM-DiI能够成功荧光标记骨髓间充质干细胞， CM-DiI作为荧光标记物稳定、可靠、标记率高、标记简便。
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