人骨髓间质干细胞中的端粒调控机制

目的：〖HT5"SS〗研究人骨髓来源间充质干细胞(MSCs)端粒长度的调控机制。方法：以贴壁培养法从人骨髓中分离MSCs并用MSCs及造血干细胞相关表面抗体作表型鉴定,用Southern blotting检测MSCs的端粒长度;应用免疫荧光染色技术检测端粒重复序列结合因子1（TRF1）和早幼粒细胞白血病蛋白小体（PML）的定位;以端粒重复序列扩增法（TRAP）和/或Western blotting法检测传代及分化成脂肪细胞的MSCs和经同步化处理被阻断在S期的MSCs的端粒酶表达。结果：与端粒酶阴性ALT细胞株WI-38-2RA细胞相比,MSCs的端粒长度较短并且端粒长度变异度不大;端粒调控相关蛋白TRF1和PML在MSCs中的定位则与端粒酶阳性细胞HeLa细胞相同,两者呈非共定位,而在端粒酶阴性WI-38-2RA细胞中两者呈共定位状态。MSCs中不存在有染色体外端粒重复序列DNA（ECTR DNA）。TRAP法检测传代培养的MSCs端粒酶呈阴性表达,但分化成脂肪的MSCs端粒酶呈阳性表达。Western blotting 检测同步化处理前MSCs端粒酶呈微弱表达,经同步化处理被阻断在S期时,MSCs的端粒酶表达明显增高,并且与S期的细胞比例呈正相关。结论：MSCs中不存在ALT相关的早幼粒细胞白血病蛋白小体(APBs)、染色体外端粒重复序列DNA(ECTR DNA)和端粒长度较长、端粒长度变异度大等ALT机制相关分子特征;非同步化在S期处理的MSCs,端粒酶呈微弱表达,但诱导向脂肪细胞分化或处在S期时,MSCs的端粒酶表达明显增高,并且与S期的细胞比例呈正相关。本研究提示MSCs是通过端粒酶机制而不是端粒延长旁路途径（ALT）机制调控其端粒末端。

Mechanism of telomere mainteance in human bone marrow mesenchymal stem cells

AIM:To study the telomere maintenance mechanism in mesenchymal stem calls(MSCs).METHODS:MSCs were isolated from healthy human bone marrow by their adherence to plastic and then were checked with CD14-FITC,CD45-FITC,CD44-FITC,HLA-DR-FITC,CD34-PE,CD29-PE and CD166-PE.Telomere length and ECTR DNA in MSCs were detected by Southern blotting.The localization of TRF1 and promyelocytic leukemia(PML)in MSCs were detected with immunofluorescence staining.TRAP protocol was performed to detect the telomerase activity in MSCs and MSCs-derived adipocytes.Western blotting and TRAP protocol were applied to measure telomerase activity of MSCs,which were synchronized by serum starvation and aphidicolin treatment.RESULTS:The telomere in length seemed shorter and relatively more homogeneous in MSCs and HeLa cells than that in WI-38-2RA cells.TRF1 did not concide with PML nuclear body in MSCs and HeLa cells while it exclusively did in WI-38-2RA cells.ECTR DNA was negative in MSCs and HeLa cells but positive in WI-38-2RA cells.Telomerase was negative in MSCs but it was positive in MSCs-derived adipocytes detected by TRAP.Moreover,a cell cycle-dependent expression profile of telomerase was found in MSCs when they were synchronized by serum starvation and aphidicolin treatment.Untreated MSCs expressed very low level of telomerase probed by Western blotting with 2C4 mAb,but the telomerase level had significantly increased when these cells were trapped in S phase.CONCLUSION:The telomere of MSCs is maintained by telomerase pathway instead of alternative lengthing of telomere(ALT)and the level of telomerase expression is associated with cell cycle stage.