早期兔骨关节炎软骨CD105＋／CD166＋细胞及其软骨分化潜能的实验研究

EXPERIMENTAL STUDY ON CD105^+/CD166^+ CELLS AND ITS CHONDROGENIC POTENTIAL IN EARLY OSTEOARTHRITIS CARTILAGE

目的探讨早期骨关节炎(osteoarthritis,OA)软骨组织中CD105+/CD166+细胞的变化规律及其软骨分化潜能,为软骨自身修复机制的研究奠定实验基础。方法 8～12月龄成年新西兰大白兔30只,建立右膝OA模型。将实验分为5组,分别获取左膝关节正常软骨细胞(A组),造模后2、4、8周右膝OA软骨细胞(分别为B、C、D组)以及兔BMSCs(E组)。将各组培养细胞标记CD105和CD166,流式细胞仪分析CD105+/CD166+细胞百分比;并采用免疫磁珠分选法将CD105+/CD166+细胞分选出来。激光共聚焦显微镜观察CD105和CD166在各组细胞中的表达分布情况;成软骨诱导培养后分别行阿尔新蓝细胞化学染色和Ⅱ型胶原免疫组织化学染色;成骨诱导培养后检测各组钙含量;成脂诱导培养后行油红O染色。结果 A、B、C、D组CD105+/CD166+细胞百分比显著低于E组(P<0.05);B、C、D组高于A组(P<0.05),D组显著高于B、C组(P<0.05),但B、C组间差异无统计学意义(P>0.05)。共聚焦显微镜观察示,A、B、C、D组软骨细胞及E组BMSCs中均可见CD105+、CD166+细胞,各组间无明显差异。各组细胞微球成软骨培养1周后,CD105+/CD166+细胞均可见蛋白多糖和Ⅱ型胶原阳性表达,各组间无明显差异。成骨诱导培养2周时,E组钙含量显著高于A、B、C、D组(P<0.05),A、B、C、D组间比较差异无统计学意义(P>0.05)。成脂诱导培养4周,E组可见较多红色脂滴;A、B、C、D组红色脂滴数量较E组少,成片状,大小不一,A、B、C、D组间无差别。结论早期兔OA软骨组织中CD105+/CD166+细胞增多,这些细胞有软骨细胞分化潜能。

Objective To study the variation of CD105＋/CD166＋ cells and its multilineage potential in early osteoarthritis （OA） cartilage so as to lay a foundation for cartilage repair and pathologic cartilage remodeling in arthritis. Methods The knee OA model was established in the right knee of 30 adult New Zealand rabbits （8-12 months old）. The chondrocytes were harvested from normal cartilage of the left knee （group A）, OA cartilage of the right knee at 2 weeks （group B）, at 4 weeks （group C）, and at 8 weeks （group D） after modeling, and BMSCs were used in group E for the expression of CD105 and CD166. The percentage of CD105＋/CD166＋ cells in each group was counted by flow cytometry, and CD105＋/CD166＋ cells were isolated and purified by magnetic-activated cell sorting. The expressions of CD105 and CD166 were observed in 5 groups by laser scanning confocal microscope. Chondrogenesis, osteogenesis, and adipogenesis were evaluated with Alcian blue cytochemistry and collagen type II immunohistochemistry, by detecting the deposition of calcium, and with oil red O staining, respectively. Results The percentage of CD105＋/CD166＋ cells in group A, B, C, and D was significantly lower than that in group E （P 〈 0.05）; it was significantly higher in groups B, C, and D than in group A （P 〈 0.05）, and in group D than in groups B and C （P 〈 0.05）, but no significant difference was found between groups B and C （P 〉 0.05）. Laser scanning confocal microscope results confirmed the expressions of CD105＋ and CD166＋ cells in groups A, B, C, D, and E, no obvious difference in expression was shown among 5 groups. At 1 week after chondrogenic induction, positive expressions of proteoglycan and collagen type II were observed in 5 groups, no obvious difference was noticed among 5 groups. At 2 weeks after osteogenic induction, calcium level in group E was significantly higher than that in groups A, B, C, and D （P 〈 0.05）, but no significant different was found among groups A, B, C, and D （P 〉 0.05）. At 4 weeks after adipogenic induction, there were more red lipid droplets in group E than in groups A, B, C, and D. Conclusion CD105＋/CD166＋ cells in early OA cartilage increase, which show chondrogenic differentiation potential.