大鼠骨髓间充质干细胞的分离与培养方法优化

摘要

目的：建立一个分离、培养、纯化大鼠的骨髓间充质干细胞的方法。方法使用贴壁法分离大鼠的骨髓间充质干细胞，通过培养初期的频繁换液和传代过程中减少胰蛋白酶的暴露时间，达到骨髓间充质干细胞的纯化。流式细胞仪检测细胞表面标志物，MTT法检测细胞生长状况，暴露于不同的诱导培养基使细胞成骨、成脂，并向肝胆系分化。结果分离所得的细胞阳性表达CD29、CD44、CD90，而对于造血细胞标志物CD45呈阴性。在成骨、成脂培养基的诱导下，细胞ALP、茜素红、油红O染色均呈阳性，并发现传至10代的细胞仍保持分化功能。将骨髓间充质干细胞贯续暴露于不同的细胞因子和激素，得到了表达肝细胞、胆管上皮细胞特异标志的子代细胞。结论通过改良后的方法可以高效、简洁地分离出形态均一的大鼠骨髓间充质干细胞，并能够向多胚层的细胞分化。%Objective To optimize the protocols for isolation and culture of mesenchymal stem cells from rat bone marrow (BMSCs). Methods BMSCs were isolated by adherence to plastic with frequent medium change and reduced trypsinization time. The cell growth curves were drawn and the surface markers of BMSCs were detected by flow cytometry. The cells were induced to differentiate into osteogenic, adipogenic, hepatic and cholic lineages. Results The cells isolated using this method were positive for CD29, CD44, and CD90 and negative for the hematopoietic surface markers CD45. The osteogenic and adipogenic differentiation of the BMSCs was verified by alkaline phosphatase staining, Alizarin red staining and Oil red staining. The cell subcultures up to passage 10 maintained capacities of differentiation into osteogenic and adipogenic lineages. The BMSCs induced with sequential addition of growth factors, cytokines and hormones differentiated into cells expressing hepatocyte-and cholangiocyte-specific markers. Conclusion The optimized method allows efficient isolation of homogenous populations of MSCs from rat bone marrow, which can be induced into multiple cell lineages.