成纤维生长因子受体2对小鼠骨髓间充质干细胞成骨分化能力的影响

Effect of fibroblast growth factor receptor 2 on osteogenic differentiation of mouse bone marrow mesenchymal stem cells

目的观察成纤维生长因子受体2(FGFR2)对小鼠骨髓间充质干细胞成骨分化能力的影响。方法过表达小鼠骨髓间充质干细胞(BMSCs)的FGFR2基因,噻唑蓝(MTT)法检测细胞24、48、72 h增殖能力。过表达FGFR2基因的小鼠骨髓间充质干细胞经成骨诱导分化14 d后,茜素红染色和碱性磷酸酶(ALP)染色法检测细胞成骨分化和矿化能力,Western blot检测成骨分化相关基因骨钙蛋白(OCN)、骨桥蛋白(OPN)、Runt相关基因2(Runx2)、ALP、骨形态发生蛋白-2(BMP-2)蛋白表达水平。建立去势骨质疏松症小鼠模型,动物水平研究上调FGFR2对小鼠骨质疏松症的影响,mircoCT评价细胞骨小梁数目和骨体积分数。结果上调FGFR2促进BMSCs在24、48、72 h细胞增殖能力,FGFR2上调组小鼠BMSCs钙结节数量(95.7±8.9)高于对照组(26.4±3.9),差异有统计学意义。ALP染色结果显示FGFR2上调组成骨分化能力强于对照组,ALP活性定量分析结果显示,FGFR2上调组ALP活性[(6.33±1.33)U/L]强于对照组[(2.54±0.75) U/L],差异有统计学意义(P<0.05),FGFR2上调组小鼠BMSCs成骨分化基因OCN、OPN、Runx2、ALP、BMP-2蛋白表达水平高于对照组。FGFR2上调组小鼠胫骨骨小梁数目(Tb.N)[(2.37±0.26)/mm]高于对照组[(1.55±0.34)/mm],FGFR2上调组小鼠胫骨骨体积分数(BV/TV)[(9.97±1.77)%]高于对照组[(5.91±0.98)%]。结论上调FGFR2可提高小鼠骨髓间充质干细胞增殖活性和成骨分化能力,可能有促进骨质疏松症恢复的作用。

Objective To observe the effect of fibroblast growth factor receptor 2 (FGFR2) on the ability of bone marrow mesenchymal stem cells (BMSCs) to differentiate into osteoblasts. Methods The FGFR2 gene of mouse BMSCs was overexpressed, and the proliferation ability at 24, 48 and 72 h was measured by methyl thiazol tetrazolium (MTT) assay. After the FGFR2 gene was overexpressed, the osteogenic differentiation and mineralization ability of the cells was examined by alizarin red staining and alkaline phosphatase (ALP) staining, and Western blotting was used to detect the expression of osteocalcin (OCN), osteopontin (OPN), runt related transcription factor-2 (Runx2), ALP and bone morphogenetic protein-2 (BMP-2) protein related genes of osteogenic differentiation after 14 days of osteogenesis induced differentiation. A mouse model of castrated osteoporosis was established, and the effect of up-regulation of FGFR2 on osteoporosis in mice was studied. The number of bone trabeculae and bone volume fraction were evaluated by mircoCT. Results The proliferation of BMSCs at 24, 48 and 72 h was promoted after up-regulation of FGFR2. The number of calcium nodules in BMSCs (95.7±8.9) in the FGFR2-up-regulated group was significantly greater than that in the control group (26.4±3.9). The results of ALP staining showed that the osteogenic differentiation ability in the FGFR2-up-regulated group was stronger than that in the control group. The quantitative analysis of ALP activity showed that the ALP activity in the FGFR2-up-regulated group [(6.33±1.33) U/L]was significantly stronger than that in the control group [(2.54±0.75) U/L]. The expression levels of OCN, OPN, UNX2, ALP and BMP-2 proteins in the FGFR2-up-regulated group were higher than those in the control group. The trabecular number (Tb.N) of bone trabecula in the FGFR2 up-regulated group [(2.37±0.26)/mm]was greater than that in the control group [(1.54±0.34)/mm], and the tibial bone volume fraction (BV/TV) in the FGFR2-up-regulated group [(9.97±1.77)%] was higher than that in the control group [(5.91±0.98)%]. Conclusion Upregulation of FGFR2 can promote the proliferation and osteogenic differentiation of mouse BMSCs, which may promote the recovery of osteoporosis.