无翅型MMTV整合位点家族成员3在大鼠牙囊及牙囊细胞成骨分化中的表达

Expression of wingless-type MMTV integration site family member 3 in rat dental follicle tissues andcells undergoing osteogenic differentiation

目的研究无翅型MMTV整合位点家族成员3 （wingless-type MMTV integration site family, member 3, Wnt3）在大鼠体内外牙囊中的表达，检测成骨诱导后Wnt3在大鼠牙囊细胞中的表达变化，探讨Wnt3在牙囊成骨分化中的作用。方法取出生后1、3、5、7、9、11、13d的SD仔鼠各1只，引颈处死，切取下颌骨，取出生后各时间点的组织切片各3张作为实验组，阴性对照组以磷酸盐缓冲液代替一抗，滴加在出生后11d的组织切片上，其余处理同实验组。免疫组化检测Wnt3在大鼠体内牙囊中的表达。间接免疫荧光检测Wnt3在牙囊细胞内的表达和分布。茜素红染色检测成骨诱导后矿化结节的形成。蛋白质印迹法检测成骨诱导1、2、3周后牙囊细胞中Wnt3和p-联蛋白（3-eatenin）表达的变化。结果Wnt3在出生后第1、3天的大鼠牙囊组织内无明显表达，第5天开始出现阳性表达，并持续表达至第13天。免疫荧光检测显示Wnt3在牙囊细胞胞质中表达。成骨诱导后牙囊细胞分化为成骨细胞，形成矿化结节，茜素红染色阳性。成骨诱导第1周Wnt3蛋白表达（2．60±0．04）较阴性对照组（1．00±0．00）显著增加（P〈0．05），并随着诱导时间的延长Wnt3表达逐渐减少，至第3周Wnt3在成骨诱导组表达水平（1．00±0．05）与阴性对照组基本相等，差异无统计学意义（P〉0．05）。β-联蛋白在成骨诱导1、2、3周后表达增加（分别为1．95±0．05、9．77±0．65、1．75±0．21），与阴性对照组（1．00±0．00）相比差异均有统计学意义（P〈0．05），并在第2周达峰值（9．77±0．65），后逐渐降低。结论Wnt3在体内外大鼠牙囊中表达，成骨诱导早期牙囊细胞中Wnt3表达明显增加，提示Wnt3可能参与牙囊细胞的早期成骨向分化；Wnt3和β-联蛋白在成骨诱导过程中表达水平的差异提示Wnt3可能与其他因子或信号通路成分相互作用，调控β-联蛋白的表达，参与牙囊细胞的成骨向分化。

Objective To investigate the expression of wingless-type MMTV integration site family, member 3 （Wnt3） in rat dental follicles and its protein level in dental follicle cells （DFC） undergoing osteogenic induction and to discuss the effects of Wnt3 on the differentiation of DFC. Methods Rats at postnatal days 1, 3, 5, 7, 9, 11 and 13 were executed, then the mandibles were immediately removed and immunohistochemistry was performed to detect the expression of Wnt3 in dental follicles of postnatal rats. The expression and distribution of Wnt3 in DFC were determined by immunofluorescence. Alizarin red-S staining was performed to assess the mineralization of DFC. Western blotting was used to evaluate Wnt3 and ±-catenin protein levels after stimulated by osteogenic medium for 1, 2 and 3 weeks, respectively. Results Immunohistochemistry revealed that the expression of Wnt3 in rat dental follicles began at day 5 and sustained to day 13. On day 1 and 3, the expression of Wnt3 in dental follicles was negative. Wnt3 was expressed in the cytoplasm of DFC. Alizarin red-S staining indicated that the osteogenic medium stimulated the differentiation of DFC into osteoblastic lineage. Western blotting demonstrated that the Wnt3 protein levelswere significantly up-regulated after stimulated with osteogenic medium for 1 weeks compared with the control （2. 60 ±0. 04 vs. 1. 00 ±0. 00, P 〈0.05）. Then the levels of Wnt3 protein were declined, and at the 3rd week, no significant difference was observed between osteo-induced group and the control （1.00 ± 0. 05 vs. 1. 00 ±_ 0. 00, P 〉 0. 05）. The levels of β-catenin were increased in osteo-induced groups compared with the control（ 1.95 ± 0.05 vs. 1.00 ± 0.00, P 〈 0.05 ;9. 77±0. 65 vs. 1.00 ± 0. 00, P 〈 0. 05 ; 1.75 ± 0. 21 vs. 1.00 ± 0. 00, P 〈 . 05 ）. Furthermore, the expression of β-catenin reached to a peak on the 2nd week （ 9. 77 ± 0. 65 ）, and then declined. Conclusions Wnt3 was expressed in the rat dental follicles both in vivo and in vitro and up-regulated during early phase of osteoblast differentiation in DFC. Wnt3 may be involved in early phase of osteoblast differentiation.