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目的 :探讨人骨髓间充质干细胞 (human mesenchymal stem cells,h MSCs)端粒酶活性的表达以及深低温冻存和向脂肪细胞诱导分化对基因表达的影响。方法 :联合应用密度梯度离心法和贴壁培养法分离人 h MSCs,传代培养。深低温冻存 h MSCs或诱导其向脂肪细胞分化 ,以 TRAP(Telomerase repeatamplification protocol assay)法分别检测新鲜的不同传代次数的 h MSCs、冻存复苏后培养的 h MSCs和诱导分化为脂肪细胞的 h MSCs的端粒酶活性。结果 :用 TRAP法检测 h MSCs(n=19)端粒酶活性 (RTA)是 (1.4 6± 0 .6 7) % ,分化成脂肪的 h MSCs(n=3)的RTA为 (11.80± 2 .5 2 ) % (P<0 .0 0 1) ;不同传代次数 MSCs端粒酶活性的比较中 ,早期 h MSCs(n=10 )的 RTA为(1.4 6± 0 .83) % ,晚期 h MSCs(n=9)的 RTA为 (1.4 6± 0 .4 7) % (P=0 .99) ;在低温冻存对 h MSCs端粒酶活性的影响中 ,新鲜的 h MSCs(n=13)的 RTA为 (1.4 1± 0 .4 4) % ,低温冻存的 h MSCs(n=6 )的 RTA为 (1.5 7± 1.0 7) %(P=0 .6 4)。结论 :h MSCs的端粒酶呈阴性 ,传代培养和低温冻存不影响基因表达水平。向脂肪细胞分化后 h MSCs端粒酶活性表达水平增高 ,其确切机制尚需进一步研究。
Objective: To investigate the expression of telomerase activity in human mesenchymal stem cells (hMSCs) and the effects of cryopreservation and differentiation into adipocytes on the gene expression. Methods: Human hMSCs were isolated and subcultured by density gradient centrifugation and adherent culture. Cryopreserved hMSCs or induced their differentiation into adipocytes. Fresh hMSCs with different passage numbers were detected by TRAP (telomerase repeatampl protocol protocol assay), h MSCs cultured and cryopreserved and adipocytes differentiated into h Telomerase activity of MSCs. RESULTS: The telomerase activity (RTA) of h MSCs (n = 19) was (1.4 6 ± 0.67)% by TRAP assay and that of hMSCs differentiated into adipocytes (n = 3) was (11.80 ± 2 .5 2)% (P <0.01). In comparison of the telomerase activity of MSCs at different passages, the RTA of early hMSCs (n = 10) was (1.4 6 ± 0.83)%, RTA of MSCs (n = 9) was (1.4 6 ± 0.47)% (P = 0.99). Among the effects of cryopreservation on telomerase activity of h MSCs, fresh hMSCs (n = 13 ) Had RTA of (1.4 1 ± 0. 4 4)%. The RTA of cryopreserved h MSCs (n = 6) was (1.5 7 ± 1.0 7)% (P = 0.64). Conclusion: The telomerase of h MSCs is negative, subculture and cryopreservation do not affect the gene expression level. The expression of telomerase activity in h MSCs differentiated into adipocytes is increased, and its exact mechanism needs further study.