摘要
目的探讨低强度脉冲超声(LIPUS)促进小鼠衰老骨髓间充质干细胞(BMMSC)成骨分化的能力,以及心肌素相关转录因子-A(MRTF-A)的作用机制。方法对5只老年小鼠以LIPUS辐照左侧股骨8周,之后以Micro CT检测及HE染色评估双侧股骨骨质状态。分离小鼠长骨骨髓细胞,培养BMMSC并分为H_(2)O_(2)+LIPUS组、H_(2)O_(2)组及对照组;以活性氧荧光染色、衰老相关β-半乳糖苷酶染色评估经H_(2)O_(2)处理3天后BMMSC衰老状态,利用免疫荧光及鬼笔环肽染色观察MRTF-A分布和细胞骨架状态。对衰老模型予MRTF-A入核抑制剂CCG-100602,以免疫荧光染色观察H_(2)O_(2)+CCG-100602+LIPUS组、H_(2)O_(2)+CCG-100602组、H_(2)O_(2)+LIPUS组及H_(2)O_(2)组MRTF-A分布;行碱性磷酸酶(ALP)、茜素红染色,评估成骨分化能力。结果老年鼠经LIPUS辐照后左侧股骨较对侧骨体积分数、骨小梁数量增加而骨小梁分离度降低(P均<0.05)。相比H_(2)O_(2)组,H_(2)O_(2)+LIPUS组细胞核内表达MRTF-A增多、细胞内刚性微丝增加。H_(2)O_(2)+CCG-100602+LIPUS组细胞核内表达MRTF-A较H_(2)O_(2)+LIPUS组明显降低;H_(2)O_(2)+LIPUS组较H_(2)O_(2)+CCG-100602+LIPUS组及H_(2)O_(2)组ALP及茜素红染色阳性区均明显增大。结论LIPUS可改善老年小鼠骨质疏松症;MRTF-A核转位是LIPUS促进衰老BMMSC成骨分化的关键调控点。
Objective To investigate the capacity of low-intensity pulsed ultrasound(LIPUS) for promoting osteogenic differentiation of senescent bone marrow mesenchymal stem cell(BMMSC) in mice,also the potential mechanism of involving myocardin-related transcription factor-A(MRTF-A) during this process.Methods Five aged mice received LIPUS irradiation on the left femur for eight weeks.Micro CT and HE staining were employed to evaluate bone status of bilateral femur.Bone marrow cells were isolated from mouse long bones,BMMSC were cultured,and the cells were divided into H_(2)O_(2)+LIPUS group,H_(2)O_(2) group and control group.Mouse BMMSC were subjected to H_(2)O_(2) for 3 days,followed by reactive oxygen species fluorescence staining and senescence-associated β-galactosidase staining to assess cellular senescence.Immunofluorescence and phalloidin staining were performed to examine MRTF-A distribution and cytoskeletal status.Based on H_(2)O_(2)-induced senescence model,MRTF-A nuclear translocation inhibitor CCG-100602 was applied,and the cells were further divided into H_(2)O_(2)+CCG-100602+LIPUS group,H_(2)O_(2)+CCG-100602 group,H_(2)O_(2)+LIPUS group and H_(2)O_(2) group.Immunofluorescence staining was used to assess MRTF-A distribution.Alkaline phosphatase(ALP) and alizarin red staining were conducted to evaluate osteogenic differentiation capacity.Results LIPUS resulted in increased trabecular bone in the left femur of aged mice compared to the right femur,with increased bone volume to total volume and trabecular number and decreased trabecular spacing(both P<0.05).Compared with H_(2)O_(2) group,H_(2)O_(2)+LIPUS group showed increased nuclear expression of MRTF-A and increased intracellular rigid actin filament density.The nuclear expression of MRTF-A in H_(2)O_(2)+CCG-100602+LIPUS group was significantly lower than that in H_(2)O_(2)+LIPUS group.Compared with the H_(2)O_(2)+CCG-100602+LIPUS group and H_(2)O_(2) group,the ALP and alizarin red staining positive areas in H_(2)O_(2)+LIPUS group were significantly larger.Conclusion LIPUS could improve osteoporosis in aged mice.Nuclear translocation of MRTF-A was a key regulator to LIPUS for promoting osteogenic differentiation of senescent BMMSC.
作者
贺诗雯
杨珂
姚欢
晏殊瑾
唐丽
王志刚
HE Shiwen;YANG Ke;YAO Huan;YAN Shujin;TANG Li;WANG Zhigang(Chongqing Key Laboratory of Ultrasound Molecular Imaging and Therapy,Ultrasound Department of the Second Affiliated Hospital,Institute of Ultrasound Imaging,Chongqing Medical University,Chongqing 400010,China;Cell Transplantation Center,Sichuan Academy of Medical Sciences&Sichuan Provincial People's Hospital,Chengdu 610072,China;Department of Ultrasound,the First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China;Department of Nuclear Medicine,the First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China;Department of Radiology,People's Hospital of Chongqing Banan District,Chongqing 401320,China)
出处
《中国医学影像技术》
北大核心
2025年第4期563-568,共6页
Chinese Journal of Medical Imaging Technology
基金
国家自然科学基金(82472107)
重庆市自然科学基金面上项目(CSTB2022NSCQ-MSX0812)。
关键词
骨质疏松
骨髓细胞
低强度脉冲超声
成骨分化
心肌素相关转录因子
osteoporosis
bone marrow cell
low-intensity pulsed ultrasound
osteogenic differentiation
myocardin-related transcription factor
