中文摘要：

取新生奶牛肋骨,剪成1 mm3大小骨片,胰蛋白酶消化5次,D-Hank’s液彻底冲洗后,骨片置于含15%FBS的DMEM中培养,获取单层细胞。采用倒置显微镜下观察细胞形态,改良钙-钴法染色,检测碱性磷酸酶(ALP)活性、骨钙素(OC)mRNA丰度及骨钙素蛋白浓度等方法,对细胞进行鉴定。通过MTT法绘制生长曲线,研究细胞增殖特性,茜素红染色观察细胞钙化功能。结果显示,采用本法所获得的细胞具有典型的成骨细胞形态和特性:ALP染色呈阳性,活性水平较高;培养7 d后可检测到骨钙素mRNA表达,12 d后细胞上清液中可检测到骨钙素蛋白;诱导培养2周后细胞可形成钙化结节。提示采用胰蛋白酶消化,组织块移行法所培养的细胞具有成骨细胞特性。本方法可获得高纯度、高活性的奶牛成骨细胞。

英文摘要：

Costal bone from a new born calf was manually fragmented into bone flaps,and digested 5 times with trypsin.Following a thorough wash to remove liberated cells,the remaining bone fragments were plated and cultured in DMEM containing 15% FBS.The shape of cultured cells was observed by inverted microscope.Then the cells were identified with modified Gomori method and alkaline phosphatase(ALP) activity assay.The level of osteocalcin(OC) and the expression of OC mRNA were analyzed by radioimmunoassay and reverse...