混合酶消化法大鼠颅盖骨细胞的原代培养和鉴定

Primary culture and identification of rats cranial cover osteocytes

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。

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 ×1mm ×1mm size. The crinial bones were digesting by mixed enzyme which con- tains enzyme and I type collagenase. The osteoblast were purified by selective plating technique. The osteoblasts were identi- fled through morphlogy observation, alkaline phosphatase dyeing, collagen I immunohistochemical staining Results The cultured cells have the typical features of osteoblast. The cells are triangle, polygonal, long spindle shape, etc. and mostly are mononuelear cells, have large pseudopodia. Alkaline phosphatase dyeing collagen I immunohistochemical 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.