CAJ | 학술논문

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(ALP)、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.
목적 탄산항매1불부가이최화이양화탄수화반응,환가이가속탄산개적형성,이개침정시신골형성적중요보취.본연구이배양세포위모형탐색탄산항매1재골형성과정중적작용.방법 용성골유도배양기(OM)유도골육류세포Saos-2개화,연후용천소홍염색법관찰세포광화결절적형성,용형광정량취합매련반응(PCR)검측세포골화표지단백핵심결합단백인자2(Runx2)、성골세포특이성전록인자(OSX)、감성린산매(ALP)、골개소화골연단백(BSP)적표체.용단백인적법화형광정량PCR검측세포개화전후탄산항매1적표체량적변화.용탄산항매억제제을선서알처리Saos-2세포,관찰세포개화과정시부피억제.2조간비교채용t검험,불동시간점적통계학분석채용중복측량적방차분석.결과 Saos-2 세포재OM유도후형성대량광화결절(0.68±0.03여2.76±0.13,P＜0.01),Runx2、ALP、골개소、OSX화BSP 전록수평명현승고,현시OM가유도Saos-2세포개화화골화.Saos-2세포개화후탄산항매1표체명현증고(0.25±0.03여0.94±0.06,P＜0.01).Saos-2세포경을선서알처리후,탄산항매1적표체량감소(1.09±0.05여0.55±0.07,P＜0.05),광화결절형성명현감소(2.76±0.13여2.19±0.07,P＜0.01),골화표지단백적표체야명현하강,설명억제탄산항매1표체가이억제Saos-2세포광화화골화과정.결론 탄산항매1재신골형성과정중기착중요작용.
Objective Carbonic anhydrase 1 (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 cells.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 CA 1 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 CA1 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.