基于“髓系骨病”理论探索Osterix阳性骨髓间充质干细胞维持骨稳态的作用  

Effect of Osterix-expressing bone marrow mesenchymal stem cells in maintaining bone homeostasis based on the“myeloid bone disease”theory

作　　者：王玉杭 王煦程 王迪 杨婧 曾庆贺 袁文华 童培建[4] 金红婷 WANG Yuhang;WANG Xucheng;WANG Di;YANG Jing;ZENG Qinghe;YUAN Wenhua;TONG Peijian;JIN Hongting(First School of Clinical Medicine,Zhejiang Chinese Medical University,Hangzhou 310053,Zhejiang,China;Third School of Clinical Medicine,Zhejiang Chinese Medical University,Hangzhou 310053,Zhejiang,China;School of Medical Technology,Zhejiang Chinese Medical University,Hangzhou 310053,Zhejiang,China;Zhejiang Provincial Hospital of Chinese Medicine,Hangzhou 310006,Zhejiang,China)

机构地区：[1]浙江中医药大学第一临床医学院,浙江杭州310053 [2]浙江中医药大学第三临床医学院,浙江杭州310053 [3]浙江中医药大学医学技术学院,浙江杭州310053 [4]浙江省中医院,浙江杭州310006

出　　处：《中医正骨》2023年第1期10-16,共7页The Journal of Traditional Chinese Orthopedics and Traumatology

基　　金：浙江省中医药科技计划项目(2021ZZ014)。

摘　　要：目的:探索Osterix阳性骨髓间充质干细胞维持骨稳态的作用。方法:Osterix-CreERT2小鼠(Osterix基因靶标小鼠)与Rosa26-tdTomato小鼠(番茄红荧光报告小鼠)交配获得子代Osterix-CreERT2/Rosa26-tdTomato小鼠,Osterix-CreERT2小鼠与Rosa26-DTA小鼠(白喉毒素A片段报告小鼠)交配获得子代Rosa26-DTA小鼠和Osterix-CreERT2/Rosa26-DTA小鼠。选取3只子代Osterix-CreERT2/Rosa26-tdTomato小鼠纳入示踪组,6只子代Rosa26-DTA小鼠纳入空白组,6只子代Osterix-CreERT2/Rosa26-DTA小鼠纳入Osterix阳性细胞剔除组。各组小鼠均于1月龄时按0.1 mg·g^(-1)体质量腹腔注射他莫昔芬,每天1次,连续注射5 d,以诱导白喉毒素表达,特异性剔除Osterix阳性细胞。他莫昔芬干预结束后2周,脱颈处死示踪组小鼠,切取左侧股骨,通过免疫荧光染色观察Osterix阳性细胞在小鼠股骨中的分布情况。他莫昔芬干预结束后6个月,脱颈处死Osterix阳性细胞剔除组和空白组小鼠,切取左侧股骨,以Micro-CT观察小鼠股骨骨微结构、阿尔新蓝-苏木精染色观察小鼠股骨组织形态、免疫组织化学染色测定小鼠股骨中碱性磷酸酶(alkaline phosphatase,ALP)和脂肪酸结合蛋白4(fatty acid-binding protein,FABP4)表达量。结果:①Osterix阳性细胞在小鼠股骨中的分布情况。Osterix阳性细胞中tdTomato蛋白的红色荧光与DAPI染色的细胞核蓝色荧光分布高度重合,均位于髓腔;Osterix阳性细胞中tdTomato蛋白的红色荧光与CD73染色的骨髓间充质干细胞细胞核绿色荧光分布高度重合,均位于髓腔。②小鼠股骨骨微结构观察结果。Osterix阳性细胞剔除组的骨松质骨密度和骨松质骨小梁厚度均低于空白组[(36.077±3.449)g·mm^(-3),(25.240±1.077)g·mm^(-3),t=2.831,P=0.031;(0.070±0.004)mm,(0.055±0.003)mm,t=2.839,P=0.014],骨松质骨小梁分离度高于空白组[(0.332±0.012)mm,(0.381±0.004)mm,t=-2.159,P=0.009];2组的骨松质骨体积分数、骨松质骨小梁数量比较,组间差Objective:To explore the effect of Osterix-expressing bone marrow mesenchymal stem cells(BM-MSCs)in maintaining bone homeostasis.Methods:Osterix-CreERT2 mice(Osterix gene target mice)were mated with Rosa26-tdTomato mice(tdTomato fluorescent reporter mice)to obtain offspring Osterix-CreERT2/Rosa26-tdTomato mice.Osterix-CreERT2 mice were mated with Rosa26-DTA mice(diphtheria toxin A fragment reporter mice)to obtain offspring Rosa26-DTA mice and Osterix-CreERT2/Rosa26-DTA mice.Three offspring Osterix-CreERT2/Rosa26-tdTomato mice were assigned to the tracing group,six offspring Rosa26-DTA mice to the blank group,and six offspring Osterix-CreERT2/Rosa26-DTA mice to the Osterix positive cells died group.Mice in each group,aging one month,were intraperitoneally injected with tamoxifen at 0.1 mg/g according to the body mass,once a day for five consecutive days to induce the expression of diphtheria toxin,followed by Osterix positive cells died.Two weeks after tamoxifen intervention,mice in the tracing group were sacrificed by cervical dislocation.The left femur was excised,and the distribution of Osterix-expressing cells in the femur was observed by immunofluorescence staining.Six months after tamoxifen intervention,mice in the Osterix positive cells died group and the blank group were sacrificed by cervical dislocation.The left femur was excised.Micro-CT was used to observe the bone microstructure of the femur.Alcian blue-hematoxylin staining was used to observe the morphology of the femur.Immunohistochemical staining was used to determine the expression of alkaline phosphatase(ALP)and fatty acid-binding protein 4(FABP4)in the femur.Results:①Distribution of Osterix-expressing cells in the femur.The red fluorescence of tdTomato protein in Osterix-expressing cells highly coincided with the blue fluorescence of DAPI-stained nuclei,both of which were located in the medullary cavity.The red fluorescence of tdTomato protein in Osterix-expressing cells highly coincided with the green fluorescence of CD73-stained nuclei of BM

关 键 词：髓 骨髓 间充质干细胞 骨生成 OSTERIX 动物实验 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]