高重力对成骨细胞骨向分化功能的影响

Effect of hypergravity on osteogenic differentiation of osteoblasts

【目的】了解力学因素在骨组织重建过程中的作用,探讨高重力对前成骨细胞(mouse embryonic osteoblast precursor cells,MC3T3-E1)成骨功能的影响,为组织工程中研究高重力对骨组织重建的影响提供理论依据。【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。ALP活性染色试剂盒检测ALP活性;ALP活性检测试剂盒检测ALP含量;实时定量PCR检测碱性磷酸酶(alkaline phosphatase,ALP)、Ⅰ型胶原(Ⅰcollagen,COLⅠ)、骨钙素(osteocalcin,OC)、runt相关转录因子(runt-related transcription factor,RUNX2)的基因表达;western blotting检测COLⅠ、OC、RUNX2蛋白表达。【结果】高重力条件下ALP活性显著增高,成骨分化相关指标ALP、COLⅠ、OC、RUNX2的基因均显著上调;COLⅠ和OC、RUNX2蛋白亦呈现高表达。各实验组细胞茜素红染色未见桔红色结节。【结论】高重力可促进成骨细胞骨向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 study the main function of mechanical factors in the process of reconstructing bone tissues, explore the effect of hypergravity on the osteogenesis function of preosteoblasts（MC3T3-E1）, and provide theoretical basis for studying the effect of hypergravity on bone-tissue reconstruction in tissue engineering.【Methods】According to the different loading forces of hypergravity,the cultured MC3T3-E1 cells were divided into 5 groups： control group, 5 G group, 10 G group, 15 G group and 20 G group. The cells in experimental groups were loaded in 30 min each time and lasted three successive days, while the cells in control group were synchronously exposed in the same surrounding except G-value. ALP activity staining kit was used to detect the ALP activity and content; real-time quantitative PCR was used to detect the gene expressions of alkaline phosphatase（ALP）, Ⅰ collagen（COL Ⅰ）,osteocalcin（OC） and runt-related transcription factor（RUNX2）; the protein expressions of COL Ⅰ, OC and RUNX2 were determined via western blot.【Result】Under the conditions of hypergravity, ALP activity obviously increased; indexes related with osteogenic differentiation including ALP, COL Ⅰ, OC and RUNX2 were significantly upregulated; the protein expressions of COL Ⅰ, OC and RUNX2 were high. Alizarin red staining method showed that no red-orange nodules appeared in the cells of each experimental group.【Conclusion】Hypergravity can promote the expressions of genes and proteins related with osteogenic differentiation in osteoblasts,and effectively stimulate the maturation and differentiation of osteoblasts.