优化酶消化法体外培养及鉴定SD大鼠的成骨细胞(英文)  

Culture and identification of SD rat osteoblasts by modified enzymatic digestion in vitro

作　　者：王双利[1] 刘宁[1] 杨淑野[1] 吴昊[1] 查振刚[1] 

机构地区：[1]暨南大学附属第一医院骨科,广东省广州市510630

出　　处：《中国组织工程研究与临床康复》2008年第15期2983-2987,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家高技术研究发展计划项目("八六三"项目)(2007AA09Z4400);广东省医学科学技术研究基金项目(B2006089);广东省科技攻关项目(2006B60501009);广州市科技资助项目(2006Z3-E5211)~~

摘　　要：背景:体外成骨细胞的培养技术已经有了很大的发展。但是,在培养过程中,如果胰蛋白酶的消化时间过长,成骨细胞膜便易受到损伤;同时,传统的培养方法存在所获得的成骨细胞数量少、纯度不高等不足。因此,有必要探求一种新的体外培养成骨细胞的方法。目的:优化成骨细胞在体外条件下的培养方法并对其进行鉴定。设计:观察性实验。单位:暨南大学附属第一医院骨科。材料:实验于2007-03/05在暨南大学附属第一医院骨科完成。选用出生24h的SD大鼠8只,由南方医科大学实验动物中心提供,雌雄不拘。实验过程中对动物的处置符合动物伦理学标准。实验用Ⅱ型胶原酶由美国Sigma公司分装,胰蛋白酶为Sigma公司产品,碱性磷酸酶试剂盒由南京建成生物制品公司生产,SABC-1021试剂盒由武汉博士德公司进口分装。方法:挑选24h之内的新生SD乳鼠麻醉后处死,无菌条件下取出颅骨,剔除附着的结缔组织及骨膜,D-Hank’s液冲洗3次。不强调对颅骨内、外膜,骨缝连接处等附着的结缔组织及颅顶的透明软骨结节等进行反复多次的剔除,避免操作时间过长影响细胞的存活率。结合0.1%Ⅱ型胶原酶来消化SD乳鼠颅骨,减少并严格控制胰蛋白酶的消化时间。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。主要观察指标:应用倒置相差显微镜、透射电子显微镜、扫描电子显微镜观察成骨细胞形态特点。采用碱性磷酸酶Gomori钙钴法染色、钙结节茜素红法染色及Ⅰ型胶原免疫组织化学染色方法进行鉴定成骨细胞生物学特性。结果:原代和传代培养的细胞具有活跃的增殖能力,细胞呈多角形或梭形,具有多个突起可互相连接,细胞核较幼稚,细胞器丰富,具有典型的成骨细胞形态特征。体外培养的成骨细胞可分泌碱�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 1 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 alkalin

关 键 词：成骨细胞 改良酶消化法 SD大鼠 

分 类 号：R329.471[医药卫生—人体解剖和组织胚胎学]