Gαi1/3蛋白介导Akt-GSK3β-NFATc1信号通路调控破骨细胞分化机制研究  

Mechanism of Osteoclast Differentiation Regulated byAkt-Gsk3β-Nfatc1 Signal Pathway Mediated by Gαi1/3 Protein

作　　者：史册 高运 杨光辉 Shi Ce;Gao Yun;Yang Guanghui(Department of Orthopaedicss,Affiliated Suqian Hospital of Xuzhou Medical University,Suqian 223800,China;Department of Orthopaedicss,Affiliated Hospital of Yangzhou University,Yangzhou 225100,China)

机构地区：[1]徐州医科大学附属宿迁医院,南京鼓楼医院集团宿迁医院骨科,江苏宿迁223800 [2]扬州大学附属医院骨科,江苏扬州225100

出　　处：《实用骨科杂志》2023年第12期1095-1099,共5页Journal of Practical Orthopaedics

基　　金：宿迁市科学技术局自然科学基金面上项目(K202016);宿迁市中医药科技项目(YB202211);徐州医科大学附属医院发展基金项目(XYFM2021043);徐州医科大学江苏省高校重点实验室开放课题(XZSYSKF2022014)。

摘　　要：目的观察G蛋白抑制性α亚单位1/3(Gαi1/3蛋白)对骨髓源性巨噬细胞(bone marrow-derived macrophages,BMMs)向破骨细胞分化的调控作用及其作用机制。方法采用慢病毒敲减及基因敲除策略,获取scr-shRNA、Gαi1/3-shRNA及Gαi1/3双敲除(Gαi1/3-double knock out,Gαi1/3-DKO)三种BMMs,加入巨噬细胞集落刺激因子(macrophage colony stimulating factor,MCSF)、核因子κB受体活化因子配体(receptor activator for nuclear factor-κB ligand,RANKL),MCSF+RANKL处理30 min,采用Western Blot对Akt-GSK3β-NFATc1信号通路中蛋白表达进行检测。MCSF和RANKL联合诱导scr-shRNA、Gαi1/3-shRNA BMMs 4 d染色后观察破骨细胞细胞核数目、破骨细胞大小及数量。予小鼠骨质疏松模型右侧股骨干骺端微量注射慢病毒scr-shRNA及Gαi1/3-shRNA,观察敲减Gαi1/3对于小鼠骨质丢失的逆转作用。结果敲减、敲除Gαi1/3对MCSF及MCSF+RANKL诱导Akt-GSK3β-NFATc1通路中关键蛋白(p-Akt473、p-GSK3β、NFATc1)的活化产生抑制作用。同时,敲减Gαi1/3抑制破骨细胞的分化以及抑制骨质疏松模型中小鼠骨质丢失。结论Gαi1/3蛋白通过介导Akt-GSK3β-NFATc1通路调控破骨细胞分化,可为临床治疗骨质疏松提供新的思路和治疗靶点。Objective To investigate the regulation of Gαi1/3 protein on osteoclast differentiation of bone marrow-derived macrophages(BMMs)and its mechanism.Methods Three BMMs,scr-shRNA,Gαi1/3-shRNA and Gαi1/3-DKO,were obtained by lentivirus knockdown and gene knockout strategies,and were treated with macrophage colony stimulating factor(MCSF),receptor activator for nuclear factor-κB ligand(RANKL)and MCSF+RANKL for 30min.Western Blot was used to detect the expression of key proteins in the Akt-GSK3β-NFATc1 signaling pathway.MCSF and RANKL were combined to induce scr-shRNA,Gαi1/3-shRNA BMMs 4 days.TRAP staining were used to observe the number of cell nuclei and the size and number of osteoclasts.Lentivirus scr-shRNA and Gαi1/3 shRNA were injected into the right epiphysis of the femoral shaft of a mouse model of osteoporosis to observe the reverse effect of Gαi1/3 knockdown on bone loss in mice.Results The activation of key proteins(p-Akt473,p-GSK3β,NFATc1)in the Akt-GSK3β-NFATc1 pathway induced by MCSF and MCSF+RANKL was inhibited by Gαi1/3.Knockdown of Gαi1/3 inhibits osteoclast differentiation and bone loss in mice in osteoporosis models.Conclusion Gαi1/3 protein regulates osteoclast differentiation by mediating Akt-GSK3β-NFATc1 pathway,which may provide a new idea and therapeutic target for the clinical treatment of osteoporosis.

关 键 词：Gαi1/3蛋白 巨噬细胞集落刺激因子 骨髓源性巨噬细胞 骨质疏松症 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]