Pellet法和Micromass法诱导软骨终板干细胞成软骨的比较  

Chondrogenic differentiation of cartilage endplate derived stem cells(CESCs) cultured in the micromass culture and in the pellet culture

作　　者：刘兰涛[1,2] 朱瑜洁[3] 黄博[1] 李长青[1] 庄颖[1] 周跃[1] 

机构地区：[1]第三军医大学附属新桥医院骨科 [2]海军92635部队 [3]四川航空股份有限公司飞行部航医室

出　　处：《中国矫形外科杂志》2013年第1期75-81,共7页Orthopedic Journal of China

摘　　要：[目的]比较Pellet法和Micromass法诱导软骨终板干细胞成软骨的能力。[方法]临床获取腰椎退变椎间盘软骨终板,采用琼脂糖筛选软骨终板干细胞后进行传代扩增,扩增后的软骨终板干细胞分别行Pellet和Micromass法进行成软骨诱导1～3周。分别于1～3周对诱导的软骨组织进行湿重和阿利新蓝密度比较,同时对诱导3周的软骨组织进行特殊染色,Ⅰ、AGG、Ⅱ型胶原免疫化学染色以及定量PCR检测软骨相关基因的比较。[结果]使用Mi-cromass法诱导软骨终板干细胞所形成的软骨组织在相应时间比Pellet法重量大(P<0.05)。阿利新蓝密度比较亦提示相应时间点Micromass法所诱导的软骨组织比Pellet法高(P<0.05)。特殊染色结果分析表明采用Micromass法所形成的软骨基质较多且均质;免疫组织化学法表明Micromass法所表达的Ⅱ型胶原、AGG较Pellet法多,而Ⅰ胶原则相反。实时定量PCR结果亦提示采用Micromass法诱导所形成的软骨组织中Ⅱ型胶原、AGG基因表达高(P<0.05),而Sox 9无明显区别(P>0.05)。[结论]采用Micromass法诱导软骨终板干细胞所形成的软骨基质更加丰富和均质,因此,Micromass法更适合对软骨终板干细胞进行成软骨相关机制及信号传导的研究。[ Objective] To compare chondrogenic differentiation of cartilage endplate derived stem cells （CESCs） cultured in the micromass culture and in the pellet culture. [ Methods] The cartilage endplate （CEP） was obtained from 5 patients who underwent posterior discectomy procedure for the lumber degenerative disease. The agarose culture was used to select CESCs. The expanded CESCs were induced into chondrogenesis using micromass and pellet culture for 1, 2 and 3 weeks, respectively. After induced for 1, 2, 3 weeks, the wet weigh and alcian blue intensity were compared. The specific staining and immunohistochemical staining of collagen Ⅱ , I and aggrecan were also performed. The chondrogenic specific genes were also analyzed using real time PCR. [ Results] The wet weigh induced by micromass culture was heavier than that with pellet culture （P 〈 0. 05） at the time of week 1, 2 and 3. The alcian blue intensity was also significantly different at the time of week 1, 2 and 3 （P 〈 0. 05 ） . The specific staining indicated that the cartilage matrix induced by micromass culture was more homogenous. The immunohistochemical staining results indicated that more collage 11 and aggrecan were expressed in the micromass culture, and more collagen I was expressed in the pellet culture. The real time PCR indicated that collage 1I and aggrecan genes were expressed much more higher in the micromass cuhue than that in the pellet culture （P 〈 0. 05 ） but no difference for Sox9 （P 〉 0. 05 ） . [ Conclusion] The cartilage matrix was more abundant and homogenous using micromass culture than that using pellet culture. Thus, the micromass culture was more suitable for mechanism and signaling pathway study in the process of CESCs differentiating into the chondrocytes.

关 键 词：Pellet法 Micromass法 软骨终板 干细胞 成软骨 

分 类 号：R68[医药卫生—骨科学]