骨关节炎患者膝关节软骨细胞的离体培养及形态学特征观察

目的建立膝关节患者关节软骨细胞体外培养体系,为其相关研究奠定基础。方法取15例人工膝关节置换术患者的软骨组织,采用组织原代培养法获得膝关节软骨细胞,传代培养。倒置相差显微镜观察细胞形态,绘制生长曲线,苏木素伊红染色、甲苯胺蓝染色及Ⅰ型胶原、Ⅱ型胶原、X型胶原、MMP-1、MMP-13、TNF-α免疫荧光染色对细胞进行鉴定。结果原代培养的人软骨细胞大量呈梭形、短梭形,少部分呈多角形,传代3次后出现树突生长细胞。形态学、免疫荧光染色显示细胞培养3代以内可保持表型稳定,多数软骨细胞维持梭形、短梭形,核为圆形或椭圆形。苏木精伊红阳性染色为细胞核呈紫蓝色,软骨细胞基质呈红色,周围胞浆呈紫红色、阿利新蓝染色后,胞质和胞膜呈深蓝色、Ⅱ型胶原免疫荧光染色可见胞质和胞膜清晰绿色荧光,细胞核区域未见明显绿色荧光。结论成功建立了的软骨细胞分离、培养体系,且3代以内细胞生长良好。

Isolate culture and morphological observation of the knee joint cartilage cells in patients with osteoarthritis

Objective To establish vivo culture system of knee joint cartilage cells in patients with osteoarthritis.Methods Cartilaginous tissue was obtained from 15 patients underwent total knee replacement,and knee joint cartilage cells was obtained by primary cultured,and then the cells were passage cultured.The cells was identificated by observing cells form,drawing the growth curve,hematoxylin and eosin staining,toluidine blue staining and collagen type Ⅰ,Ⅱ,Ⅹ,and MMP-1,MMP-13,TNF alpha immunofluorescence stain.Results Most of the cartilage cells showed spindle and short spindle,and small amount showed polygons,dendritic cells were found after three generations.Morphology and immunofluorescence stain showed cell culture within three generations maintain the stability of the phenotype,most cartilage cells maintain spindle,short spindle,nuclear round or oval.Hematoxylin and eosin staining showed the nuclei were violet blue,matrix of cartilage cells was red dye,alcian blue staining showed the cytoplasm and cell membrane was deep blue,and collagen type Ⅱ immunofluorescence staining showed the cytoplasm and cell membrane was clear green fluorescent,the nuclear area was not see obvious green fluorescent.Conclusions The isolation,culture system of cartilage cells can be successfully set up,and cells grow well within three generations.