中国组织工程研究与临床康复2008，Vol.12Issue(15)：2983-2987,5.
优化酶消化法体外培养及鉴定SD大鼠的成骨细胞
Culture and identification of SD rat osteoblasts by modified enzymatic digestion in vitro
摘要
Abstract
BACKGROUND: The skill to culture osteoblasts primarily has been well developed. However, trypsinase can affect membrane protein of osteoblasts if the time of digestion is long. Therefore, it is of great significance to select an ideal method to avoid the damage from trypsinase to cells as possible when culturing osteoblasts.OBJECTIVE: To explore a novel method to isolate and culture SD rat osteoblasts in vitro, and identify the functions of the cells.DESIGN: Observational study.SETTING: Department of Orthopaedics, First Affiliated Hospital of Jinan University.MATERIALS: This experiment was carried out in the Department of Orthopaedics, First Affiliated Hospital of Jinan University from March to May in 2007. Eight SPF 24-hour old SD rats were used in the experiment. The rats, irrespective of gender, were provided by the Experimental Animal Center of Nanfang Medical University. The experimental animals were disposed according to ethical criteria. The main reagents were detailed as follows: collagenase Ⅱ (Sigma Company);trypsin (Sigma Company); alkaline phosphatase (ALP) kit (Nanjing Jiancheng Biological Products Company); SABC-1021(Wuhan Boster Biotechnology Company).METHODS: 24-hour old SD rats were chosen for experiment. The newly born SD rats were sacrificed by anesthesia and the cranial bones of the rats were obtained cleanly, erased completely of the periosteum and cut to blocks of I mm3. The cranial bones were digested by 0.25 % trypsinase for 20 minutes, then by 0. 1% type Ⅱ collagenase for 60 minutes. The digestive time of trypsinase was controlled in the process of digestion to avoid to harm the cells. The liquid was gathered and centrifuged. The cells were cultured in culture flask and were purified by many times adhered.MAIN OUTCOME MEASURES: Morphology observations under the inverted phase contrast microscope, transmission electron microscope, and scanning electron microscope were performed. The phenotype, calcium tuberculation and the expression of alkaline phosphatase were studied with alizarin red staining and modified Gomori Ca-Co assays respectively.The cells were also evaluated with collage Ⅰ immunohistochemical staining.RESULTS: The cultured cells had active proliferation ability. Cells showed multi-angle or fusiform shape. Nucleus was immature and organell was plentiful. Therefore, they had typical morphological characters of osteoblasts. Moreover, they showed the osteoblastic phenotypes such as their synthesis of alkaline phosphatase, collage Ⅰ and formation of calcium tuberculations.CONCLUSION: The cells cultured by our modified enzymatic digestion method had typical morphological and biological characteristics of osteoblasts.关键词
成骨细胞/改良酶消化法/SD大鼠分类
基础医学引用本文复制引用
王双利,刘宁,杨淑野,吴昊,查振刚..优化酶消化法体外培养及鉴定SD大鼠的成骨细胞[J].中国组织工程研究与临床康复,2008,12(15):2983-2987,5.基金项目
Supported by: the Grant from the National High-Tech Research and Development program of China (863 Program), No.2007AA09Z440 （863 Program）
the Medical Scientific and Technological Research Projects of Guangdong Province,No. B2006089 （）
the Scientific and Technological Research Projects of Guangdong Province,No.2006B60501009 （）
the Scientific and Technological Assistance Program of Guangzhou City,No. 2006Z3-E5211 国家高技术研究发展计划项目("八六三"项目)(2007AA0924400) （"八六三"项目）
广东省医学科学技术研究基金项目(B2006089) （B2006089）
广东省科技攻关项目(2006860501009) （2006860501009）
广州市科技资助项目(2006Z3-E5211) （2006Z3-E5211）
