Zmpste24^(-/-)小鼠骨髓间充质干细胞的增殖凋亡及其TRP通道的表达研究

Proliferation / apoptosis and TRP channele xpression of BMMSCs of Zmpste24^(-/-)mice

目的:探讨Zmpste24^(-/-)小鼠骨髓间充质干细胞(BMMSCs)的增殖凋亡情况及瞬时性受体电位通道(TRP通道)表达水平与野生型(WT)小鼠的区别。方法:体外分离培养4月龄Zmpste24^(-/-)和WT小鼠的BMMSCs,经干细胞表面分子鉴定后,分别采用β-半乳糖苷酶试剂盒检测两种小鼠细胞的衰老情况;流式细胞仪检测小鼠细胞的增殖、凋亡情况;实时定量RT-PCR检测两种小鼠细胞的TRP通道表达水平;激光共聚焦显微镜检测两种小鼠的细胞内Ca2+浓度。结果:Zmpste24^(-/-)和WT小鼠的BMMSCs均阳性表达Sca-1,阴性表达CD34、CD45。Zmpste24^(-/-)小鼠BMMSCs的衰老细胞数目和凋亡水平明显高于WT小鼠(P<0.05),而其增殖能力则明显低于WT小鼠(P<0.05);TRPC1、TRPM4、TRPM8、TRPV1、TRPV2、TRPV3、TRPV5通道的表达水平均明显高于WT小鼠(P<0.05);胞内Ca^(2+)浓度明显高于WT小鼠(P<0.05)。结论:Zmpste24^(-/-)小鼠BMMSCs的增殖率降低、凋亡率升高可能与其TRP通道高表达、胞内钙离子浓度升高有关。

AIM： To investigate the difference of the proliferation, apoptosis and TRP channel expression of bone marrow mecenchymal stem cells （BMMSCs） between Zmpste24-/- and wild type（WT） mice. METHODS： BMMSCs were isolated and cultured from 4 - month - old Zmpste24-/- and WT mice respectively. BMMSCs were identified with MSC-related monoclonal antibodies by flow cytometry. The senium level of the ceils was determined by SA-13-gal staining kit. Flow cytometry was performed to examine the proliferation and apoptosis of the ceells. TRP channel expression was tested by Real - Time RT-PCR and intracellular Ca2 ＋ i was measured by laser scanning confocal microscope. RESULTS：Positive expression of Sca - 1 and negative expression of CD34 and CIM5 were identified in BMMSCs of Zmpste24-/- and WT mice. The aged ceils of Zmpste24-/- mice were more than those of WT （ P 〈 0.05 ）. The BMMSCs of Zmpste24 -/- mice showed reduced proliferation ability （ P 〈 0.05 ） and increased apop-tosis （P 〈0.05） than those of WT. The BMMSCs of Zmpste24-/- mice exhibited an increase of TRPC1, TRPM4, TRPM8, TRPV1, TRPV2, TRPV3 and TRPV5 channel＇s expression （ P 〈 0.05） and Ca2 ＋ i （P 〈 0.05 ） as compared with those of WT. CONCLUSION： The higher expression level of TRP channels and the elevated intracellular Ca2 ＋ i may play an important role in the decreased proliferation ability and increased apoptosis of BMMSCs of Zmpste24-/- mice.