体外培养豚鼠膝关节软骨细胞的生物学特性及其鉴定

Biological Characteristics of Guinea Pig Chondrocytes Cultured In Vitro

目的探索豚鼠膝关节软骨细胞的分离、培养和鉴定方法,观察豚鼠膝关节软骨细胞的生物学特性。方法无菌条件下,从2周龄豚鼠的膝关节面削取软骨片,采用机械-酶消化法分离软骨细胞,经台盼蓝拒染计数,将细胞按1×106个/孔接种于25 cm2细胞培养瓶,传代培养,描绘生长曲线。利用相差显微镜及透射电镜观察细胞形态。应用甲苯胺蓝染色、1,9-二甲基亚甲蓝(1,9-Dimethylmethylene blue,DMB)染色、阿利新蓝染色、Ⅱ型胶原免疫组化及透射电镜对细胞进行鉴定。结果甲苯胺蓝染色、DMB染色或阿利新蓝染色时均可异染;II型胶原免疫组化染色时,软骨细胞胞浆被染成棕黄色,胞核不着色。透射电镜分析显示细胞核圆形,有丰富的粗面内质网、高尔基体及分泌的基质成分。生长曲线分析显示第3、4代细胞生长速度稳定,细胞传至第5代以后,出现"成纤维细胞样"。结论本研究建立了简单易行的豚鼠膝关节软骨细胞分离、培养和鉴定方法;第3、4代细胞生长速度稳定,适合于实验研究;软骨细胞5代培养后,细胞表型发生改变。

Objective To explore the methods for isolation,culture and identification of articular chondrocyte of guinea pig and observe the biological characteristics of chondrocytes of guinea pig cultured in vitro.Methods Cartilage was harvested in small slices with a sharp knife under sterile conditions from knee joint of guinea pigs of 2 weeks old.The slices were cut into small pieces and digested in enzyme solution.After suspension,viable cells were counted in a hemocytometer after trypan blue dye exclusion.Chondrocytes（cell density of 1×106/bottle） were placed in DMEM containing 10% FBS in 25 cm2 cell culture bottles.The cells were sub-cultured till they became confluent.Cell growth curve was depicted.Cultured cells were indentified with Toluidine blue staining,DMB staining,Alcian Blue dye,typeⅡcollagen immuno-histochemistry dye and transmission electron microscope（TEM）.Results The chondrocytes were stained with toluidine blue,DMB and Alcian Blue.Ⅱtype collagen immunohistochemistry showed that the cytoplasm of chondrocyte was stained yellow but with no color in cell nucleus.The cells displayed around,eccentrically located nucleus,clusters of ribosome,rough endoplasmic reticulum and a prominent golgiosome.Growth curve showed growth speed of cells of P3 and P4 was stable.When chondrocyte subcultured to P5,phenotype of chondrocytes became like fibroblasts.Conclusion The present methods for isolation,culture and identification of chondrocytes were simple and feasible.Growth speed of P3,P4 was stable.Chondrocytes of P3,P4 was suitable for experimental study.After P5 of chondrocyte,phenotype was changed.