大鼠骨髓间充质干细胞原代培养及成骨成脂分化能力染色鉴定

摘要

目的：实验旨在建立一套简便有效的骨髓间充质干细胞原代培养、诱导分化及染色方法，观测骨髓间充质干细胞生物学特性，及其成骨、成脂分化潜能，为后续进行骨髓间充质干细胞基因修饰实验做好准备。方法本实验通过全骨髓培养法分离骨髓间充质干细胞，并用诱导分化培养液做定向诱导培养，观察骨髓间充质干细胞向成骨及成脂肪细胞分化过程中的细胞形态学变化，进行成骨成脂细胞染色鉴定，以探讨骨髓间充质干细胞的分离培养方法、生长规律及向成骨成脂细胞分化的条件。结果(1)通过全贴壁法成功进行了骨髓间充质干细胞原代及传代培养并绘制出第4代细胞生长曲线；(2)通过茜素红染色验证了骨髓间充质干细胞的成骨分化能力；(3)通过油红O染色验证骨髓间充质干细胞的成脂分化能力。结论全贴壁法提取的大鼠骨髓间充质干细胞经传代4次左右可达到一定纯度，在一定诱导条件下，经特定染色方法鉴定，可分化为成骨细胞，成脂细胞。%Objective Aim to build a simple and effective method of primary cell culture induced differentiation and staining of bone marrow mesenchymal stem cells(BMSC) and observe the biological characteristics and potential capacity of BMSC to differentiate to osteoblasts and adipocytes, thus we can make ready for the gene modiifcation of BMSC in the future.Methods BMSC was separated by whole bone marrow culture and induced by differentiation induced solution. To investigate the methods of separation, culture, the growth law, the conditions of osteoblasts and adipocytes differentiation by observing the form change of BMSC, staining identiifcation of osteoblasts and adipocytes during differentiation.Results (1) BMSC was successfully separated, cultured and the growth curve of BMSC of the 4th generation was drew, (2) Identiifed the osteogenic potential by alizarin red staining, (3) Identiifed the osteogenic potential by Oil red O staining.Conclusion The purity of BMSC separated by whole bone marrow culture can reached a certain level after subcultured for 4 times and the BMSC can differentiate to osteoblasts and adipocytes under special conditions which can be identiifed by certain stainings.