骨髓间充质干细胞体外诱导和端粒酶活性的研究

Study on in vitro induction and telomerase activity of bone marrow mesenchymal stem cell

目的:建立骨髓间充质干细胞(MSCs)体外分离、培养、纯化的方法,探讨其体外诱导条件和端粒酶活性。方法:取正常成人骨髓用含10%小牛血清的LG-DMEM培养液培养、扩增。流式细胞仪行细胞表面抗原检测,细胞化学染色鉴定其生物学特性,在特定培养条件下检测其向成骨和脂肪细胞分化的能力,采用TRAP(Telomerase repeat amplification protocol assay)-银染法测定其端粒酶的活性。结果:分离培养获得的贴壁细胞,碱性磷酸酶染色阳性。流式细胞仪检测CD34、CD45表达阴性,CD29、CD44、CD115、CD166表达阳性。经向成骨和脂肪细胞诱导3周后,可得到成骨和脂肪细胞,经茜素红染色、油红O染色得到证实。TRAP-银染法证实MSCs表达一定的端粒酶活性。结论:体外分离培养的MSCs可以向成骨、脂肪诱导分化,表达一定的端粒酶活性,具有干细胞的生物学特性。

Objective：To establish the method for in vitro isolating, cultivating, puring of bone marrow-derived mesenchymal stem cells （MSCs） and explore their induction condition and telomerase activity. Method： Bone marrow derived MSCs were cultured in Dulbecco＇s Modified Eagle＇s Medium with low glucose containing 10% new bovine serum. FACS was performed to examine the expression of cell surface molecules. Cytochemical staining was used to identify their characteristics. Osteogenic differentiation and adipogenic differentiation were assessed in different medium. TRAP（Telomerase repeat amplification protocol assay）-argentation method was carried out to determine the telomerase activity. Result： MSCs could be isolated and cultured from bone marrow. These MSCs were negative for CD34,CD45, and positive for CD29,CD44,CD115,CD166. After 3 weeks of osteoblast inducing, it was positive for alizarin red staining. And after 3 weeks of adipocytes inducing, the cells displayed accumulation of lipid vacuoles and was vertified by Red Oil O staining. They expressed telomerase activity. Conclusion： MSCs isolated and cultured from adult human bone marrow can be induced into osteoblast and adipocytes, They expressed telomerase activitv. Thev have stem cell biologic feature.