流体剪切力影响成骨细胞生物学功能及分化机制的研究

Study on the influences of fluid shear stress on the biological functions and differentiation mechanism of osteoblast

摘要:

摘 要：目的 探讨流体剪切力（FSS）作用下成骨细胞生物学功能及分化机制。方法 4 组大鼠成骨细胞分别施 加 0、30、60 和 120 min FSS（12 dyn/cm2 ）刺激，免疫荧光染色后观察FSS刺激成骨细胞不同时间后的细胞骨架变化，采 用Image J软件计算细胞分形维数，比较细胞内部复杂程度；使用Ca2+试剂盒检测钙响应，碱性磷酸酶（ALP）测试盒检 测ALP活性；Real-time PCR检测FSS刺激不同时间后成骨细胞的BMP2、Runx2、Osterix（Osx）基因表达；Western blot 检测FSS刺激不同时间后成骨细胞的BMP2、Runx2、Osx蛋白表达水平，采用Quantity one进行光密度分析。结果 12 dyn/cm2 FSS刺激成骨细胞可引起细胞骨架形态、微丝丰富度及细胞复杂程度的变化，以 60 min最明显。与 0 min组相比， 细胞Ca2+浓度在FSS刺激 30 min后明显升高，延续至 60 min为高峰平台期（P<0.05），120 min 后Ca2+浓度回落；60 min 组的BMP2、Runx2、Osx基因和蛋白表达水平均明显上调（P<0.01）；ALP活性升高，在刺激 60 min时最为明显（P<0.05）。 结论 12 dyn/cm2 FSS刺激能改善成骨细胞生物学功能，促进BMP2、Runx2、Osx基因及其蛋白表达上调；能通过改变成 骨细胞骨架促进Ca2+浓度升高，进而激活与分化高度相关的BMP2-Runx2-Osx信号通路，提高成骨细胞分化标志物ALP 活性。

Abstract:

Abstract: Objective To investigate the biological function and differentiation mechanism of osteoblast under fluid shear force (FSS). Methods Four groups of OB were subjected to FSS stimulation for 0, 30, 60 and 120 min, respectively. The cytoskeleton changes of OB after FSS stimulation for different time were observed by using immunofluorescence staining. The fractal dimension of the cells was calculated by Image J software to compare the intracellular complexity. Ca2+ response was detected by related kit and changes of cell cycle were detected by flow cytometry. ALP activity was detected by Alkalin phosphatase (AKP/ALP) test kits. The expressions of BMP2, Runx2 and Osterix (Osx) were determined by reverse transcription-PCR, real time PCR and Western blot after different time of FSS stimulation. Quantity one was applied for optical density analysis. Results Osteoblasts stimulated by 12 dyn/cm2 FSS can induce changes in cytoskeleton morphology, microfilament richness and cell complexity, the most obvious at 60 min. Compared with the 0 min group, the cellular Ca2+ concentration increased significantly after 30 min of FSS stimulation, and continued to reach the peak plateau stage at 60 min (P<0.05), and the Ca2+ concentration decreased after 120 min. The expression levels of BMP2, Runx2 and Osx genes and proteins in 60 min group were significantly up-regulated (P<0.01). The activity of ALP was increased and the most obvious at 60 min of stimulation (P<0.05). Conclusions FSS stimulation with 12 dyn/cm2 can improve the cellular biological functions of OB, promote the expressions of BMP2, Runx2 and Osx in mRNA and protein levels. FSS stimulation promotes the increase of Ca2+ concentration by changing the OB skeleton, and then activates the BMP2-Runx2-Osx signaling pathway, which is highly related to differentiation, improve the activity of ALP, a marker of OB differentiation.