摘要
目的 观察CD105-生物素-亲和素系统促进滑膜间充质干细胞的黏附和CD105阳性细胞的筛选,探讨软骨形成的效果.方法 酶消化获取滑膜干细胞,应用流式细胞仪检测细胞表面CD29、CD34、CD44、CD45、CD90、CD105的表达.分3组（A：未处理＋干细胞;B：亲和素化＋生物素化干细胞;C：亲和素化＋ CD105-生物素化干细胞）,通过细胞吸光度值和显微镜下随机视野计数分别测定不同时间点细胞黏附数量;免疫荧光检测CD105阳性细胞的筛选.成软骨诱导3周后,通过苏木素-伊红（HE）染色、阿尔新蓝染色和Ⅱ型胶原免疫荧光观察3组软骨形成.结果 滑膜干细胞贴壁生长,呈星形或梭形;CD29、CD44、CD90阳性表达,CD34、CD45阴性表达,CD105阳性率约为42％.C组和B组早期黏附细胞数量增加,与A组比较在1、2h差异有统计学意义（P＜0.05）,B组虽然多于C组,但差异无统计学意义（P＞0.05）.免疫荧光显示C组黏附的CD105阳性细胞在1、2h均明显高于B组和A组.软骨诱导培养后,3组均合成了蛋白聚糖和Ⅱ型胶原,但是C组软骨特异性细胞外基质最多.结论 CD105-生物素-亲和素系统既增加了滑膜干细胞的黏附,又筛选了CD105阳性细胞,更好的促进了软骨形成.
Objective To observe the CD105-biotin-avidin promoting synovium-derived mesenchymal stem cells （SMSCs） adhesion and selecting CD105-positive cells in the cartilage tissue engineering,and evaluate its effects on cartilage formation.Methods SMSCs were obtained by enzyme digestion.Flow cytometry was used to detect markers on cell surface,including CD29,CD34,CD44,CD45,CD90 and CD105.With divided 3 groups （A：untreated ＋ cells;B：avidin ＋ biotin-cells;C：avidin ＋ CD105-biotin-cells）,adhesion cell number was counted by absorbance and random field count at different time point.CD105-positive cells were tested through immunofluorescence.After 3 weeks chondrogenic induction,hematoxylin-eosin （HE）,alcian blue staining and immunofluorescence were used to assess chondrogenesis.Results SMSCs showed star or spindle shape with adherent growth.The expression of CD29,CD44 and CD90 was positive,while CD34 and CD45 negative.The positive rate of CD105 was about 42%.Compared with Group A,Group B and C significantly increased cells adhesion at 1 and 2 h （P 〈 0.05）.Although Group B had more cells than C,there was no statistical difference.Immunofluorescence showed CD105-positive cells in Group C were significantly higher than that in Group B and A.After chondrogenic induction,while proteoglycan and type Ⅱ collagen were observed in all groups,there was most cartilage specific extracellular matrix in Group C.Conclusion CD105-biotin-avidin system could not only enhance cells adhesion,but also select CD105-positive cells,which may promote chondrogenesis better.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2015年第5期1107-1110,共4页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目（81071468）
