p75神经营养受体在胎鼠颌突外胚间充质干细胞体外矿化过程中表达变化的研究  

Expression of p75 neurotrophin receptor during the mineralization of ectomesenchymal stem cells in vitro

作　　者：鞠迎新 温秀杰[1] 杨琨[1] 李刚- 刘骏宇 刘鲁川[1] 

机构地区：[1] 第三军医大学大坪医院野战外科研究所口腔科,重庆400042 [2]第三军医大学附属新桥医院口腔科,重庆400037

出　　处：《中华口腔医学杂志》2016年第7期426-431,共6页Chinese Journal of Stomatology

基　　金：国家自然科学基金（81271097）

摘　　要：目的 探讨不同胚龄SD大鼠胎鼠颌突外胚间充质干细胞（ectomesenchymal stem cells,EMSC）的体外矿化能力,以及p75神经营养受体（p75 neurotrophin receptor,p75NTR）在矿化过程中的表达变化,揭示p75NTR在牙齿硬组织矿化中的调控作用与机制.方法 分离培养孕12.5 d （E12.5 d）、E15.5 d和E18.5 d的SD大鼠胎鼠颌突EMSC,通过流式检测技术对比各组细胞群的细胞表型;分别对E12.5 d、E15.5 d和E18.5 d的EMSC进行体外矿化诱导,7d后进行碱性磷酸酶（alkaline phosphatase,ALP）活性检测,21d后茜素红染色观察矿化结节形成情况,同时采用实时定量PCR和蛋白质印迹法检测0、7、14和21 d的Runt相关转录因子2（Runt-related transcription factor2,RUNX2）、Ⅰ型胶原、ALP和p75NTR的表达情况.结果 细胞表面特异性标志物CD29、CD146和p75NTR在E12.5 d、E15.5 d和E18.5 d的EMSC中均呈阳性表达,CD45为阴性表达,其中p75NTR在E18.5 d的EMSC中阳性表达率为84.04％,高于E12.5 d（22.53％）和E15.5 d（81.43％）;诱导21 d后茜素红染色显示,E18.5 d的EMSC实验组矿化能力较强,ALP活性变化趋势与茜素红染色结果一致;蛋白质印迹法结果显示：RUNX2、Ⅰ型胶原表达量在E18.5d的EMSC实验组（RUNX2：1.92±0.20,Ⅰ型胶原：1.85±0.66）显著高于E12.5 d（RUNX2：0.38±0.02,Ⅰ型胶原：0.33±0.94）和E15.5 d组（RUNX2：0.72±0.22,Ⅰ型胶原：0.64±0.07）（P＜0.05）,p75NTR的表达在诱导21d的实验组（E12.5 d：0.79±0.23、E15.5 d：0.84±0.29、E18.5 d：1.35±0.22）均显著高于相同时间点对照组（E12.5 d：0.42±0.12、E15.5 d：0.43±0.13、E18.5 d：0.48±0.15） （P＜0.05）,其余各组间差异无统计学意义;实时定量PCR结果进一步印证了蛋白质印迹法结果.结论 p75NTR参与EMSC的矿化过程,E18.5 d的EMSC矿化能力最强,p75NTR表达量最高,是牙组织工程较理想的种子细胞.Objective To investigate the mineralized capacities of ectomesenchymal stem cells （EMSC） from facial process of Sprague Dawley（SD） rat embryo of different age in vitro and the expression of p75 neurotrophin receptor（p75NTR） in this process.Methods The stem cell surface antigens of EMSC from 12.5 d,15.5 d and 18.5 d SD rat embryonic facial process were tested by flow cytometry technology.E12.5 d EMSC,E15.5 d EMSC and E18.5 d EMSC were incubated under mineralization induction and analysed by alkaline phosphatase（ALP） staining on day 7（d7） and alizarin red staining on day 21（d21）.Expression changes of Runt-related transcription factor-2（RUNX2）,collagen Ⅰ （Col Ⅰ） and p75NTR in each group were measured using Western blotting and real time（RT）-PCR on day 0（d0）,day 7（d7）,day 14（d14） and day 21（d21）.Results The expression of the special substances CD29,CD146 and p75NTR in E12.5 d EMSC,E15.5 d EMSC and E18.5 d EMSC were positive,and the expression of CD45 was negative.The expression level of p75NTR in E18.5 d EMSC（84.04%） was much higher than that of E12.5 d EMSC （22.53%） and E15.5 d EMSC（81.43%）.The mineralized capacities of E18.5 d EMSC was stronger than that of E12.5 d EMSC and E15.5 d EMSC.The higher expression of RUNX2,Col Ⅰ in E18.5 d EMSC（RUNX2：1.92±0.20,Col Ⅰ：1.85±0.66） was found compared with E12.5 d EMSC（RUNX2：0.38±0.02,Col Ⅰ：0.33±0.94）and E15.5 d EMSC（RUNX2：0.72±0.22,Col Ⅰ：0.64±0.07）（P〈0.05）,and p75NTR in the E18.5 d EMSC experimental group（E12.5 d：0.79±0.23,E15.5 d：0.84±0.29,E18.5 d：1.35±0.22） was significantly higher than the in control group（E12.5 d：0.42±0.12,E15.5 d：0.43±0.13,E18.5 d：0.48±0.15）（P〈0.05）.RTPCR further proved the results of the Western blotting.Conclusions p75NTR participated in the mineralization differentiation of EMSC.E18.5 d EMSC had a higher expression of p75NTR and stronger mineralization capacity and was the ideal engineering seed cells.

关 键 词：间质干细胞 受体 神经生长因子类 颌突 矿化 

分 类 号：R783[医药卫生—口腔医学]