混合酶消化法大鼠颅盖骨细胞原代培养及鉴定.doc

混合酶消化法大鼠颅盖骨细胞原代培养及鉴定基金项目：福建省自然科学基金项目（编号：2908） 作者单位：350004 福建医科大学省立临床学院（孙玲）；福建省立医院（侯建明） 通讯作者：侯建明 【摘要】 目的 通过不断的摸索，寻找一种获得细胞数量较多且纯的原代成骨细胞的体外培养方法，为下一步实验研究奠定基础。方法 取出生24 h内的SD大鼠脱臼处死，取出颅盖骨，剪成1 mm×1 mm×1 mm大小的组织片；采用胰酶和Ⅰ型胶原酶混合酶消化法分离获得原代成骨细胞；采用“差速贴壁法”进行纯化。通过对细胞形态学的镜下观察，碱性磷酸酶染色及Ⅰ型胶原免疫组化法等对细胞进行鉴定。结果 镜下观察所获得的细胞具有成骨细胞的典型特征，细胞多为单核，呈三角形、多边形、长梭形等形态，并伸有粗大伪足；碱性磷酸酶染色及Ⅰ型胶原免疫组化染色呈阳性。结论 采用混合酶消化法可获得数量较多且活性较好的细胞，再采用“差速贴壁法”可以去除成纤维细胞等杂质细胞，获得纯度较高的成骨细胞。 【关键词】 成骨细胞； 原代培养； 大鼠； 混合酶消化法 Primary culture and identification of rats cranial cover osteocytes SUN Ling,HOU Jian-ming.Fujian Medical Uniersity, Fuzhou 350004, China 【Abstract】 Objective Through continuous exploration, we want to search for a method of cultivating the original generation osteoblasts in vitro which can gets a larger quantity and pure the original generation of osteoblasts, to prepare for the next experiment research.Methods 24 hours old of SD rats were executed by dislocating.The cranial cover bones of rats were removing and cuting into the blocks of 1mm x 1mm x 1mm size. The crinial bones were digesting by mixed enzyme which contains enzyme andⅠtype collagenase.The osteoblast were purified by selective plating technique. The osteoblasts were identified through morphlogy observation, alkaline phosphatase dyeing, collagenⅠimmunohistochemical staining.Results The cultured cells have the typical features of osteoblast.The cells are triangle, polygonal, long spindle shape, etc.and mostly are mononuclear cells, have large pseudopodia. Alkaline phosphatase dyeing collagenⅠimmunohistochemical staining are both positive.Conclusion A large quantity and activity of the good cells were obtained by mixed enzyme digestion method. High purity osteoblasts were obtained by selective plating technique that can remove impurities such as fibroblast cells. 【Key words】 Osteoblast； Primary culture； Rat； Mixed enzyme digestion 随着中国人口老龄化的到来，骨质疏松的发病率呈上升趋势，骨质疏松的发病机制和治疗的研究也越来越引起人们的重视。为了探讨各种基因在成骨细胞中的表达以及外源性基因对成骨细胞的影响，建立一种简单，高效的成骨细