氯喹对骨髓单核细胞向破骨细胞分化的抑制作用及其机制  

Inhibitory effect of chloroquine on differentiation of mouse bone marrow monocytes into osteoclasts and its mechanism

作　　者：林立营 张文 史要鹏 姜梅荣 张冬燕 LIN Liying;ZHANG Wen;SHI Yaopeng;JIANG Meirong;ZHANG Dongyan(Department of Stomatology&Precision Biomedical Key Laboratory,Liaocheng People’s Hospital,Liaocheng 252000,China;不详)

机构地区：[1]聊城市人民医院口腔科聊城市人民医院精准生物医学重点实验室,山东聊城252000 [2]聊城市东昌府区中医院口腔科

出　　处：《山东医药》2022年第20期35-40,共6页Shandong Medical Journal

基　　金：山东省医药卫生科技发展计划项目(202008020069)。

摘　　要：目的观察氯喹对骨髓单核细胞(BMMs)向破骨细胞分化的抑制作用,并基于T细胞免疫调节因子1(TCIRG1)基因调控探讨相关机制。方法获取小鼠BMMs,分为对照组、氯喹1组、氯喹2组,分别加入0、5、10μmol/L的氯喹,用核因子κB受体激活因子配体和巨噬细胞集落刺激因子刺激BMMs分化;采用抗酒石酸酸性磷酸酶(TRAP)染色观察破骨细胞分化情况,RT-PCR法检测破骨细胞分化相关基因TCIRG1、T细胞活化核因子1(NFATc1)、Dc-stamp、MMP9、Oscar、IP3受体(IP3R)1、IP3R2、IP3R3 mRNA,Westernblotting法检测TCIRG1、NFATc1、IP3R蛋白,免疫组化法观察NFATc1的亚细胞定位,流式细胞术检测溶酶体酸化情况,RT-PCR法检测空泡型质子泵相关亚基ATP6 V0d2、ATP6 V1c1 mRNA。将BMMs分为A、B、C、D组,A组转染过表达TCIRG1的腺病毒并以10μmol/L氯喹刺激,B组转染空病毒并以10μmol/L氯喹刺激,C组仅给予10μmol/L氯喹,D组不转染也不添加氯喹;采用Westernblotting法检测细胞中的TCIRG1、NFATc1、IP3R2蛋白,RT-PCR法检测TCIRG1、NFATc1、IP3R1、IP3R2、IP3R3 mRNA。结果氯喹2组TRAP阳性细胞体积小于氯喹1组;氯喹1组和氯喹2组细胞中NFATc1、Oscar、IP3R1、IP3R2、IP3R3 mRNA相对表达量低于对照组,氯喹2组Dc-stamp、MMP9 mRNA表达低于对照组(P均<0.05);对照组、氯喹1组、氯喹2组细胞中TCIRG1、NFATc1、IP3R2蛋白表达依次降低(P均<0.05),氯喹2组细胞核中NFATc1表达减少;氯喹1组、氯喹2组相较对照组溶酶体酸度降低;对照组、氯喹1组、氯喹2组TCIRG1、ATP6 V0d2、ATP6 V1c1 mRNA相对表达量依次降低(P均<0.05)。与B、C组相比,A组出现了一些体积较大的TRAP阳性细胞,但是数量和体积未超过D组;A组NFATc1、IP3R2 mRNA及蛋白表达高于B、C组,但低于D组(P均<0.05)。结论氯喹对BMMs向破骨细胞分化有抑制作用,TCIRG1过表达可部分逆转氯喹对分化的抑制作用;氯喹的作用机制可能与调控TCIRG1、IP3R、NFATc1表达有关。Objective To observe the inhibitory effect of chloroquine on the differentiation of bone marrow mono-cytes(BMMs)into osteoclasts,and to explore the related mechanism based on the gene regulation of T-cell immune regula-tor gene 1(TCIRG1).Methods Mouse BMMs were obtained and induced to differentiate into osteoclasts by nuclear fac-torκB receptor activating factor ligand(RANKL)and macrophage colony stimulating factor(M-CSF).At the same time,cells were divided into the control group,chloroquine group 1,and chloroquine group 2,which were added with 0,5 and 10μmol/L chloroquine,respectively.Tartrate-resistant acid phosphatase(TRAP)staining was used to detect the differen-tiation of osteoclasts.RT-PCR was used to detect the mRNA expression levels of osteoclastogenesis-related genes,TCIRG1,nuclear factor of activated T cell 1(NFATc1),DC-stamp,MMP9,Oscar,IP3 receptor(IP3R)1,IP3R2 and IP3R3.Western blotting was used to detect the protein expression levels of TCIRG1,NFATc1 and IP3R.Immunohisto-chemistry was used to observe the subcellular localization of NFATc1.Flow cytometry was used to detect lysosome acidifi-cation.The mRNA of ATP6 V0d2 and ATP6 V1c1 which were related to vacuolar proton pump was detected by RT-PCR.BMMs were divided into groups A,B,C and D.BMMs in the group A were transfected with adenovirus overexpressing TCIRG1 and were stimulated with 10μmol/L chloroquine,BMMs in the group B were transfected with empty virus and were stimulated with 10μmol/L chloroquine,BMMs in the group C were only stimulated with 10μmol/L chloroquine,and BMMs in the group D were neither transfected nor added with chloroquine.Western blotting was used to detect TCIRG1,NFATc1 and IP3R2 proteins,and RT-PCR was used to detect TCIRG1,NFATc1,IP3R1,IP3R2 and IP3R3 mRNAs.Results The volume of TRAP positive cells in the chloroquine group 2 was smaller than that in the chloroquine group 1.The mRNA relative expression levels of NFATc1,Oscar,IP3R1,IP3R2 and IP3R3 in the chloroquine group 1 and chloroquine group 2 were lower than those in

关 键 词：破骨细胞 骨髓单核细胞 细胞分化 氯喹 T细胞活化核因子1 T细胞免疫调节因子1 IP3受体2 

分 类 号：R782[医药卫生—口腔医学]