Wnt3a蛋白影响矿化液诱导牙髓干细胞分化能力的研究  

Study on the influence of Wnt3a on osteogenetic differentiation ability of dental pulp stem cells induced by mineralizing medium

作　　者：孙艳艳[1,2] 袁梦桐[1] 史欣[1] 刘明月[1] 胡伟平[1] 

机构地区：[1]哈尔滨医科大学附属第二医院口腔修复科,150086 [2]徐州市口腔医院口腔修复科,江苏221002

出　　处：《中华口腔医学杂志》2016年第10期634-639,共6页Chinese Journal of Stomatology

基　　金：黑龙江省自然科学基金（H2013101）

摘　　要：目的：研究Wnt3a蛋白对牙髓干细胞（dental pulp stem cells，DPSC）增殖及成骨向分化的影响。方法对培养液和矿化液分别加入不同质量浓度的Wnt3a蛋白（0、5、20、50、100μg/L）作用于DPSC，应用甲基噻唑基四唑法于不同作用时间（1、3、5、7 d）检测DPSC的增殖情况及碱性磷酸酶活性；茜素红染色检测矿化结节形成情况；通过实时荧光定量PCR（real-time quantitative PCR，RT-PCR）法检测骨涎蛋白、Ⅰ型胶原、Runt相关转录因子2（Runt-related transcription factor 2，RUNX-2）及骨钙蛋白4种骨源性基因的表达情况。结果5μg/L的Wnt3a蛋白对DPSC的促增殖作用最强；培养7 d后含有5μg/L Wnt3a蛋白的矿化液组碱性磷酸酶活性（0.47±0.04）显著强于其他组（矿化液组：0.39±0.05；20μg/L组：0.34±0.03；50μg/L组：0.27±0.07；100μg/L组：0.20±0.03）；28 d后茜素红染色结果表明该组促进矿化作用最强，产生的矿化结节大小及数量明显多于其他组。7 d后，5μg/L Wnt3a矿化液组骨涎蛋白和Ⅰ型胶原基因阳性表达水平（分别为154.10±23.57和13.74±3.80）均显著高于矿化液组（分别为16.22±6.08和1.38±0.27，P＆lt;0.05）。结论适宜浓度的Wnt3a蛋白可以促进DPSC增殖；Wnt3a蛋白与矿化液联合作用于DPSC可以促进其成骨向分化。Objective To investigate the effects of Wnt3a protein on proliferation and osteogenic differentiation of human dental pulp stem cells（DPSC）. Methods Intact human permanent teeth extracted for orthodontic reasons were collected and used as study models. The biological effects of Wnt3a on DPSC were investigated using methyl thiazolyl tetrazolium（MTT）, alkaline phosphatase（ALP） activity assay, alizarin red S staining and realtime fluorescence quantitative PCR. Osteogenic-related gene expression of induced DPSC was examinedby using tests of bone sialoprotein（BSP）, osteocalcin（OCN）, collagen typeⅠ（COL-Ⅰ） and Runt-related transcription factor 2（RUNX-2）. Results Wnt3a proteininduced an increase of cell growth and treatment of DPSC with Wnt3a induced a highest increase in cell growth at the concentration of 5μg/L. 5 μg/L Wnt3a proteins combined with the osteogenic medium treatment caused up-regulated osteogenic differentiation, ALP activity and express of osteogenic-related genes of DPSC, and the ALP activity（0.47 ± 0.04） was significantly stronger than the other groups（osteogenic medium： 0.39 ± 0.05;20μg/L：0.34 ± 0.03;50 μg/L： 0.27 ± 0.07; 100 μg/L： 0.20 ± 0.03）. Conclusions Exogenous Wnt3a protein treatment on DPSC could affect the proliferation and osteogenic differentiation.

关 键 词：WNT蛋白质类 细胞增殖 牙髓干细胞 成骨向分化 

分 类 号：R781[医药卫生—口腔医学]