IL-1β诱导新西兰大白兔膝关节退变软骨细胞的体外培养及鉴定

In vitro Culture and Identification of IL-1β Induced Degeneration of Cartilage Cells in New Zealand White Rabbits Knee Joint

目的探讨并鉴定IL-1β诱导新西兰大白兔膝关节软骨细胞退变的方法,为从体外培养退变软骨细胞的中医药研究骨关节炎提供实验依据。方法无菌条件下取4周龄新西兰大白兔双侧膝关节软骨,采用Ⅱ型胶原酶消化并机械吹打的方法分离关节软骨细胞,细胞传至第2代时,随机分为正常对照组(Z组)与IL-1β诱导的模型组(M组),Z组不加任何干预,M组加入含10 ng/mL IL-1β的10%FBS培养基,两组均传至第3代。采用形态学、甲苯胺蓝染色、Ⅱ型胶原免疫组织化学染色及流式细胞仪技术比较鉴定。结果倒置显微镜下见Z组第2、3代关节软骨细胞表型稳定,增殖力良好,绝大部分细胞变为梭形、铺路石样,M组第2、3代关节软骨细胞变为长梭形或不规则形状;甲苯胺蓝染色及免疫组织化学技术显示Z组第2、3代关节软骨细胞染色后阳性表达均优于M组;流式细胞仪分析显示,M组第2代软骨细胞凋亡率与Z组比较,差异有统计学意义(P<0.01)。结论采用IL-1β诱导新西兰大白兔膝关节软骨细胞体外培养模型,细胞明显退变,可用于骨关节炎的相关实验研究。

Objective To explore and identify the method for IL-1β induced New Zealand rabbit knee chondrocyte degeneration, thus providing experimental bases for Chinese medical research on os- teoarthritis from in vitro cultured chondrocytes. Methods Under aseptic conditions, bilateral knee joint cartilage was collected from 4-week old New Zealand rabbits. Chondrocytes were separated by type 11 collagenase digestion and mechanical blowing method. They were randomly divided into two groups when passaged to the 2nd generation, the normal control group （group Z） and the IL-1β induced model group （group M）. No intervention was given to those in group Z. 10% FBS culture media containing 10 ng/mL IL-1β was added to group M. All cells were passaged to the 3rd generation. They were compared using mor- phological observation, toluidin blue staining, type H collagen immunohistochemical staining, and flow cytometry. Results Under inverted microscope, the second and the 3rd generation chondrocytes＇ phe- notype of group Z was stable with good proliferation. Most cells turned into fusiform and slabstone shaped. In group M, most cells turned into long spindle shape or irregular shape. Results of toluidine blue staining and immunohistochemistry showed that the positive expression of chondrocytes after staining in group Z was superior to that in group M. Results of flow cytometry showed that there was statistical difference in the apoptosis rate of the second generation chondrocytes between group M and group Z （P 〈0.01 ）. Con- clusion It was obviously seen that chondrocytes in IL-1β induced New Zealand rabbit knee chondrocyte model obviously degenerated, which could be used in related experimental researches on osteoarthritis.