高表达碳酸酐酶1参与骨化过程的研究  

Over-expression of carbonic anhydrase 1 is involved in bio-mineralization process

作　　者：郑亚冰[1] 王林[3] 赵丹[2] 王月俭[3] 安琨[4] 韩金祥[3] 常晓天[1] 

机构地区：[1]山东省千佛山医院医学研究中心中心实验室,济南250014 [2]山东省千佛山医院骨科,济南250014 [3]山东省医药生物技术研究中心 [4]山东省医学科学院研教中心

出　　处：《中华风湿病学杂志》2012年第12期804-808,I0002,共6页Chinese Journal of Rheumatology

基　　金：基金项目：国家自然科学基金（30671949,30972720）;国家基础研究发展计划（2010CB529105）

摘　　要：目的碳酸酐酶1不仅可以催化二氧化碳水化反应，还可以加速碳酸钙的形成，而钙沉淀是新骨形成的重要步骤。本研究以培养细胞为模型探索碳酸酐酶1在骨形成过程中的作用。方法用成骨诱导培养基（OM）诱导骨肉瘤细胞Saos-2钙化，然后用茜素红染色法观察细胞矿化结节的形成，用荧光定量聚合酶链反应（PCR）检测细胞骨化标志蛋白核心结合蛋白因子2（Runx2）、成骨细胞特异性转录因子（OSX）、碱性磷酸酶（AIJP）、骨钙素和骨涎蛋白（BSP）的表达。用蛋白印迹法和荧光定量PCR检测细胞钙化前后碳酸酐酶1的表达量的变化。用碳酸酐酶抑制剂乙酰唑胺处理Saos-2细胞，观察细胞钙化过程是否被抑制。2组间比较采用t检验，不同时间点的统计学分析采用重复测量的方差分析。结果Saos-2细胞在OM诱导后形成大量矿化结节（0．68±0．03与2．76±0．13，P〈0．01），Runx2、ALP、骨钙素、OSX和BSP转录水平明显升高，显示OM可诱导Saos-2细胞钙化和骨化。Saos-2细胞钙化后碳酸酐酶1表达明显增高（0．25±0．03与0．94±0．06，P〈0．01）。Saos-2细胞经乙酰唑胺处理后，碳酸酐酶1的表达量减少（1．09±0．05与0．55±0．07，P〈0．05），矿化结节形成明显减少（2．76±0．13与2．19±0．07，P〈0．01），骨化标志蛋白的表达也明显下降，说明抑制碳酸酐酶1表达可以抑制Saos-2细胞矿化和骨化过程。结论碳酸酐酶1在新骨形成过程中起着重要作用。Objective Carbonic anhydrase I （CA1） not only enhances the hydration reaction but also promotes the formation of CaCO3, which is an essential step for new bone formation in vitro. However, there is no direct evidence to demonstrate the involvement of CA1 in bio-mineralization in cells and tissues. This study is aimed to evaluate the important role of CA1 in bio-mineralization and ossification in cultured ceils. Methods Calcification in Saos-2 cells was induced using osteogenic medium （OM） and the calcification was determined by Alizarin Red-S staining. The expressions of ossification protein marker Runt-related transcription factor-2 （Runx2）, osterix （OSX）, alkaline phosphatase （ALP）, osteocalcin （OCN） and bone sialoprotein （BSP） were detected in the process of bone formation by real-time PCR. The expression of CA1 in the calcified cells were measured using real-time PCR and Western blotting. We also detected calcification in Saos-2 cells in the presence of acetazolamide, an anti-carbonic anhydrase drug to CA1, to determine the role of CA1 in biomineralization in culture cells. T test analysis was used to compare the two groups, M-ANOVA of repeated measurs was conducted for different time point. Results Following the stimulation of OM, Saos-2 cells produced a great amount of calcium-rich deposits [0.68±0.03 vs 2.76±0.13, P〈0.01 ]. Increased transcriptions of ossification protein markers were also detected in these stimulated Saos-2 cells, indicating that the OM launched the process of bone formation in the cells. CA1 had a significantly increased expression during this process [0.25±0.03 vs 0.94±0.06, P〈0.01 ]. Following treatment with acetazolamide, the expression of CAI evidently declined [ 1.09±0.05 vs 0.55±0.07, P〈0.05], and the mineralized nodule formation was declined [2.76±0.13 vs 2.19±0.07, P〈0.01]. Conclusion These findings indicate that CA1 participates in the biomineralization and ossification, and may play an important roles in bone formation.

关 键 词：碳酸酐酶1 骨生成 成骨诱导 生物矿化 

分 类 号：R363[医药卫生—病理学]