成纤维细胞生长因子受体2持续增强对骨髓间充质干细胞软骨成骨的影响

Continuously enhanced function of fibroblast growth factor type Ⅱ receptor mutation affects the endochondral ossification of bone mesenchymal stem cells in mice

目的利用基因敲人技术建立模拟人Apert综合征的成纤维细胞生长因子受体2（FGFR2）$252W小鼠模型，探讨FGFR2功能持续增强的小鼠骨髓间充质干细胞（BMSCs）成软骨诱导分化后表达特征。方法于出生后6周获取BMSCs增殖进行检测。取生长良好的第2代细胞成软骨诱导，对诱导后14d进行阿利新蓝、茜素红染色，对成软骨、成骨基因进行定量分析，并对信号通路细胞外信号调节激酶（Erk）1／2磷酸化观察。结果FGFR2功能突变小鼠BMSCs增殖速度减慢。体外成软骨诱导条件下，突变组BMSCs阿利新蓝和茜素红染色程度下降。定量分析突变组成软骨相关基因是野生组80％（Colx）、88％（ColⅡ）（P〈0．05），成骨表达基因是野生型1．23（OC）、1．38（OP）倍（P〈0．05）。FGFR2功能持续增强使BMSCsErkl／2磷酸化增强。结论FGFR2可能不仅具有调控成骨细胞株发育的功能，还具有调控软骨细胞株发育的重要功能。FGFR2功能持续增强导致小鼠BMSCs增殖减慢，成软骨发育障碍，虽对成骨分化增强，却抑制矿化过程，这其中机制可能与Erk1／2通路增强有关。

Objective To study the chondrogenic ability of bone mesenchymal stem cells （BMSCs） of wild type mice and fibroblast growth factor type I1 （FGFR2） S252W mutant type and to ex- plore the influence of persistent enhanced FGFR2 function by a new knock-in mouse model with the FGFR2 S252W. Methods Six week-old mice were sacrificed and BMSCs were isolated and cultured in vitro. Pro- liferation, and chondrogenic and osteogenic ability were measured by Alcian blue and Alizarin red staining in chondrogenic differentiation medium. Reverse transcription-polymerase chain reaction （RT-PCR） was applied to analyze differentiation of chondrocytes and osteoblasts. The level of extracellular signal regulated kinase 1/2 （Erkl/2） phosphorylation was detected by using Western blotting. Results The proliferation of second-generation BMSCs of mutant type was slower. After been cultured in chondrogenic differentiation medium, both Alcian blue and Alizarin red staining showed decreased staining cells in cultured FGFR2 S252W BMSCs compared with wild type BMSCs on the day 14. The mRNA expression rate of chondrocytes differentiation gene in mutant group was 80% （ Col X） and 88% （ Col II） of wild group, while that of osteo- blast differentiation gene showed 1.23 （OC）, and 1.38 （OP） times, respectively. The activity of Erkl/2 pathway of FGFR2 $252W was enhanced. Conclusion FGFR2 may have the function to regulate both chondrocyte and osteoblast lineages development. Continuously enhanced FGFR2 function may inhibit the proliferation of BMSCs and suppress their chondrogenic differentiation, however the differentiation of osteo- blasts in FGFR2S252W mice was accelerated, and the mineralization was decreased. More importantly, the results indicated that these effects are probably mediated by the Erkl/2 signaling pathway.