小鼠颅骨成骨细胞的分离培养及生物学鉴定  

Culture and identification of osteoblast from mouse skull

作　　者：王钦[1] 李辉[1] 张磊[1] 覃琦丽[1] 薛林涛[1] 滕维中[2] 石德顺[1] 

机构地区：[1]广西大学动物繁殖研究所,广西南宁530005 [2]广西大学物理科学与工程学院,广西南宁530005

出　　处：《广西农业生物科学》2008年第4期325-328,387,共5页Journal of Guangxi Agricultural and Biological Science

基　　金：广西区科技攻关项目(桂科转0718005-3B);广西大学博士启动基金(DD052009)

摘　　要：对小鼠成骨细胞的体外分离培养方法进行了摸索，并对其生物学特性进行了鉴定。手术法分离新生小鼠颅骨骨片，用0．25％胰蛋白酶＋0．02％乙二胺四乙酸（EDTA）预消化20～30min，而后用0．08％胶原酶＋0．05％胰蛋白酶＋0．004％EDTA的复合酶多次消化（每次10min）收集细胞，然后接种于细胞培养瓶中进行培养，并通过苏木精-伊红（HE）染色、BCIP／NBT Kit碱性磷酸酶（ALP）染色、Von Kossa法钙结节染色和四甲基偶氮唑盐（MTT）微量酶反应比色法对培养细胞进行生物学鉴定。结果发现，分离得到的细胞92％为存活细胞，差速贴壁培养后可筛选得到高纯度的成骨细胞，原代培养8d后可形成单层细胞，传代培养6～7d后可形成单层细胞。这些细胞有ALP活性，并具有体外钙化能力。结果表明，本研究建立的小鼠成骨细胞分离培养与鉴定方法是可行的。Methods for isolating and culturing osteoblasts from osteocranium of mouse were explored, and the biological characters of osteoblasts were identified. New-born mouse skull was separated by surgery, then pre-digested with 0.25% trypsin＋ 0.02% ethylene-diamine-tetra-acetic acid （EDTA） for 20 - 30 min and digested with 0.08% collagenase ＋ 0.05% trypsin ＋ 0. 004% EDTA in three or four times （10 min each time） for harvesting osteoblasts. Cells isolated were placed into the culture flask for in vitro culture. The biology characters of osteoblasts were identified by Haematoxylin ＆ Eosin （HE） staining, alkaline phosphatase （ALP） staining with BCIP/NBT kit, Von Kossa method and methyl thiazolyl tetrazolium （MTT） colorimetric assay method. Ninety-two percent of cells isolated were survival, and high purified osteoblasts were obtained after growing speed difference selection. Osteoblasts confluented monolayers after 8 d of primary culture, and 6-7 d of passage culture. Osteoblasts displayed ALP activity and ability of calcification in vitro. These results indicate that the protocols for isolation, culture and identification of osteoblasts are practicable.

关 键 词：成骨细胞 分离培养 鉴定 碱性磷酸酶 小鼠 

分 类 号：Q813[生物学—生物工程]