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

发布时间：2019-05-08 08:43

【摘要】：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。
[Abstract]:Aim: to investigate the effect of follicle-stimulating hormone 尾 (FSH 尾) on osteoblastic differentiation of mouse embryonic fibroblasts (mouse embryonic fibroblasts,MEFs) induced by bone morphogenetic protein 9 (BMP 9). Methods: recombinant adenoviruses (Ad-GFP,Ad-FSH 尾 and Ad-BMP9) expressing green fluorescent protein (green fluorescent protein,GFP), FSH 尾 and BMP9 were constructed by Ad Easy system. The experimental groups were AdGFP group, Ad-BMP9 group, Ad-FSH 尾 group and Ad-BMP9 Ad-FSH 尾 group. The recombinant adenoviruses were divided into three groups: AdGFP group, Ad-BMP9 group, Ad-FSH 尾 group and Ad-BMP9 Ad-FSH 尾 group. The protein expression levels of transcription factors (Runx2 and DLX5) in the early stage of osteogenic differentiation were detected by chemiluminescence quantitative analysis and histochemistry staining, respectively. Alkaline phosphatase (alkaline phosphatase,ALP) activity; Western blot was used to detect the expression of transcription factors (Runx2 and DLX5) in the early stage of osteogenic differentiation. RT-PCR was used to detect the expression of bone troponin (osteocalcin,OCN) and osteopontin (osteopontin,OPN) in the late osteogenic differentiation stage, alizarin red staining was used to detect calcium salt deposition, and flow cytometry was used to detect the cell cycle distribution in each experimental group. Results: in MEFs, overexpression of FSH 尾 significantly promoted BMP9 induced MEFs ossification on the 3rd day (6 874.67 卤224.93 vs.11 935.67 卤568.76, P < 0 000) and the fifth day (43 434.67 卤2 047.57 vs.98 913.33 卤4 606.26, P < 0 000). On the 7th day, ALP activity, transcription factor Runx2 (1.171 卤0.105 vs.1.350 卤0.053, P < 0.01), DLX5 (1.382 卤0.123 vs.1.738 卤0.286), ALP activity (38 198.00 卤2 376.43 vs.78 534.67 卤2 114.56, P < 0.05), and transcription factor Runx2 (1.171 卤0.105 vs.1.350 卤0.053, P < 0.05), DLX5 (1.382 卤0.123 vs.1.738 卤0.286) were detected. OCN (1.300 卤0.045 vs.1.471 卤0.046, 0.002), OPN (1.300 卤0.084 vs.1.479 卤0.038, P = 0.016) and calcium deposition. In addition, FSH 尾 and / or BMP9 had no significant effect on the cell cycle of MEFs (G1: F = 0.226, P = 0.876 / G _ 2: F = 0.147, P = 0.929 / S: F = 0.027, P = 0.994). Conclusion: without affecting the cell cycle of MEFs, FSH 尾 combined with BMP9 can induce osteoblastic differentiation of MEFs.
【作者单位】：重庆医科大学附属第一医院生殖健康与不孕症中心;
【基金】：重庆市科学技术委员会资助项目(编号:cstc2012jj A10113)
【分类号】：R580

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