不同因子对大鼠牙源性间充质细胞成牙本质分化的影响

上海口腔医学 ›› 2014, Vol. 23 ›› Issue (6): 661-668.

不同因子对大鼠牙源性间充质细胞成牙本质分化的影响

赵征¹, 刘洪臣², 黄征难¹, 鄂玲玲², 杨海青¹

1.解放军济南军区第401医院口腔科,山东青岛 266071; 2.解放军总医院口腔科,北京 100853

收稿日期:2013-09-26 修回日期:2014-04-12 出版日期:2014-12-20 发布日期:2015-01-08
通讯作者: 赵征,Tel:0532-51870540,E-mail:zhaozheng170@sohu.com
作者简介:赵征（1973-）,女,口腔医学博士后,副主任医师,硕士研究生导师
基金资助:
中国博士后科学基金特别资助课题（201003774）

The effect of different factors on forming-dentin differentiation of rat dental mesenchymal cells

ZHAO Zheng, LIU Hong-chen, HUANG Zheng-nan, E Ling-ling, YANG Hai-qing

1.Department of Stomatology, The 401st Hospital of Jinan Military Area, PLA. Qingdao 266071,Shandong Province;
2.Department of Stomatology, PLA General Hospital. Beijing 100853, China

Received:2013-09-26 Revised:2014-04-12 Online:2014-12-20 Published:2015-01-08
Supported by:
Chinese Post-Doctoral Special Science Foundation (201003774)

摘要/Abstract

摘要： 目的：探讨bFGF、IGF1、BMP4、TGF-β1联合应用对大鼠牙源性间充质细胞（rat dental mesenchymal cells, rDMCs）成牙本质分化的影响。方法：应用酶消化法和差速消化法分离、培养大鼠牙源性上皮细胞（rDECs）和rDMCs,经cytokeratin、vimentin免疫荧光染色鉴定其来源。应用茜素红染色、Gomori钙-钴法检测矿化液诱导下rDMCs的矿化能力。应用免疫组织化学染色、图像分析、实时荧光定量PCR和Western 印迹法检测在bFGF+IGF1（组1）、TGF-β1+BMP4（组2）、bFGF+IGF1+TGF-β1+BMP4（组3）分别诱导下 rDMCs中DSPP、CAP、OPN、OCN的表达差异。采用SPSS14.0软件包对数据进行统计学分析。结果：成功分离培养、鉴定rDECs和rDMCs,经矿化液诱导后的rDMCs钙结节和ALP染色阳性。组1、2中,DSPP、CAP、OPN、OCN mRNA和蛋白的表达水平均显著高于空白对照组4,差异显著（P<0.01）,其中组1 CAP、OCN和组2 DSPP、OPN的表达水平最高。结论：rDMCs经矿化诱导后具有成骨特性。bFGF+IGF1显著促进CAP、OCN的表达,加速rDMCs向成牙骨质细胞、成骨细胞分化与牙骨质基质、骨基质的矿化;TGF-β1+BMP4显著促进DSPP、OPN的表达,加速rDMCs向成牙本质细胞、成骨细胞分化与牙本质基质、骨基质的矿化,显示其成骨趋向。4种因子联合应用,并不具备显著的协同作用。

关键词: 大鼠牙源性间充质细胞, 碱性成纤维细胞生长因子, 胰岛素样生长因子1, 骨诱导形成蛋白4, 转化生长因子β, 1

Abstract: PURPOSE: To investigate the effects of combined use of bFGF, IGF1, BMP4 and TGF-β1 on forming-dentin differentiation of rat dental mesenchymal cells (rDMCs). METHODS: Enzyme and differential digestions were performed to isolate and culture rDECs and rDMCs, and immunofluorescence staining against cytokeratin and vimentin were carried out to identify cell sources. Then alizarin red staining and Gomori calcium-cobalt method were used to detect the mineralization ability of rDMCs after mineralized induction. Immunohistochemistry, image analysis, real-time PCR and Western blot were utilized to determine the expression differences of DSPP/CAP/OPN/OCN in rDMCs after induction by bFGF+IGF1 (group 1), TGF-β1+BMP4 (group 2) and bFGF+IGF1+TGF-β1+BMP4 (group 3), respectively. Statistical analysis was performed with SPSS 14.0 software package. RESULTS: The rDECs and rDMCs were isolated, cultured and identified successfully. Calcium nodus and ALP staining were positive in cytoplasms of rDMCs after being induced by mineralization liquid. In groups 1 and 2, the expression levels of DSPP/CAP/OPN/OCN mRNA and protein were notably higher than those of control group, significant differences were found between groups (P<0.01). Among them, the expression levels of CAP/OCN in group 1 and DSPP/OPN in group 2 were the highest, respectively. CONCLUSIONS: The rDMCs possess osteogenesis property after mineralization induction. bFGF+IGF1 can notably promote the expressions of CAP/OCN, and accelerate rDMCs to differentiate into cementoblast and osteoblast, and the mineralization of cementum matrix and bone matrix. TGF-β1+BMP4 can markedly increase the expressions of DSPP/OPN, and quicken rDMCs to differentiate into odontoblast and osteoblast, and the mineralization of dentinal matrix and bone matrix which display osteogenesis trend. Combined use of four factors had no significant synergism.

Key words: rDMCs, bFGF, IGF1, BMP4, TGF-β1

中图分类号:

R782

赵征, 刘洪臣, 黄征难, 鄂玲玲, 杨海青. 不同因子对大鼠牙源性间充质细胞成牙本质分化的影响[J]. 上海口腔医学, 2014, 23(6): 661-668.

ZHAO Zheng¹, LIU Hong-chen², HUANG Zheng-nan¹, E Ling-ling², YANG Hai-qing¹. The effect of different factors on forming-dentin differentiation of rat dental mesenchymal cells[J]. Shanghai Journal of Stomatology, 2014, 23(6): 661-668.

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