人骨髓间充质干细胞分离培养及生物学特性研究

Study of human bone marrow mesenchymal stem cells separation and its biological characteristic

目的探讨体外培养骨髓间充质干细胞的一些生物学特性,为利用MSCs进行疾病的细胞治疗提供实验基础。方法密度梯度离心,贴壁培养和消化时间控制相结合,分离纯化人骨髓间充质干细胞,并用马血清诱导分化为脂肪细胞。结果分离获得了高纯度贴壁生长的MSCs。MSCs原代培养呈均匀分布的集落样生长,呈梭形,细胞传代稳定,在体外连续传代培养9代,未发生形态学改变,无衰老征象;传代培养MSCs(P3)在20%马血清的L-DMEM培养液中分化为脂肪细胞。结论采用Percoll细胞分离液密度梯度离心,贴壁培养和消化时间控制相结合,可以获得纯度较高和活性骨髓MSC,是简便有效使用可行的方法;骨髓MSCs体外增殖和传代能力强,通过离体培养可使体内环境下低丰度的MSCs实现数量扩增;

[Objective ] To study on human bone marrow MSCs isolating, purified, amplified method and biological character in vitro. [Method] Isolated and purified MSCs from bone marrow of human rib by density gradient centrifugation and adhering to the culture plastic. After successive subculture and amplification, the growth curve were drawn, the morphology were observed under microscope. Passage 3 were induced in 20% horse serum L- DMEM, observed MSCs morphological change and differentiated character. [Result] The isolated MSCs were mononuclear cells in bone marrow, their living behaviors was quite stable in L-DMEM containing 10% FBS and the growth curves of passage 1, 3, 5 were quite similar, exhibiting a large expensive potential and the typical fibroblastlike morphology. Passage 3 differentiated to adipocyte in L-DMEM containing 20% horse serum, Red O staining result was positive. [Conclusion] A method for isolation and purification in vitro of MSCs from bone marroe has been established. MSCs are easy to be isolated, purified and amplified in vitro.