人脐血造血干/祖细胞体外定向诱导分化过程中端粒酶的表达

探讨脐血造血干/祖细胞体外诱导分化过程中端粒酶的表达,为造血干细胞产品的临床应用提供一个监测细胞增殖潜能和安全性的指标。用源自人脐血的CD34~+细胞及不同细胞因子组合(SCF+IL-3+IL-6+G-CSF,SCF+IL-3+IL-6+EPO)在体外进行诱导分化;用TRAP聚丙烯酰胺凝胶电泳法、TRAP-ELISA法检测CD34~+细胞及诱导分化细胞的端粒酶活性;用Western印迹法在蛋白水平检测端粒酶催化亚基hTERT的表达,用RT-PCR在细胞转录水平检测端粒酶催化亚基hTERT mRNA的表达。结果显示,在体外诱导分化14-21天为细胞生长峰值,细胞总数可增加(1006.4±103.2)倍,随着培养时间的延长,细胞数量不再增加。CD34~+细胞低度表达端粒酶活性和hTERT基因,在细胞诱导分化过程中端粒酶活性及hTERT表达逐渐升高,细胞诱导14天后端粒酶活性及hTERT表达下降,28天端粒酶活性及hTERT基因检测不出。结论:利用CD34~+细胞在体外定向诱导分化出的大量细胞,不具有无限增殖的特性,可安全地应用于临床,且利用端粒酶的检测可为临床应用诱导分化细胞的时机提供依据。

Expression of Telomerase during Induction of Committed Differentiation of Human Cord Blood Hematopoietic Stem/Progenitor Cells In Vitro

To investigate the expression of telomerase in cord blood hematopoietic stem/progenitor cells during their committed differentiation in vitro and provide an index of monitoring the proliferating potential of the hematopoietic stem/progenitor cells and security for clinical application.Human CD34-positive cells were isolated from umbilical cord blood by using magnetic cell sorting system (MACS),and were induced to differentiation with hematopoietic growth factors (SCF+ IL-3 + IL-6 + G-CSF and SCF + IL-3 + IL-6 + EPO) in a liquid culture system.The telomerase activity and the cytalytic subunit of telomerase (hTERT) of the cells were analysed during different periods of culture by using TRAP-PCR,TRAP-ELISA,Western blot and RT-PCR techniques ,respectively.The results showed that peak of cell growth was achieved on day 14-21 during induction of differentiation in vitro.Total cell number could increase 1006.4 + 103.2 times and could not increase there after.Telomerase activity and hTERT expression were low in freshly isolated cord blood CD34 + cells and increased after about 7 days of culture in addition of cytokine combinations of SCF + IL-3 + IL-6 + G-CSF and SCF+ IL-3 + IL-6 + EPO,respectively.The telomerase activity and hTERT decreased after 14 days of culture and were not dectected after 28 days of culture.It was concluded that the hematopoietic stem/progenitor cells can be expanded in large number in vitro and do not have the character of immortality and the telomerase activity could be a useful index in hematopoiesis regulation.