【摘要】 目的观察成体大鼠骨髓间充质干细胞（MSC）体外培养过程中生物学特性的改变,为骨髓间充质干细胞的应用研究提供实验依据。方法成体大鼠骨髓 MSC 的分离培养,流式细胞仪观察 MSC 免疫表型及细胞周期,检测细胞的诱导分化能力以及细胞的生长曲线,TRAP-ELISA 方法检测其端粒酶活性。结果体外培养的成体大鼠 MSC,随着传代次数的增加,长梭形细胞的体积逐渐增大。第4代时免疫表型阳性细胞率分别为 CD29:（94.75±3.68）%,CD71:（95.43±2.23）%,CD90:（98.08±3.88）%;当传到第7代时,阳性细胞率仅为 CD29:（50.00±3.35）%,CD71:（50.70±2.43）%,CD90:（48.60±2.83）%;第9代时 MSC 检测不到任何阳性免疫表型。前5代 MSC 增殖较快,第3代时处于 S 期和 G₂/M 期的细胞比例为（38.36±2.01）%,处于 G₀/G₁期细胞为（61.64±2.13）%;第7代以后细胞增殖能力明显降低,第12代时处于 S 期和 G₂/M 期的细胞为（10.83±1.63）%,而 G₀/G₁期的细胞为（89.17±1.96）%,此时 MSC 已经基本停止增长。当体外培养的 MSC传到第9代以后,在成骨和成脂肪诱导体系作用下,细胞丧失了分化为 Von Kossa 法染色和油红 O 染色阳性细胞的能力;同时其端粒酶活性也随着传代次数的增多由最初的（52.7±0.78）%逐渐降低为阴性。结论体外培养的成体大鼠骨髓 MSC 随着传代次数的增加,其生物学特性发生明显改变。更多还原

【Abstract】 Objective To investigate biological characteristics of rat bone marrow mesenchymal stem cells（MSC）cultured in vitro and to explore their potential applications.Methods MSC were isolated from rat bone marrow by density gradient centrifugation and were induced to differentiation.Flow cytometry was used to characterize their surface antigen expression,cell cycle status and cell growth parameters. Telomerase activity was determined by TRAP-ELISA assay.Results Fusiform MSC became larger and flattener with increasing passages of culture.After the fourth passage,the MSC showed an immunophenotype of CD29（94.75%±3.68%）,CD71（95.43%±2.23%）,and CD90（98.08%±3.88%）.After the seventh passage,MSC with such immunophenotype decreased with CD29:50.00%±3.35%,CD71: 50.70%±2.43%,and CD90:48.60%±2.83%.Cells with such immunoprofile completely disappeared after passage 9.Overall,MSC grew faster during the first 5 passages.The number of MSC in S and G₂/M phases were 38.36%±2.01% and those in G₀/G₁ phase were 61.64%±2.13% after 3 passages.The cell growth decreased after passage 7.Percentage of MSCs in S and G₂/M phases was 10.83%±1.63% and that in G₀/G₁was 89.17%±1.96% after passage 12,after which the cells failed to further divide.After passage 9,MSCs lost their ability to differentiate to Von Kossa and oil red 0 positive staining cells.In addition,telomerase activity of MSC also gradually decreased with the prolonged passages,from the original 52.7%±0.78% to no telomerase activity.Conclusion The biological and immunophenotypical characteristics of cultured MSC showed obvious alterations with increasing numbers of passage of culture.更多还原