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目的探讨高重力对前成骨细胞MC3T3-E1形态和成骨功能的影响。方法将MC3T3-E1细胞按不同高重力分为对照组、5 g组、10 g组、15 g组、20 g组,实验组每次加载30 min连续加载3 d,对照组同步置于除g值外相同的环境中。鬼笔环肽染色考察细胞骨架蛋白形态;碱性磷酸酶(alkaline phosphatase,ALP)活性检测试剂盒检测ALP含量;实时定量PCR检测ALP、Ⅰ型胶原(collagenⅠ,ColⅠ)、骨钙素(osteocalcin,OC)、runt相关转录因子(runt-related transcription factors,Runx2)的基因表达;Western blotting检测ColⅠ、OC蛋白表达。结果高重力条件下成骨细胞胞体变薄,细胞表面积明显增大;细胞骨架排列结构变得疏松、松散,微丝肌动蛋白束状结构减少,微丝呈弥散状,有序性降低。成骨分化相关指标ALP、ColⅠ、OC、Runx2的基因均显著上调;ColⅠ和OC蛋白亦呈现高表达。对照组茜素红染色只有极少量面积很小的桔红色结节,高重力加载后各组细胞明显形成面积大小不一的桔红色结节。结论高重力下成骨细胞可通过骨架结构发生重排引起细胞形态的改变,并进一步通过上调分化相关基因和蛋白表达,有效刺激成骨细胞成熟分化。
Objective To investigate the effects of high gravity on the morphology and osteogenesis of pre-cultured osteoblasts MC3T3-E1. Methods MC3T3-E1 cells were divided into control group, 5 g group, 10 g group, 15 g group and 20 g group with different high gravity. Value outside the same environment. Phalloidin staining was used to examine the morphology of cytoskeletal proteins. ALP activity was detected by alkaline phosphatase (ALP) activity assay kit. ALP, collagenⅠ (ColⅠ), osteocalcin OC) and runt-related transcription factors (Runx2). Western blotting was used to detect the expression of Col I and OC. Results Under high gravity conditions, the osteoblasts became thinner and the cell surface area increased obviously. The arrangement of cytoskeleton became loose and loose, the actin bundles decreased, while the microfilaments showed diffuse and orderly decrease. The genes related to osteogenic differentiation ALP, ColⅠ, OC, Runx2 were significantly up-regulated; Col Ⅰ and OC proteins also showed high expression. Alizarin red staining of the control group only a very small amount of very small orange nodules, high gravity loading of each group of cells was significantly different size of the orange-red nodules. Conclusion The osteoblasts under high gravity can cause cell morphological changes by rearrangement of skeletal structure, and further stimulate differentiation and maturation of osteoblasts by up-regulating differentiation-related genes and protein expression.