椎间盘纤维环细胞的培养

Research of cell culture of anulus fibrosus in rat

目的 通过对椎间盘纤维环细胞的培养 ,观察细胞的演变 ,为研究椎间盘纤维环细胞的生物学特性、退变机理、组织工程、基因治疗等提供方法和体外模型。方法 采用胰蛋白酶、胶原酶分离大鼠椎间盘纤维环细胞 ,选择DMEM(体积分数 2 0 %的小牛血清 )培养基体外培养 ,行HE染色 ,甲苯胺蓝 ,免疫细胞化学染色分析细胞的生物学特性 ,透射电镜观察细胞的超微结构。结果 采用粗品胶原酶能充分消化纤维环组织 ;在含体积分数为 2 0 %小牛血清的DE TA培养液中细胞生长良好 ;原代纤维环细胞多为梭形 ,有伪足伸出 ,甲苯胺蓝染色 ,胞浆染为深蓝色 ;透射电镜下纤维环细胞内多见长条状粗面内质网 ,部分囊池扩张。线粒体较少 ,可见平行于细胞膜方向的微丝。免疫细胞学方法检测表明纤维环细胞有Ⅰ、Ⅱ型胶原表达。结论 本实验为椎间盘纤维环细胞的培养提供了简单、有效的方法 ,为进一步研究椎间盘纤维环细胞生物学功能和调控研究打下了基础。

Objective To establish the anulus fibrosus(AF) cell lines and to observe their continuous changes, in order to provide the methods and the cell model for studying the biologic specificity、degenerat ion、 tissue engineering and gene therapy. Methods The isolation of cells from the anulus fibrosus of rat was digested respectively with the elzyme and the collagenase; then selecting the DMEM (20% calf serum) as the culture medium to be cultured in vitro. After staining with the haematineeosin and the toluidine blue, the immunohistochemical method was used to study the synthesis of type Ⅰand type Ⅱ collagen. The ultrastructure of anulus fibrosus cell was observed under the transmission electronic microscope. Results The cells were released from the tissue after 20 min processing with collagenase at 37 ℃, and were growing well in the DMEM culture medium. The primary cells mostly appeared as flattend and spindle shape, with the pseudopodia being stretching out. The cytoplasm was profoundly blue after stained with the toluidine blue. There were many ribbonlike rough endoplasmic reticula viewed under the transmission eletronic microscope and the microfilaments were parallel to the cytomembrane.The immunohistochemical test revealed that there existed the positive expression of typeⅠcollagen and typeⅡcollagen on the cells. Conclusion The experimental study provides a simple and effective method for the culture of anulus fibrosus cell in vitro. It also form a firm foundation for facilitating the furthur study of the manipulation and modulation of biologic function and biologic treatments of disc cell.