钙敏感受体缺失对小鼠成骨细胞增殖与分化的影响及机制研究  

Effect of lacking calcium-sensing receptor on proliferation and differentiation of mouse osteoblasts and its mechanism

作　　者：张崛[1] 冯玉旭[2] 徐勇[1] 刘欢[1] 苗登顺[3] 任永信[1] 范卫民[1] 

机构地区：[1]南京医科大学第一附属医院骨科,210029 [2]南京市浦口区中心医院骨科 [3]南京医科大学骨与干细胞研究中心

出　　处：《中华创伤骨科杂志》2013年第5期430-434,共5页Chinese Journal of Orthopaedic Trauma

基　　金：国家自然科学基金（30901528）

摘　　要：目的探讨钙敏感受体（CaSR）在小鼠成骨细胞增殖与分化中的作用，以及Wnt信号通路在其促进成骨细胞增殖及分化过程中的调节作用。方法取新生同窝野生型小鼠（对照组）和CaSR敲除纯合子小鼠（实验组）各4只，分离培养颅骨成骨细胞并传第3代，分别于培养2、4、6、8d时采用CCK-8法测定细胞的吸光度（OD）值，检测成骨细胞碱性磷酸酶（ALP）的活性。取培养6d的细胞行实时荧光定量-聚合酶链反应（RT-qPCR）检测核心结合因子（Runx2）、ALP、骨钙素（OCN）、核激活因子受体配体（RANKL）、骨保护素（OPG）及β-链蛋白的mRNA基因表达水平；采用Weste Blot检测β-链蛋白、Wnt-5a、胰岛素样生长因子-1（IGF-1）、Runx2的蛋白表达水平。结果培养6d时对照组和实验组成骨细胞的OD值（1.55±0.05、1.26±0.02）和ALP活性[（0.023±0.002）、（0.017±0.001）U/mg·prot] 均达到峰值，与其他时间点比较差异均有统计学意义（P〈0.05）;同一时间点实验组成骨细胞的OD值和ALP活性均低于对照组，差异有统计学意义（P 〈0.05）。对照组比较，培养6d时实验组成骨地细胞的Runx2、ALP、OCN、RANKL/OPG、β-链蛋白的mRNA表达水平，以及β-链蛋白、Wnt-5a、IGF-1、Runx2的蛋白表达水平无显著降低，两组比较差异均有统计学意义（P〈0.05）。结论CaSR缺失将导致成骨细胞的增殖和分化障碍，其机制与经典的Wnt信号通路抑制有关。Objective To investigate the effect of calcium sensing receptor （CaSR） on proliferation and differentiation of mouse osteoblasts and the regulatory role of Wnt signaling pathway in promotion of proliferation and differentiation of mouse osteoblasts. Methods Four wild-type mice （control group）and 4 CaSR receptor knockout homozygous mice （experimental group） were used for passage of skull osteoblasts. The third generation of osteoblasts was harvested to measure cellular optical density （OD） and alkaline phosphatase （ALP）activity using Cell Counting Kit-8（CCK-8） on days2,4,6 and 8, respectively. At the end of day 6,the mRNA levels of core binding factor （Runx2）, ALP, osteocalcin （OCN）, receptor activator of NF-KB ligand （RANKL）, osteoprotegerin （OPG） andβ-catenin were determined by RT-qPCR, and the protein expressions of Runx-2, β-catenin, IGF-1 and Wnt-5a were determined by Western blot. Results The OD values（1.55±0.05、versus 1.26±0.02）and ALP activities （0.023±0.002 U/mg·prot versus 0.017±0.001 U/mg·prot ）of the osteoblasts reached the peak in the 2 groups on day 6, significantly higher than those at the other time points（P 〈0.05）. However, the OD value and ALP activity in the experimentaly lower than in the control group at the same time point（P 〈0.05）. Compared with the control group, the mRNA expressions of IGF-1, OCN, Runx2, ALP, RANKL/OPG andβ-catenin and the protein expressions ofβ-catenin, Wnt-5a, IGF-1 and Runx2 were all significantly reduced in the experimental group（P〈0.05）.Conclusions Lack of CaSR may result in obstacles to proliferation and differentiation of osteoblasts. The mechanism may be related to the inhibition of Wnt signaling pathway.

关 键 词：成骨细胞 受体 G-蛋白偶联 细胞增殖 细胞分化 信号传导 

分 类 号：R363[医药卫生—病理学]