中华口腔医学杂志
中華口腔醫學雜誌
중화구강의학잡지
Chinese Journal of Stomatology
2013年
7期
423-428
,共6页
刘燕%杜宇%凌均棨%项露赛
劉燕%杜宇%凌均棨%項露賽
류연%두우%릉균계%항로새
WNT蛋白质类%牙囊%β-联蛋白%成骨分化
WNT蛋白質類%牙囊%β-聯蛋白%成骨分化
WNT단백질류%아낭%β-련단백%성골분화
Wnt proteins%Dental Sac%β-catenin%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、13 d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β-联蛋白(β-catenin)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.
目的 研究無翅型MMTV整閤位點傢族成員3(wingless-type MMTV integration site family,member 3,Wnt3)在大鼠體內外牙囊中的錶達,檢測成骨誘導後Wnt3在大鼠牙囊細胞中的錶達變化,探討Wnt3在牙囊成骨分化中的作用.方法 取齣生後1、3、5、7、9、11、13 d的SD仔鼠各1隻,引頸處死,切取下頜骨,取齣生後各時間點的組織切片各3張作為實驗組,陰性對照組以燐痠鹽緩遲液代替一抗,滴加在齣生後11d的組織切片上,其餘處理同實驗組.免疫組化檢測Wnt3在大鼠體內牙囊中的錶達.間接免疫熒光檢測Wnt3在牙囊細胞內的錶達和分佈.茜素紅染色檢測成骨誘導後礦化結節的形成.蛋白質印跡法檢測成骨誘導1、2、3週後牙囊細胞中Wnt3和β-聯蛋白(β-catenin)錶達的變化.結果 Wnt3在齣生後第1、3天的大鼠牙囊組織內無明顯錶達,第5天開始齣現暘性錶達,併持續錶達至第13天.免疫熒光檢測顯示Wnt3在牙囊細胞胞質中錶達.成骨誘導後牙囊細胞分化為成骨細胞,形成礦化結節,茜素紅染色暘性.成骨誘導第1週Wnt3蛋白錶達(2.60±0.04)較陰性對照組(1.00±0.00)顯著增加(P＜0.05),併隨著誘導時間的延長Wnt3錶達逐漸減少,至第3週Wnt3在成骨誘導組錶達水平(1.00±0.05)與陰性對照組基本相等,差異無統計學意義(P＞0.05).β-聯蛋白在成骨誘導1、2、3週後錶達增加(分彆為1.95±0.05、9.77 ±0.65、1.75±0.21),與陰性對照組(1.00 ±0.00)相比差異均有統計學意義(P＜0.05),併在第2週達峰值(9.77±0.65),後逐漸降低.結論 Wnt3在體內外大鼠牙囊中錶達,成骨誘導早期牙囊細胞中Wnt3錶達明顯增加,提示Wnt3可能參與牙囊細胞的早期成骨嚮分化；Wnt3和β-聯蛋白在成骨誘導過程中錶達水平的差異提示Wnt3可能與其他因子或信號通路成分相互作用,調控β-聯蛋白的錶達,參與牙囊細胞的成骨嚮分化.
목적 연구무시형MMTV정합위점가족성원3(wingless-type MMTV integration site family,member 3,Wnt3)재대서체내외아낭중적표체,검측성골유도후Wnt3재대서아낭세포중적표체변화,탐토Wnt3재아낭성골분화중적작용.방법 취출생후1、3、5、7、9、11、13 d적SD자서각1지,인경처사,절취하합골,취출생후각시간점적조직절편각3장작위실험조,음성대조조이린산염완충액대체일항,적가재출생후11d적조직절편상,기여처리동실험조.면역조화검측Wnt3재대서체내아낭중적표체.간접면역형광검측Wnt3재아낭세포내적표체화분포.천소홍염색검측성골유도후광화결절적형성.단백질인적법검측성골유도1、2、3주후아낭세포중Wnt3화β-련단백(β-catenin)표체적변화.결과 Wnt3재출생후제1、3천적대서아낭조직내무명현표체,제5천개시출현양성표체,병지속표체지제13천.면역형광검측현시Wnt3재아낭세포포질중표체.성골유도후아낭세포분화위성골세포,형성광화결절,천소홍염색양성.성골유도제1주Wnt3단백표체(2.60±0.04)교음성대조조(1.00±0.00)현저증가(P＜0.05),병수착유도시간적연장Wnt3표체축점감소,지제3주Wnt3재성골유도조표체수평(1.00±0.05)여음성대조조기본상등,차이무통계학의의(P＞0.05).β-련단백재성골유도1、2、3주후표체증가(분별위1.95±0.05、9.77 ±0.65、1.75±0.21),여음성대조조(1.00 ±0.00)상비차이균유통계학의의(P＜0.05),병재제2주체봉치(9.77±0.65),후축점강저.결론 Wnt3재체내외대서아낭중표체,성골유도조기아낭세포중Wnt3표체명현증가,제시Wnt3가능삼여아낭세포적조기성골향분화；Wnt3화β-련단백재성골유도과정중표체수평적차이제시Wnt3가능여기타인자혹신호통로성분상호작용,조공β-련단백적표체,삼여아낭세포적성골향분화.
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 β-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 levels were 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 ＜ 0.05).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.
