摘要
目的基因敲入技术建立成纤维细胞生长因子受体2（FGFR2）功能持续增强小鼠模型（FGFR2S252W/＋），观察p38信号通路在FGFR2功能持续增强对软骨内成骨过程的影响。方法获取出生后6周小鼠骨髓间充质干细胞（BMSCs）进行体外培养并进行成软骨诱导。Westernblot检测p38信号蛋白，逆转录．聚合酶链反应（RT-PCR）比较野生型及突变型Ⅱ型胶原（Col2）、X型胶原（Col10）、OC、OP基因表达，加入p38信号通路阻滞剂SB203580后再次比较相关基因表达。体外胚胎骨培养观察p38信号通路在FGFR2功能持续增强对软骨内成骨过程的影响。结果体外BMSCs成软骨诱导后，FGFR2功能突变小鼠BMSCs表达p38蛋白磷酸化增强，表达Col2、Col10减弱，但OC、OP基因表达强于野生型；加入p38信号通路阻滞剂SB203580后，BMSCs基因表达量明显增高，表达c012、Col10为原来1．30±0．07、1．94±0．13，表达OC、OP为原来1．97±0．17、1．50±0．10。胚胎骨体外培养可见SB203580治疗能纠正软骨内成骨发育障碍，使胫骨的总长度及钙化组织长度明显增长。结论FGFR2下游p38信号通路对软骨内成骨过程影响巨大，其信号通路阻滞剂对软骨内成骨发育障碍有救治作用。
Objective To study the role of p38 signal pathway in the endochondral ossification of bone mesenchymal stem cells （BMSCs） of wild type mice and mutant type fibroblast growth factor receptor 2 （FGFR2）s252w/＋ ）, and to explore the mechanism of p38 signal pathway in persistent enhanced FGFR2 function on development of mice BMSCs by a knock-in mouse model with the FGFR2sa52w/＋. Methods The BMSCs were isolated from 60week-old mice. Western blotting was used to compare the level of P-p38 and p38, and the reverse transcription-polymerase chain reaction （RT-PCR） was applied to detect the genes of Co12, CollO, OC and OP in chondrogenic differentiation medium of BMSCs. The cultured BMSCs were treated with SB203580, the expression of genes was compared, and the in vitro culture of long bones was utilized to detect the role of p38 signal pathway in the endochondral ossification by FGFR2 mu tant. Results The activity of p38 signal pathway of FGFR2s252w/＋ was enhanced. After culture in chondrogenic differentiation medium, the mRNA expression of collagentype Ⅱ （Co12） and collagentype X （CollO） in BMSCs from mutant group was decreased, and that of OC and OP was increased. After treatment with SB203580, the expression of these genes was increased, SB203580 group express Co12, Coll0 were 1.30 ±0. 07 and 1.94 ± 0. 13 times the MT group, expres OC, OP were 1.97 ±0. 17,1.50 ±0. 10 times the MT group. Using in vitro culture of long bones, it was found the retardation of total length growth of long bones was rescued by SB203580 treatment, suggesting that p38 signal pathways was responsible for the retarded long bone development in FGFR2s252w/ mice. Conclusion The results indicated that these effects are mediated by the p38 signal pathway. Furthermore, the retardation of long bones has also been rescued by SB203580 treatment, suggesting that p38 signal pathway was responsible for the retarded long bone development in FGFR2s252w/＋ mice.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2014年第7期1402-1405,共4页
Chinese Journal of Experimental Surgery
基金
国家重点基础研究发展计划（973计划）资助项目（2011CB964701）
