FSHβ在骨形态蛋白9诱导小鼠胚胎成纤维细胞成骨分化中的作用研究

Role of FSHβ in BMP9-induced osteogenic differentiation in mouse embryonic fibroblasts

目的:探讨卵泡刺激素β亚基(follicle-stimulating hormoneβ,FSHβ)对骨形态发生蛋白9(bone morphogenetic protein 9,BMP9)诱导小鼠胚胎成纤维细胞(mouse embryonic fibroblasts,MEFs)成骨分化中的作用。方法:采用Ad Easy系统构建表达绿色荧光蛋白(green fluorescent protein,GFP)、FSHβ和BMP9的重组腺病毒(Ad-GFP、Ad-FSHβ和Ad-BMP9),实验分组为:AdGFP组、Ad-BMP9组、Ad-FSHβ组和Ad-BMP9+Ad-FSHβ组。运用化学发光定量和组织化学染色法分别检测各组3、5、7 d碱性磷酸酶(alkaline phosphatase,ALP)活性;Western blot检测成骨分化早期转录因子(Runx2和DLX5)的蛋白表达水平;RT-PCR检测成骨分化晚期指标骨钙蛋白(osteocalcin,OCN)和骨桥蛋白(osteopontin,OPN)的m RNA表达水平;茜素红染色检测钙盐沉积;流式细胞技术检测各实验组细胞的周期分布。结果:在MEFs中,过表达FSHβ能明显促进BMP9诱导MEFs骨化过程中第3天(6 874.67±224.93 vs.11 935.67±568.76,P=0.000)、第5天(43 434.67±2 047.57 vs.98 913.33±4 606.26,P=0.000)、第7天(38 198.00±2 376.43 vs.78 534.67±2 114.56,P=0.000)的ALP活性,转录因子Runx2(1.171±0.105 vs.1.350±0.053,P=0.026)、DLX5(1.382±0.123 vs.1.738±0.286,P=0.001)和晚期成骨指标OCN(1.300±0.045 vs.1.471±0.046,P=0.002)、OPN(1.300±0.084 vs.1.479±0.038,P=0.016)的表达水平,以及钙盐沉积。此外,FSHβ和(或)BMP9对MEFs细胞周期没有明显影响(G1期:F=0.226,P=0.876;G2期:F=0.147,P=0.929;S期:F=0.027,P=0.994)。结论:在不影响MEFs细胞周期的情况下,FSHβ协同BMP9诱导MEFs成骨分化。

Objective：To study the effect of follicle-stimulating hormone β（FSHβ） on bone morphogenetic protein 9（BMP9）- induced osteogenic differentiation in mouse embryonic fibroblasts（MEFs）. Methods：AdEasy system was used for generating target re- combinant adenoviruses （Ad-GFP, Ad-FSHβ, and Ad-BMP9）, and our investigation was designed into four groups ： Ad-GFP group, Ad-BMP9 group, Ad-FSHβ group, and Ad-BMP9＋Ad-FSHβ group. Chemiluminescence technique and histochemical staining were conducted to assay alkaline phosphatase（ALP） activity on day 3,5 ,and 7 ,Western blot to test the protein expression of Runx2 and Dlx5, RT-PCR to measure the mRNA expression of osteocalcin（OCN） and osteopontin（OPN）, and Alizarin Red S staining to detect the matrix mineralization. In addition, Flow cytometry was utilized for analysing the cell cycle of different groups. Results： Exogenous expression of FSHβ dramatically enhanced the ALP activity on day 3 （6 874.67 ± 224.93 vs. 11 935.67 ± 568.76,P=0.000）,day 5 （43 434.67±2 047.57 vs. 98 913.33±4 606.26,P=0.000）,and day 7（38 198.00 ± 2 376.43 vs. 78 534.67 ± 2 114.56,P=0.000）, and the protein expression of Runx2（ 1.171 ± 0.105 vs. 1.350 ± 0.053,P=0.026） and DLX-5（1.382 ± 0.123 vs. 1.738 ± 0.286,P=-0.001 ）,and the mRNA expression of late osteogenic makers,namely OCN（1.300 ± 0.045 vs. 1.471 ± 0.046,P=0.002） and OPN（ 1.300± 0.084 vs. 1.479± 0.038, P=0.016）, and matrix mineralization induced by BMP9 in MEFs. Moreover, efficient infection of Ad- FSHI3 or/and Ad-BMP9 remains no apparent influence on cell cycle in MEFs （ G1 -phase ： F=0.226, P=0.876 ; G2-phase ： F=0.147, P=0.929 ; S-phase： F=0.027, P=0.994）. Conclusion ： FSHβ promotes BMP9-induced osteogenic differentiation without substan- tial effect on cell cycle in MEFs.