骨形态发生蛋白7在高糖环境下对成骨细胞分化的影响

目的:研究对于重组人骨形态发生蛋白7(bone morphogenetic protein7,BMP7)在持续稳定高糖环境下对成骨细胞生物学性能及成骨活性的影响。方法:细胞取小鼠颅顶前成骨细胞亚克隆14(MC3T3-E1Subclone 14,MC3T3),分为4组:正常生理糖浓度组(葡萄糖浓度为5.5 mmol/L)为对照组,生理糖浓度+BMP7组高糖组(葡萄糖浓度为5.5 mmol/L,BMP7浓度为100 μg/L)(葡萄糖浓度为25 mmol/L),高糖+BMP7组(葡萄糖浓度为25 mmol/L,BMP7浓度为100 μg/L)。甲基噻唑基四唑(MTT)法检测不同条件培养后1 d、3 d、5 d、7 d后的细胞增殖情况,成骨诱导检测细胞碱性磷酸酶(alkaline phosphatase,ALP)活性。罗丹明-鬼笔环肽染色后以激光共聚焦显微镜观察MC3T3细胞骨架于BMP7作用24h后的形态变化,荧光实时定量PCR(quantitative real-time PCR,RT-qPCR)检测BMP7作用48 h后成骨基因特异性转录因子2(Runx2),骨钙素(osteocalcin,OCN)、碱性磷酸酶(ALP)的mRNA表达。结果:MTT实验显示25 mmol/L葡萄糖可抑制MC3T3增殖(P<0.05),加入BMP7后可提高细胞增值率(P<0.05)。并可上调高糖导致的ALP活性下降。细胞骨架观察结果显示,对照组细胞骨架成束状铺开,交织成网,较为均匀。高糖组细胞微丝解聚成团状,模糊不清。RT-qPCR结果显示BMP7可促进MC3T3细胞的ALP、OCN及Runx2(P<0.05)的表达。结论:持续稳定高糖环境能够抑制成骨细胞的增殖及ALP活性,影响细胞骨架结构,抑制成骨基因表达,添加BMP7可以在不同程度上反转该趋势,但仍较正常水平低。

Effects of Bone Morphogenetic Protein 7 on MC3T3-E1 Osteoblasts in High Glucose Condition

Objective: To investigate the effects of bone morphogenetic protein7(BMP7) on biological properties of osteoblasts in high glucose circumstance. Methods: MC3T3-E1 Sub-clone 14 cells were cultured and divided into four groups according to the different culture medium:normal glucose concentration of 5.5 mmol/L;normal glucose concentration of 5.5 mmol/L and BMP7 (100 μg/L); high glucose concentration of 25 mmol/L;high glucose concentration of 25 mmol/L and BMP7(100 μg/L),respectively. The cell proliferation was measured by MTT assay 1d,3d,5d and 7d after exposure. The alkaline phosphatase (ALP) activity was used to detect the differentiation of MC3T3-E1 cells. The Alizarinred dye wasused to observe the number of calcium nodes at 21d. The F-actin cytoskeleton of MC3T3-E1 was stained with Rhodamine,then examined under a confocal laser scanning microscope 24h after exposure to BMP7. The mRNA expressions of ALP, osteocalcin(OCN) and Runx2 were quantified by real-time PCR(RT-qPCR) 48h after exposure to BMP7. Results: The MTT, ALP assays showed that high glucose(25mmol/L) inhibited MC3T3-E1 proliferation and ALP activity(P＜0.05),also decreased mRNA expression of ALP, OCN and Runx2,the addition of BMP7 significantly increased ALP, OCN and Runx2 expression. In the cells exposed to high glucose, F-actin cytosksleton started to change with disruptive structures. Conclusion: High glucose concentration significantly inhibited the proliferation andALP activity,destroyed F-actin cytoskeleton structure,and inhibited the expression of osteoblasticgene. BMP7 could promote the cell mineralization under such high glucose concentration environment.