摘要
目的探讨microRNA-34a(miR-34a)在绝经后骨质疏松症(postmenopausal osteoporosis,PMOP)发生发展中的作用及机制。方法应用抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色及F-肌动蛋白的罗丹明鬼笔环肽染色检测miR-34a对破骨细胞分化的影响;应用碱性磷酸酶(alkaline phosphatase,ALP)染色及ALP活力测定、茜素红染色等方法观察过表达/抑制miR-34a的破骨细胞的条件培养基(conditional medium,CM)对成骨细胞分化的影响;应用实时荧光定量PCR技术(qRT-PCR)及蛋白质免疫印迹法(Western blot),分析miR-34a对破骨细胞分化相关指标[TRAP和活化T细胞核因子c1(nuclear factor-activated T cells type c1,NFATc1)]、成骨细胞分化相关指标[runt相关转录因子2(runt-related transcription factor 2,Runx2)、转录因子Osterix(Osx)和ALP]以及炎症因子[白细胞介素(interleukin,IL)-1α、IL-1β和IL-6]表达的影响;应用微计算机断层扫描技术(Micro-CT)观察miR-34a对去卵巢小鼠股骨远端骨量及骨微结构的影响。结果miR-34a抑制了核因子κB受体活化因子配体(receptor Activator for Nuclear Factor-κB Ligand,RANKL)诱导的破骨细胞分化。破骨细胞过表达miR-34a后,其CM能够促进成骨细胞分化;而抑制miR-34a的破骨细胞,其CM抑制了成骨细胞分化。进一步研究发现,miR-34a使破骨细胞产生骨形态发生蛋白(bone morphogenetic protein,BMP2)增加,从而促进成骨细胞分化。此外,TNF-α刺激RAW264.7细胞后,IL-1α、IL-1β和IL-6等炎症因子水平升高,而miR-34a使这些炎症因子水平降低。结论miR-34a通过促破骨细胞产生BMP2间接促进成骨细胞的分化,并抑制炎症因子的产生,从而缓解骨质疏松。
Objective To investigate the role and mechanisms of microRNA-34a(miR-34a)in postmenopausal osteoporosis(PMOP).Methods Tartrate-resistant acid phosphatase(TRAP)staining and phalloidin staining were applied to observe the effects of miR-34a on osteoclasts differentiation.Alkaline phosphatase(ALP)staining and ALP activity assay,alizarin red staining were employed to investigate the effects of conditional medium(CM)from osteoclasts over-expressed or inhibited with miR-34a on osteoblasts differentiation.Western blot and real-time quantitative reverse transcription PCR(qRT-PCR)were used to test the effects of miR-34a on the expressions of osteoclasts marker genes[TRAP and nuclear factor-activated T cells type cl(NFATcl)],osteoblasts marker genes[runt-related transcription factor 2(Runx2),Osterix(Osx)and ALP],and pro-inflammatory cytokines[interleukin(IL)-1α,IL-1β and IL-6].Effect of miR-34a on bone mass and bone microstructure of distal femur in ovariectomized mice was observed by micro computed tomography.Results miR-34a inhibited RANKL-induced osteoclasts differentiation.The differentiation of osteoblasts was further enhanced when cells were cultured in CM from miR-34a-overexpressed osteoclasts.Conversely,osteoblasts differentiation was decreased when osteoblasts were cultured in CM from miR-34a-silenced osteoclasts.Further studies found that miR-34a induced osteoclasts to produce morphogenetic protein(BMP2),promoting osteoblast differentiation.In addition,the increasing inflammatory factors(IL-lα,IL-1β,and IL-6)level was observed in RAW264.7 cells stimulated with TNF-α,which could be alleviated by miR-34a.ConclusionnmiR-34a indirectly enhances osteoblast differentiation by promoting osteoclasts to produce BMP2,and downregulates the production of inflammatory cytokines,effectively alleviating PMOP.
作者
褚晓杰
吕文涛
包忠蕾
朴椿洁
张欣媛
贾佳
张玉玺
CHU Xiao-jie;LYU Wen-tao;BAO Zhong-lei;PIAO Chun-jie;ZHANG Xin-yuan;JIA Jia;ZHANG Yu-xi(Department of Clinical Pharmacy,Daqing Oilfield General Hospital,Daqing 163001,Heilongjiang,China;Department of Pharmacy and Laboratory Medicine,Daqing Medical College,Daqing 163312,Heilongjiang,China;School of Clinical Medicine,Harbin Medical University,Haerbin 150081,China)
出处
《中华骨质疏松和骨矿盐疾病杂志》
CSCD
北大核心
2023年第2期133-143,共11页
Chinese Journal Of Osteoporosis And Bone Mineral Research