大鼠椎间盘纤维环细胞的培养及鉴定

背景：目前关于椎间盘纤维环细胞的培养方面还没有形成统一的标准,导致实验的操作性和重复性存在缺陷。目的：通过对椎间盘纤维环细胞的体外培养,观察细胞的演变,以期为组织工程研究椎间盘纤维环提供方法和体外模型。方法：采用酶消化法分离SD大鼠椎间盘纤维环细胞,进行单层培养,实验在原代培养中使用体积分15%胎牛血清的DMEM培养基,细胞传代培养一代后可换成体积分数10%胎牛血清的DMEM培养基。倒置相差显微镜观察其形态结构和生物学特性,通过甲苯胺蓝染色和免疫细胞化学染色等方法对其细胞表型进行鉴定。结果与结论：培养的椎间盘纤维环细胞形态为圆形或梭形;原代细胞生长较慢,传代细胞生长增快;细胞具有甲苯胺蓝易染性;免疫细胞学方法检测表明纤维环细胞有Ⅰ,Ⅱ型胶原表达。结果证实,实验成功在体外培养了大鼠椎间盘纤维环细胞。

Culture and identification of rat annulus fibrosus cells

BACKGROUND:There is not a criterion for culture of annulus fibrosus cells, thus, there is deficiency in operability and repetitiveness. OBJECTIVE:To establish the annulus cell lines and to observe their continuouschanges, in order to provide the methods and cell model for studying molecular biology in tissue engineering field.METHODS:The annulus fibrosus cells were isolated with enzyme digestion and grew as monolayer. DMEM containing 15% fetal calf serum was used in the primary culture, which was replaced by DMEM containing 10% fetal calf serum after cell passage. Morphology, structure and biological characteristics of cells were detected by an inverted phase contrast microscope. The phenotype was identified with the toluiding blue staining and immunocytochemistry.RESULTS AND CONCLUSION:The disc fibrinous ring cells were cultured in monolayer with the round or fusiformis morphology. The primary cells grew more slowly than the subculture cells. The cells displayed intense toluiding blue metachromasia; the immunohistochemical test revealed that the positive expression of type Ⅰ and Ⅱ collagen on the cells. The experiment successfully culture annulus fibrosus cells in vitro.