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目的观察体外培养人骨髓间质干细胞(mesenchymal stem cells,MSCs)成脂分化过程中端粒酶活性的变化规律,并探讨其可能的机制。方法通过贴壁培养法从人骨髓中分离MSCs,利用造血干细胞相关表面抗体对MSCs进行表型鉴定,利用相应诱导分化剂,诱导MSCs向成骨细胞、脂肪细胞和神经元样细胞分化,分析其多向分化潜能。以端粒重复序列扩增法(TRAP)检测MSCs及MSCs诱导成脂分化过程中端粒酶活性;以Western印迹法检测MSCs及MSCs诱导成脂分化过程中端粒酶反转录酶(hTERT)及端粒相关蛋白[端粒重复序列结合因子1(TRF1)、端锚聚合酶1(Tankyrase 1)]的表达水平。结果 TRAP法检测传代培养的MSCs端粒酶呈阴性表达,而MSCs诱导成脂分化过程中,端粒酶活性明显升高,以第7天最高(P<0.05),其后呈下降趋势;Western印迹法检测显示,hTERT在MSCs中有微弱表达,在MSCs诱导成脂分化过程中,hTERT和Tankyrase 1表达水平升高,以第7天最高,此后逐渐下降,而TRF1表达水平保持不变。结论体外培养的MSCs端粒酶呈微弱表达,MSCs在成脂诱导分化过程中,端粒酶活性先升高,其后逐渐下降,而Tankyrase 1可能在其中发挥重要作用。
Objective To observe the changes of telomerase activity during the process of adipogenic differentiation of mesenchymal stem cells (MSCs) cultured in vitro and to explore its possible mechanism. Methods MSCs were isolated from human bone marrow by adherent culture method. MSCs were identified by hematopoietic stem cell-associated surface antibodies. MSCs were induced to differentiate into osteoblasts, adipocytes and neuron-like cells by using corresponding differentiation mediators. Multi-directional differentiation potential. The telomerase activity in MSCs and MSCs induced by adipogenic differentiation was detected by telomeric repeat amplification (TRAP). The expression of telomerase reverse transcriptase (hTERT) in MSCs and MSCs was detected by Western blotting. And telomere-associated protein [telomeric repeat binding factor 1 (TRF1), Tankyrase 1]. Results The TRAP method was used to detect the telomerase activity of MSCs in subcultured cells. However, the telomerase activity of MSCs induced by adipogenic differentiation was significantly increased (P <0.05) on the 7th day, and then decreased. Western blotting showed that hTERT was weakly expressed in MSCs. During the induction of adipogenic differentiation, hTERT and Tankyrase 1 expression increased, reaching the highest level on day 7, then decreased gradually, whereas the expression level of TRF1 remained unchanged. CONCLUSION: Telomerase activity of MSCs cultured in vitro is weakly expressed. During the process of adipogenic differentiation, MSCs first increase in telomerase activity, and then gradually decrease. However, Tankyrase 1 may play an important role in this process.