一种获取具有旺盛增殖力的大鼠软骨细胞培养方法

目的探讨大鼠软骨细胞的培养方法。方法采用单纯胶原酶消化配合吹打法,从新生(出生24 h内)SD大鼠的膝、肩、肘关节和股骨头处的软骨中提取软骨细胞进行原代培养,在显微镜下观察并对甲苯胺蓝染色、阿利新蓝染色、II型胶原免疫荧光染色和增殖细胞核抗原免疫荧光染色进行鉴定;针对不同批次的实验观察结果,进行一致性检验。结果原代细胞培养第3天即可传代,前3代细胞表型稳定,增殖力良好;第4代开始出现细胞分化的迹象。不同批次实验的一致性检验结果稳定。结论采用单纯胶原酶消化配合吹打法能够在短时间内获取大量纯化且增值能力旺盛的软骨细胞。

A method for cultivating high proliferative chondrocytes in rats

Objective To explore a new method for cultivating chondrocytes in rats.Methods Chondrocytes were extracted form cartilages of knee,shoulder,elbow and thighbone in newly born(within 24 hours) SD rats,and cultivated by digesting with collagenase alone and shaking by pipette;the chondrocytes were observed by microscope,stained by toluidine blue and alcian blue,and differentiated by type II collagen immunofluorescence and proliferating cell nuclear antigen immunofluorescence.A consistency test was performed for different batches of experimental outcomes.Results Primary cells could passage since cultured from the 3rd day,the first three generations of cells were stable and with good proliferation;the 4th generation began to show signs of cell differentiation.Experimental consistency of different batches were stable.Conclusion Combined method of collagenase digestion with shaking by pipette can culture large number of highly purified and proliferative chondrocytes in a short time.