牙髓干细胞向成牙本质细胞诱导分化过程中Oc t4可变剪接体表达改变的研究

摘要

AIM:To examine the expression of Oct4 isoforms during odontoblastic differentiation of human dental pulp stem cells (hDPSCs).METHODS:hDPSCs were cultured in odontogenic induction medium .Expression of Oct4 isoforms (Oct4A and Oct4B),stem cells markers Sox2,Klf4 and DSPP were detected by RT-PCR.The pro-tein location of Oct4A in hDPSC was detected by confocal laser scanning microscope (CLSM).RESULTS:hDPSCs positively expressed CD146,CD105,CD90 and CD29,but negatively expressed CD45 and CD34.Oct4A isoform and Sox2 and Klf4 were downregulated after odontogenic induction (P<0.05),while Oct4B isoform was not detected be-fore and after induction of hDPSCs.TIP1 10 showed the same dynamic changes with Oct4A during odontoblastic differ-entiation.Moreover,Oct4A was translocated from nuclear to cytoplasm after odontogenic induction.CONCLUSION:The stemness of hDPSC are reduced during odontogenic induction.Downregulation and translocation of spliced variant Oct4A indicates that it may play a role in maintaining stemness of hDPSC.%目的：研究多能性转录因子Oct4可变剪接体在人牙髓干细胞（hDPSCs）向成牙本质细胞定向分化过程中的表达改变。方法：矿化液诱导hDPSCs向成牙本质细胞分化，RT－PCR检测未经分化诱导、分化诱导7、14 d时Oct4可变剪接体（Oct4A，Oct4B）以及干性分子Sox2、Klf4和DSPP的表达改变；激光共聚焦显微镜（CLSM）检测Oct4A在分化过程中的表达和定位。结果：hDPSCs高表达CD146、CD105、CD90、CD29；CD45及CD34表达阴性。矿化液诱导细胞分化后牙本质涎磷蛋白（DSPP）的总蛋白和mRNA表达阳性。在hDPSCs未经分化诱导、分化诱导7、14 d时，Oct4A和Sox2、Klf4均有表达，分化诱导后表达量明显降低（P＜0．05），14 d时hDPSCs表达均低于7 d（P＜0．05）；Oct4B在hDPSCs未经分化诱导、分化诱导7、14 d 时表达均为阴性， TIP110与Oct4A在hDPSCs成牙本质分化过程中表达趋势相同。未经分化诱导hDPSCs剪接体Oct4A主要表达于细胞核，而分化诱导21 d后主要表达于细胞质。结论：hDPSCs向成牙本质细胞分化过程中伴随着其干性降低、剪接体Oct4A表达降低和核浆穿梭效应的发生，提示Oct4A在维持hDPSCs干性中发挥着一定作用。