力生长因子对破骨细胞活性的影响及其机制  

Effect of mechano-growth factor on osteoclast activity and its mechanism

作　　者：佟雁翔[1] 王斌 贾燕飞[1] 冯卫[1] 张立峰[1] 李亚光[1] 薛飞[1] 于成涌[1] 张哲汉 王文选[1] 贾文超 王祎 杨又玮 Tong Yanxiang;Wang Bin;Jia Yanfei;Feng Wei;Zhang Lifeng;Li Yaguang;Xue Fei;Yu Chengyong;Zhang Zhehan;Wang Wenxuan;Jia Wenchao;Wang Yi;Yang Youwei(Department of Orthopedics,Second Affiliated Hospital of Inner Mongolia Medical University,Hohhot 010030,China;Inner Mongolia Medical University,Research Student Academy,Hohhot 010030,China)

机构地区：[1]内蒙古医科大学第二附属医院骨科,呼和浩特010030 [2]内蒙古医科大学研究生学院,呼和浩特010030

出　　处：《中华创伤杂志》2021年第11期1034-1041,共8页Chinese Journal of Trauma

基　　金：内蒙古自治区自然科学基金(2018MS08019)。

摘　　要：目的探讨力生长因子(MGF)对破骨细胞活性的影响及其机制。方法使用诱导剂25 ng/ml巨噬细胞集落刺激因子(M-CSF)及30 ng/ml核因子-κB受体活化因子配体(RANKL)对RAW264.7前体破骨细胞系进行诱导培养,培养7 d后通过抗酒石酸酸性磷酸酶(TRAP)染色法进行鉴定。取培养的破骨细胞,采用Western blot法测定45 ng/ml的MGF对破骨细胞中磷脂酰肌醇-3-激酶/蛋白激酶B(PI3K/AKT)信号通路及其活性的影响,即AKT、磷酸化(p)-AKT、哺乳动物雷帕霉素靶蛋白(mTOR)、p-mTOR和TRAP在0,4,8和12 h的表达水平,同时采用RT-PCR法从分子水平测定破骨细胞TRAP在0,4,8和12 h的表达水平。用20μmol/L PI3K/AKT磷酸化抑制剂LY294002联合45 ng/ml MGF作用于破骨细胞,采用Western blot法检测AKT、p-AKT、mTOR、p-mTOR和TRAP在0,4,8和12 h的表达水平。结果M-CSF和RANKL对RAW264.7细胞培养7 d后,能够得到大量TRAP染色阳性的破骨细胞。Western blot结果显示,MGF作用于破骨细胞后,AKT和mTOR的表达水平随作用时间无明显变化(P>0.05),p-AKT和p-mTOR的表达水平随着作用时间的延长持续增高,分别由0 h的(2.18±0.34)pg/ml和(0.83±0.10)pg/ml增加至12 h的(3.86±0.36)pg/ml和(1.56±0.19)pg/ml(P<0.05),TRAP的表达水平随作用时间显著降低,由0 h的(5.66±0.47)pg/ml降至12 h的(3.76±0.38)pg/ml(P<0.05)。RT-PCR法测定破骨细胞TRAP表达结果显示,MGF抑制破骨细胞TRAP的表达,由0 h的1.02±0.06降至12 h的0.53±0.11(P<0.05)。Western blot结果显示,LY294002联合MGF作用于破骨细胞后,AKT和mTOR的表达水平随作用时间无明显变化(P>0.05),p-AKT和p-mTOR的表达水平显著降低,分别由0 h的(3.28±0.18)pg/ml和(3.29±0.22)pg/ml降至12 h的(2.06±0.34)pg/ml和(2.04±0.20)pg/ml(P<0.05),而TRAP的表达水平随MGF作用时间则差异无统计学意义(P>0.05)。结论MGF通过PI3K/AKT信号途径抑制破骨细胞TRAP的表达,进而抑制破骨细胞活性;LY294002抑制破骨细胞PI3K/AKT信号通路的表达,进Objective To investigate the effect of mechano-growth factor(MGF)on osteoclast activity and its mechanism.Methods The RAW264.7 precursor osteoclast cell line was cultured with 25 ng/ml macrophage-colony stimulating factor(M-CSF)and 30 ng/ml receptor activator of NF-κB ligand(RANKL),and identified by tartrate resistant acid phosphatase(TRAP)staining after 7 days of culture.Western blot anslysis was used to determine the effect of 45 ng/ml MGF on the phosphoinositide-3-kinase/protein kinase B(PI3K/AKT)signaling pathway in separated osteoclasts,including levels of AKT,phosphorylation(p)-AKT,lactation mammalian target of rapamycin(mTOR),p-mTOR and TRAP at 0,4,8 and 12 hours.Real-time fluorescence quantitative PCR was used to expressions of TRAP in osteoclasts at 0,4,8 and 12 hours.The PI3K/Akt phosphorylation inhibitor LY294002(20μmol/L)combined with MGF(45 ng/ml)was used to act on osteoclasts,and expression levels of Akt,p-Akt,mTOR,p-mTOR and TRAP were detected by Western blot at 0,4,8 and 12 hours.Results After culturing RAW264.7 cells with M-CSF and RANKL for 7 days,a large number of osteoclasts with positive TRAP staining can be obtained.Western blot analysis showed expression levels of Akt and mTOR did not change significantly over time(P>0.05),expression levels of p-Akt and p-mTOR increased continuously from(2.18±0.34)pg/ml and(0.83±0.10)pg/ml at 0 hour to(3.86±0.36)pg/ml and(1.56±0.19)pg/ml at 12 hours(P<0.05),and expression level of TRAP decreased significantly over time,from(5.66±0.47)pg/ml at 0 hour to(3.76±0.38)pg/ml at 12 hours(P<0.05).Real-time fluorescence quantitative PCR analysis of expression of TRAP in osteoclasts showed that MGF inhibited the expression of TRAP in osteoclasts,which decreased from 1.02±0.06 at 0 hour to 0.53±0.11 at 12 hours(P<0.05).After acting LY294002 combined with MGF on osteoclasts,Western blot analysis showed expression levels of Akt and mTOR did not change significantly over time(P>0.05),expression levels of p-AKT and p-mTOR decreased significantly from(3.28±0.18)p

关 键 词：破骨细胞 磷脂酰肌醇3-激酶 蛋白激酶类 力生长因子 

分 类 号：R580[医药卫生—内分泌]