人正常软骨细胞和骨性关节炎软骨细胞体外培养对照研究

目的:对人正常软骨细胞和骨关节炎软骨细胞进行体外培养,并就其2组在传代过程中生物学特性变化进行比较和评价。方法:酶两步顺序消化法消化关节软骨分离细胞;体外传代培养观察细胞形态、生长,MTT法测定增殖,甲苯胺蓝和Ⅱ型胶原免疫组化染色,从蛋白和基因水平检测Ⅱ型胶原、蛋白多糖的变化,以及用流式细胞仪检测细胞凋亡。结果:①骨性关节炎软骨细胞形态似成纤维细胞,生长速度明显较正常软骨细胞慢。②MTT检测骨关节炎组2、4、6代软骨细胞增殖均低于正常组(P0.05),Ⅱ型胶原免疫组化和甲苯胺蓝O染色均较弱,传至第4代软骨细胞生物学特性基本消失。③2、4代骨关节炎软骨细胞凋亡率明显高于正常软骨细胞(P0.05)④各代骨性关节炎软骨细胞Ⅱ型胶原和蛋白多糖表达量都不如正常软骨细胞。结论:体外培养的人骨性关节炎软骨细胞符合软骨细胞退变的表现,能很好的为骨关节炎细胞水平的研究提供最佳实验对象。

Comparison of Chondrocytes Isolated from Healthy People and Osteoarthritis Patients in vitro

Objective：To study the differences of cultivation and biological characteristics of chondrocytes isolated from health people and osteoarthritis patients.Methods： Two-step enzyme digestion was used to isolate chondrocytes.During the cultivation in vitro,the shape and growth changes of the chondrocytes were observed.MTT assay was performed to observe cell proliferation.Toluidine blue staining and immunohistochemical staining were applied to observe the expression of type II collagen.Flow cytometry was performed to detect cell apoptosis.Results： ①Osteoarthritis chondrocytes was similar to fibroblasts in shape,and grew slower than normal chondrocytes.② MTT showed that the OD value of osteoarthritis chondrocytes of the 2nd,4th and 6th generation were lower than that of normal chondrocytes（P〈0.05）.When in the 2nd generation,type II collagen immunohistochemical staining and toluidine blue O staining were weakly positive.The biological characteristics of cartilage cells disappeared in 4th generation.③ The percentage of apoptotic cells of healthy people were significantly lower than that of osteoarthritis patients in the 2nd and 4th generations（P〈0.05）.④ The expressions of type II collagen and proteoglycan in normal chondrocytes were higher than those of osteoarthritis patients.Conclusion：In vitro cultivation of human osteoarthritis chondrocytes are in accordance with cataplasia,thus it can be a good cell model for osteoarthritis study.