3D培养牙周膜干细胞生物学特性的初步研究

发布时间：2018-03-07 13:59

本文选题：牙周膜干细胞　切入点：D培养　出处：《牙体牙髓牙周病学杂志》2014年05期 　论文类型：期刊论文

【摘要】：目的:研究悬滴法3D培养和2D贴壁培养的牙周膜干细胞(PDLSCs)生物学特性的差异。方法:改良酶消化组织块法分离培养PDLSCs,分别行2D贴壁培养及3D培养(3D培养按不同细胞接种数分为2.5×104,5×104及1×1053组),培养96 h后分别用Live/Dead染色法分析细胞活性;流式细胞术检测干细胞表面标记物;然后对2D、3D培养的PDLSCs进行矿化诱导,茜素红染色及荧光定量PCR检测矿化结节形成情况以及矿化相关基因ALP和OPN mRNA的表达水平。结果:3D培养的PDLSCs可形成致密的细胞聚合物,2.5×104组PDLSCs细胞聚合物中心未见坏死现象,5×104组和1×105组则有较为明显坏死现象;3D和2D培养的PDLSCs间充质干细胞表面标记物的阳性表达率均无显著差异(P0.05);茜素红染色和荧光实时定量PCR结果显示,3D培养的PDLSCs形成矿化结节的数量及其矿化相关基因ALP OPN mRNA的表达水平均显著高于2D培养的细胞(P0.05)。结论:3D悬滴培养法是一种理想的PDLSCs培养方法。
[Abstract]:Objective: to study the biological characteristics of periodontal ligament stem cells (PDLSCs) in 3D and 2D adherent culture. Methods: PDLSCs were isolated and cultured by modified enzyme digestion tissue mass method, and were cultured by 2D adherent culture and 3D culture respectively. The cell number was divided into 2.5 脳 10 4 5 脳 10 4 and 1 脳 10 53 groups. After 96 h culture, the cell activity was analyzed by Live/Dead staining. Flow cytometry was used to detect the surface markers of stem cells, and then the PDLSCs cultured in 3D culture was mineralized. Alizarin red staining and fluorescence quantitative PCR were used to detect the formation of mineralized nodules and the expression levels of mineralization-related genes ALP and OPN mRNA. Results the polymer centers of PDLSCs cells in 2.5 脳 10 ~ 4 group of PDLSCs cells were not found to be damaged in the PDLSCs cultured with PDLSCs in 1: 3 D culture. There was no significant difference in the positive expression rate of surface markers of PDLSCs mesenchymal stem cells cultured in 3D and 2D. The results of alizarin red staining and real-time quantitative PCR showed that there was no significant difference between 3D and 2D cultured PDLSCs mesenchymal stem cells. The results of alizarin red staining and fluorescence real-time quantitative PCR showed that there was no significant difference in the expression of surface markers between 3D and 2D cultured PDLSCs mesenchymal stem cells. The number of mineralized nodules formed by PDLSCs and the expression level of mineralization-related gene ALP OPN mRNA were significantly higher than those of 2D cultured cells (P0.05).
【作者单位】：南方医科大学南方医院;南方医科大学口腔医学院;南方医科大学附属广东省口腔医院;
【基金】：国家自然科学基金面上项目(81371137)
【分类号】：R780.2

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