改进酶消化法培养SD大鼠成骨细胞及其鉴定  

Identification of SD rat osteoblasts cultured by modified enzymatic digestion

作　　者：王双利[1] 查振刚[1] 刘宁[1] 杨淑野[1] 吴昊[1] 姚平[2] 张嘉晴[3] 

机构地区：[1]暨南大学附属第一医院骨科,广州市510632 [2]暨南大学医学院生理教研室,广州市510632 [3]暨南大学医学院生化教研室,广州市510632

出　　处：《实用医学杂志》2008年第6期915-918,共4页The Journal of Practical Medicine

基　　金：国家高技术研究发展计划项目(“863”项目)(编号2007AA09Z4400);广东省医学科学技术研究基金项目(编号B2006089);广东省科技攻关项目(编号:2006B60501009);广州市科技资助项目(编号:2006Z3-E5211)

摘　　要：目的:改进SD大鼠成骨细胞的体外分离、培养方法并进行功能鉴定。方法:将新生SD大鼠处死,无菌条件下取出颅骨,剔净骨膜后剪成1mm×1mm×1mm大小组织块。组织块经0.25%胰蛋白酶消化20min,继以0.1%Ⅱ型胶原酶消化60min,收集上清离心,所得成骨细胞接种于培养瓶中并行"多次贴壁法"纯化。观察细胞形态学,选用碱性磷酸酶Gomori钙钴法染色,钙结节茜素红法染色及Ⅰ型胶原免疫组化染色等方法进行鉴定。结果:培养的细胞具有典型的成骨细胞形态特征,在体外培养时可维持其在体内的功能,即合成碱性磷酸酶、形成矿化结节,Ⅰ型胶原染色阳性。结论:用改进后的酶消化法分离、培养SD大鼠成骨细胞,更能减少胰蛋白酶在消化过程中对细胞造成的损伤,所获成骨细胞量多、纯度高,操作简易可行,可作为骨组织工程种子细胞常规的培养方法。Objective To explore a novel method for isolating and culturing SD rat osteoblasts in vitro, and identify their functions. Methods The newly born SD rats were sacrificed, and then the cranial bones of the rats were obtained cleanly with the periosteum erased completely, and cut into blocks of 1 mm × 1 mm × 1 mm. It was digested by 0.25% trypsinase for 20 minutes and by 0.1% collagenase Ⅱ for 60 minutes, and then was gathered and centrifuged. The cells were cultured in culture flask and purified by many times adhered. Morphology observation under the microscope was performed. The phenotype, calcium tuberculation and the expression of alkaline phosphatase were studied with alizarin red staining and modified Gomori Ca-Co assays. Collage Ⅰ immunohistochemical staining was also applied. Results The cultured cells had typical morphological characters of osteoblasts, showing the osteoblastic phenotypes such as their synthesis of alkaline phosphatase and formation of mineralized nodes. Collagenase I was stained positive. Conclusion The study indicates that it is easy to purify plentiful SD rat osteoblasts with normal functions by modified enzymatic digestion, which can be used as a routine method in seeding cell culture for bone tissue engineering.

关 键 词：成骨细胞 大鼠 改进酶消化法 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]