牙髓干细胞向成牙本质细胞诱导分化过程中Oct4可变剪接体表达改变的研究

Expressions of Oct4 isoforms during odontoblastic differentiation of human dental pulp stem cells

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。

AIM： To examine the expression of Oct4 isoforms during odontoblastic differentiation of human dental pulp stem cells（ h DPSCs）. METHODS： h DPSCs were cultured in odontogenic induction medium. Expression of Oct4 isoforms（ Oct4 A and Oct4B）,stem cells markers Sox2,Klf4 and DSPP were detected by RT- PCR. The protein location of Oct4 A in h DPSC was detected by confocal laser scanning microscope（ CLSM）. RESULTS： h DPSCs positively expressed CD146,CD105,CD90 and CD29,but negatively expressed CD45 and CD34. Oct4 A isoform and Sox2 and Klf4 were downregulated after odontogenic induction（ P〈0. 05）,while Oct4 B isoform was not detected before and after induction of h DPSCs. TIP110 showed the same dynamic changes with Oct4 A during odontoblastic differentiation. Moreover,Oct4 A was translocated from nuclear to cytoplasm after odontogenic induction. CONCLUSION：The stemness of h DPSC are reduced during odontogenic induction. Downregulation and translocation of spliced variant Oct4 A indicates that it may play a role in maintaining stemness of h DPSC.