兔纤维环细胞培养及生物学特性的实验研究

Study of culture of rabbit annulus fibrosus cells and biological feature of annulus fibrosus cells in vitro

目的建立兔椎间盘纤维环细胞体外培养体系,并对其生物学特性进行鉴定。方法采用酶消化法分离兔椎间盘纤维环细胞,进行单层培养,实验在原代培养中使用体积分数为20%FBS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、免疫细胞化学染色和碱性磷酸酶染色等方法对其细胞表型进行鉴定,MTT法绘制髓核细胞生长曲线。结果在相差显微镜下,单层培养的椎间盘纤维环细胞呈贴壁生长的梭形或多角形细胞,细胞具有甲苯胺蓝异染性;Ⅰ型胶原和Ⅱ型胶原的表达阳性;细胞碱性磷酸酶染色阳性。结论采用酶消化法可成功培养兔椎间盘纤维环细胞,生物学特性鉴定符合纤维环细胞特征。

Objective To establish the culture system of the annulus fibrosus cells of rabbit in vitro and identify the cell phenotype. Methods The annulus fibrosus cells of rabbit were isolated by enzyme digestion and cultured in monolayer. DMEM containing 20% fetal bovine serum was used in the primary culture. The morphology, structure and biological characteristics of cells were detected by an inverted microscope. The cell pbenotype was identified by toluidine blue stai ning, immunocytochemistry and alkaline phospbatase detection. The growth curve was drawn through MTT. Results Under inverted microscope, annulus fibrosus cells were polygonal and spindle-like in monolayer culture. The cells displayed intense toluidine blue metachromasia. The cells showed positive expression of collagen Ⅰ and Ⅱ as well as alkaline phosphatase. Conclusion The enzyme digestion method can be successfully applied to culture of rabbit annulus fibrsus cells which are accordance with the characteristics of annulus fibrsus cells by phenotypic identification.