CAJ | 학술논문

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至第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Western 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).与对照组比较,培养6d时实验组成骨细胞的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的蛋白表达水平均显著降低,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,其机制可能与经典的Wnt信号通路抑制有关.
목적 탐토개민감수체(CaSR)재소서성골세포증식여분화중적작용,이급Wnt신호통로재기촉진성골세포증식급분화과정중적조절작용. 방법 취신생동와야생형소서(대조조)화CaSR고제순합자소서(실험조)각4지,분리배양로골성골세포병전지제3대,분별우배양2、4、6、8d시채용CCK-8법측정세포적흡광도(OD)치,검측성골세포감성린산매(ALP)적활성.취배양6d적세포행실시형광정량-취합매련반응(RT-qPCR)검측핵심결합인자(Runx2)、ALP、골개소(OCN)、핵격활인자수체배체(RANKL)、골보호소(OPG)급β-련단백적mRNA기인표체수평；채용Western Blot검측β-련단백、Wnt-5a、이도소양생장인자-1(IGF-1)、Runx2적단백표체수평. 결과 배양6d시대조조화실험조성골세포적OD치(1.55±0.05、1.26±0.02)화ALP활성[(0.023±0.002)、(0.017±0.001)U/mg· prot]균체도봉치,여기타시간점비교차이균유통계학의의(P＜0.05)；동일시간점실험조성골세포적OD치화ALP활성균저우대조조,차이유통계학의의(P＜0.05).여대조조비교,배양6d시실험조성골세포적Runx2、ALP、OCN、RANKL/OPG、β-련단백적mRNA표체수평,이급β-련단백、Wnt-5a、IGF-1、Runx2적단백표체수평균현저강저,량조비교차이균유통계학의의(P＜0.05). 결론 CaSR결실장도치성골세포적증식화분화장애,기궤제가능여경전적Wnt신호통로억제유관.
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 days 2,4,6 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) and β-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 experimental group were significantly 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 and β-catenin and the protein expressions of β-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.