干细胞诱导、细胞系诱导和原代分离3种提取成骨细胞的方法比较  

Three methods to isolate osteoblasts: stem cell induction, cell line induction and primary isolation

作　　者：邓享誉 陈胜[1] 邵增务[1] 郑东[1] Deng Xiang-yu Chen Sheng Shao Zeng-wu Zheng Dong(Department of Orthopedics, Wuhan Union Hospital, Tongji Medical College, Huangzhong University of Science and Technology, Wuhan 430000, Hubei Province, Chin)

机构地区：[1]华中科技大学同济医学院附属协和医院骨科,湖北省武汉市430000

出　　处：《中国组织工程研究》2017年第17期2729-2734,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金资助(81472134)~~

摘　　要：背景:成骨细胞是骨组织构建的重要组织细胞之一。成骨细胞取材困难,不同方法获得的成骨细胞纯度及钙化能力不尽相同。目的:比较小鼠骨髓间充质干细胞成骨诱导、细胞系MC3T3-E1成骨诱导和小鼠颅骨原代细胞培养3种方法获得的成骨细胞的纯度及钙化能力。方法:采用贴壁纯化法分离出小鼠骨髓间充质干细胞,传至3代后用成骨诱导培养基诱导21 d;将前成骨细胞系MC3T3分别用成骨诱导培养液1和成骨诱导培养液2诱导21 d;使用Ⅰ型胶原酶消化法取得乳鼠颅骨原代成骨细胞。对3种方法获得的成骨细胞进行茜素红钙结节染色,观察其成骨特性。结果与结论:(1)骨髓间充质干细胞诱导后钙结节平均为16.3个/低倍镜视野;(2)前成骨细胞系MC3T3诱导培养液1组可观察到稀疏的钙结节,平均为1.7个/低倍镜视野,诱导培养液2组可观察到密集的钙结节,平均为44.6个/低倍镜视野,两种诱导液相比钙结节形成能力差异有显著性意义(P<0.01);(3)原代成骨细胞钙结节平均为0.6个/低倍镜视野;(4)除MC3T3诱导液1组与原代成骨细胞相比差异无显著性意义外,其余两两相比成骨能力差异均有显著性意义(P<0.01);(5)由于骨髓间充质干细胞分离培养具有更多不可控因素,因此MC3T3细胞用含有地塞米松的诱导培养液2进行成骨诱导方法较好。BACKGROUND： Osteoblasts have become a kind of important seed cells in bone tissue engineering. However, it is difficult to harvest osteoblasts, and the purity and calcification ability of osteoblasts isolated by different methods are inconsistent. OBJECTIVE： To compare the purity and calcification ability of osteoblasts induced from mouse bone marrow mesenchymal stem cells, MC3T3 cell lines, and cultured primarily from the neonatal mouse cranium. METHODS： Mouse bone marrow mesenchymal stem cells were isolated by differential adhesion method, and after passaing, passage 3 cells were cultured in osteogenic induction medium for 21 days. MC3T3 cell lines were cultured in osteogenic induction media 1 and 2 for 21 days. Osteoblasts were cultured primarily from neonatal mouse cranium by type Ⅰ coll agenase digestion method. Calcium nodules of osteoblasts obtained by three methods were observed by Alizarin red staining to detect osteogenic activity of cells. RESULTS AND CONCLUSION： （1） There were average 16.3 calcium nodules per low-power field after osteogenic induction of bone marrow mesenchymal stem cells. （2） There were sparsely distributed calcium nodules in MC3T3 cells after induction with osteogenic induction medium 1, accounting for 1.7 calcium nodules per low-power field, while there were dense calcium nodules in MC3T3 cells after induction with osteogenic induction medium 2, accounting for 44.6 calcium nodules per low-power field. There was a significant difference in the calcium nodule formation ability between the two groups （P 〈 0.01）. （3） After primary culture, there was only 0.6 calcium nodule per low-power field. （4） Except for the insignificant difference between osteogenic induction medium 1 and primary culture groups, there were significant differences in pair-wise comparison of any other two groups. Except the insignificant difference between group I of MC3T3 inducing conditional media and primary culture osteoblasts, there were significant differences in the osteogenic

关 键 词：干细胞 分化 骨髓间充质干细胞 MC3T3 成骨细胞 成骨诱导 钙结节染色 吉姆萨染色 国家自然科学基金 

分 类 号：R394.2[医药卫生—医学遗传学]