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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 investigate the effect of follicle-stimulating hormone β (FSHβ) on osteogenic differentiation of mouse embryonic fibroblasts (MEFs) induced by bone morphogenetic protein 9 (BMP9) effect. Methods Ad-GFP, Ad-BMP9 and Ad-BMP9 were transfected into Ad-BMP9 cells by using Ad Easy system. AdGFP group and Ad-BMP9 Group, Ad-FSHβ group and Ad-BMP9 + Ad-FSHβ group. The activity of alkaline phosphatase (ALP) on the 3rd, 5th, 7th day was detected by chemiluminescence quantification and histochemical staining. The protein expression of early osteogenic differentiation factors (Runx2 and DLX5) The mRNA expression levels of osteocalcin (OCN) and osteopontin (OPN), a marker of osteogenic differentiation, were detected by RT-PCR. Calcium deposition was detected by alizarin red staining and flow cytometry The periodic distribution. RESULTS: Overexpression of FSHβ in MEFs significantly promoted BMP9-induced ossification of MEFs on day 3 (6874.67 ± 224.93 vs.11 935.67 ± 568.76, P = 0.000), on day 5 (43 43.67 ± 2.047.57 vs. 98 913.33 ± 4 606.26, P = 0.000), ALP activity on day 7 (38 198.00 ± 2 376.43 vs.78 534.67 ± 2 114.56, P = 0.000), the transcription factor 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), and late osteogenic index 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), as well as calcium salt deposition. In addition, FSHβ and / or BMP9 had no significant effect on MEFs cell cycle (G1 phase: F = 0.226, P = 0.876; G2 phase: F = 0.147, P = 0.929; S phase: F = 0.027, P = 0.994). CONCLUSIONS: FSHβ cooperates with BMP9 to induce osteoblast differentiation of MEFs without affecting the cell cycle of MEFs.