用酶消化-连续组织块法培养SD大鼠成骨细胞的研究  

Study on Method of Enzymatic Digestion-Serial Explant Culturing SD Rat Osteoblasts

作　　者：刘江涛[1] 王永清[1] 张卫国[1] 葛保健[1] 叶锋[1] 梁新军[1] 夏仁云[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院骨科,武汉430030

出　　处：《中国中医骨伤科杂志》2006年第6期46-48,共3页Chinese Journal of Traditional Medical Traumatology & Orthopedics

基　　金：内蒙古科技厅科技合作项目(2004006)

摘　　要：目的采用酶消化-连续组织块法进行新生SD大鼠颅骨成骨细胞(OB)的体外培养,以获得较多的原代成骨细胞。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1-2mm2组织块。0.25%胰蛋白酶消化20min,0.1%胶原酶反复消化组织块3次,每次60min,收集第2、3次的液体,离心接种于培养瓶中培养;并将组织块贴壁以连续组织块法培养3-4次。采用“多次贴壁法”纯化成骨细胞。从形态学、碱性磷酸酶染色等方面鉴定。结果培养的细胞具有典型的成骨细胞形态特征,碱性磷酸酶呈阳性染色。结论用酶消化-组织块法培养的成骨细胞具有典型的成骨细胞形态特征;并可以充分利用材料,获得较多的成骨细胞。Objective：To harvest more primary osteoblasts（OB） derived from newly born SD rat calvarias by culture technique of enzymatic digestion-serial explant in vitro. Method：5 newly born SD rats were killed , the upper skull tissues of them were isolated by aseptic technique. Then erased completely the periosteum and snipped to 1--2 mm2 pieces. Digested by 0.25% trypsin for 20 minutes, then by O. 1% collagenase for 60 minutes three times. The liquid of second and third time was gathered and centrifuged. The cells were sub cultured in culture flask. The tissue blocks were repeatedly cultured by serial explant method for three or four times. The osteoblasts were purified by many times adhered. The osteoblasts were identified by morphology and Alkaline phosphatase （ALP） staining in cell. Result：The cultured cells had typical mor- phological characters of osteoblast, and ALP staining was positive. Conclusion：The osteoblasts cultured by technique of enzymatic dlgestion-serial explant in vitro have typical characteristics of osteoblast. The rats can be sufficiently utilized, and more osteoblasts can be harvested.

关 键 词：成骨细胞 细胞培养 

分 类 号：R782.2[医药卫生—口腔医学]