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 the control group,and the mRNA relative expression levels of Dc-stamp and MMP9 in the chloroquine group 2 were lower than those in the control group(all P<0.05).The protein expression lev-els of TCIRG1,NFATc1 and IP3R2 in the control group,chloroquine group 1 and chloroquine group 2 decreased succes-sively(all P<0.05),and the nuclear expression of NFATc1 in chloroquine group 2 decreased.The acidity of lysosomes in the chloroquine group 1 and chloroquine group 2 was lower than that in the control group.The mRNA relative expression levels of TCIRG1,ATP6 V0d2 and ATP6 V1c1 in the control group,chloroquine group 1,and chloroquine group 2 de-creased successively(all P<0.05).Compared with groups B and C,some larger TRAP positive cells appeared in the group A,but the number and volume of TRAP positive cells did not exceed group D.The mRNA and protein expression levels of NFATc1 and IP3R2 in the group A were higher than those in the groups B and C,but were lower than those in the group D(all P<0.05).Conclusion Chloroquine can inhibit the differentiation of BMMs into osteoclasts,and TCIRG1 overexpression can partially reverse the inhibitory effect of chloroquine on osteoclast differentiation;the mechanism may be related to the regulation of chloroquine on the expression of TCIRG1,IP3R and NFATc1.
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)
出处
《山东医药》
CAS
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)。
作者简介
第一作者:林立营(1987-),男,主治医师,主要研究方向为颌骨再生。E-mail:llyrmb@126.com;通信作者:张冬燕(1986-),女,副主任医师,主要研究方向为颌骨免疫微环境。E-mail:dongyan_1126@126.com。
