骨折修复过程中骨形态发生蛋白2对趋化因子12表达的调控及其意义

Regulation mechanism of BMP2 on CXCL12 expression in fracture repair and its clinical significance

目的探讨骨形态发生蛋白2(bone morphogenetic protein,BMP2)对趋化因子12(chemokine C-X-C motif-ligand-12,CXCL12)表达调节及其在骨折修复过程中的意义。方法构建BMP2敲除鼠BMP2^(cKO/+)和BMP2^(cKO/cKO)。免疫组化分析正常小鼠、BMP2^(cKO/+)小鼠及BMP2^(cKO/cKO)小鼠胫骨骨折模型中骨内膜细胞CXCL12的表达及其随时间的变化。实时定量PCR(RT-q PCR)比较对照组与BMP2^(cKO/+)小鼠骨内膜细胞及其分化细胞CXCL12、骨钙蛋白、α-SMA表达差异。结果BMP2^(cKO/+)和BMP2^(cKO/cKO)小鼠BMP2显著低于对照组。小鼠骨折修复过程中骨内膜细胞和成骨细胞CXCL12表达明显升高,且表达CXCL12细胞先增多再减少;BMP2^(cKO/+)小鼠骨折修复过程中表达CXCL12的骨内膜细胞和成骨细胞逐渐增加。BMP2^(cKO/+)小鼠分离的骨内膜细胞CXCL12表达显著高于正常小鼠。在诱导分化的小鼠骨内膜细胞中添加rh BMP2,CXCL12的表达减少,骨钙蛋白和α-SMA表达显著增加。诱导分化的BMP2^(cKO/cKO)骨内膜细胞CXCL12表达显著高于正常小鼠,而骨钙蛋白的表达则显著降低。CXCL12受体拮抗剂AMD3100处理诱导分化的BMP2^(cKO/cKO)骨内膜细胞,骨钙蛋白和α-SMA表达显著增加。结论骨折修复过程中,BMP2下调CXCL12的表达有助于成骨细胞的分化从而促进骨折的修复。

Objective To investigate regulation mechanism of bone morphogenic protein 2（ BMP2） on chemokine C-X-C motif-ligand-12（ CXCL12） expression in the fracture repair and its clinical significance. Methods Knockout mice of BMP2（ BMP2 ^cKO/＋ and BMP2 ^cKO/cKO） were constructed. Immunohistochemistry methods were used to analyze the cells with CXCL12 expression and the timely changes in the controlled, BMP2^cKO/＋ and BMP2^cKO/cKO mice bone fracture repair models. Real time-q PCR was used to compare the expression changes of CXCL12, osteocalcin and α-SMA gene in endosteal cells and differential endosteal cells isolated from the controlled, BMP2 ^cKO/＋and BMP2 ^cKO/cKO mice. Results The expression of BMP2 was significantly lower in BMP2 ^cKO/＋ and BMP2 ^cKO/cKO mice than the controlled mice. CXCL12 was expressed in the endosteal cells and some osteocytes during mice fracture repair, and the cells with CXCL12 expression increased at the beginning and then decreased. The endosteal cells and osteocytes which expressed CXCL12 increased gradually in BMP2^cKO/＋ mice. CXCL12 expression in endosteal cells isolated from BMP2 ^cKO/＋ mice was higher than that cells from the controlled mice. In isolated endosteal cells, rh BMP2, while inducing osteoblastic differentiation, stimulated expression of osteocalcin and α-SMA that was coupled with a decrease of CXCL12 expression significantly. In isolated BMP2 ^cKO/cKO endosteal cells, high CXCL12 expression and low osteocalcin expression were observed. In isolated BMP2^cKO/cKO endosteal cells with AMD3100（ an antagonist of CXCL12 receptor） treatment, osteocalcin and α-SMA e xpression increased. Conclusions In fracture repair process, BMP2 leads to the downregulation of CXCL12 expression that would facilitate endosteal cells differentiating into osteoblasts and promote fracture healing.