[1]王玉杭,王煦程,王迪,等.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[J].中医正骨,2023,35(01):10-16.
　WANG Yuhang,WANG Xucheng,WANG Di,et al.Effect of Osterix-expressing bone marrow mesenchymal stem cells in maintaining bone homeostasis based on the“myeloid bone disease”theory[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2023,35(01):10-16.
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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()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第35卷
期数:
2023年01期
页码:
10-16
栏目:
基础研究
出版日期:
2023-01-20

文章信息/Info

Title:
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1; 王煦程2; 王迪3; 杨婧1; 曾庆贺1; 袁文华4; 童培建4; 金红婷1
(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)
Author(s):
WANG Yuhang1; WANG Xucheng2; WANG Di3; YANG Jing1; ZENG Qinghe1; YUAN Wenhua1; TONG Peijian4; JIN Hongting1
1.First School of Clinical Medicine,Zhejiang Chinese Medical University,Hangzhou 310053,Zhejiang,China 2.Third School of Clinical Medicine,Zhejiang Chinese Medical University,Hangzhou 310053,Zhejiang,China3.School of Medical Technology,Zhejiang Chinese Medical University,Hangzhou 310053,Zhejiang,China4.Zhejiang Provincial Hospital of Chinese Medicine,Hangzhou 310006,Zhejiang,China
关键词:
髓;  骨髓;  间充质干细胞;  骨生成;  Osterix;  动物实验
Keywords:
marrow;  bone marrow;  mesenchymal stem cells;  osteogenesis;  Osterix;  animal experimentation
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,组间差异均无统计学意义[(6.902±2.216)%,(2.531±0.399)%,t=1.848,P=0.129;(0.950±0.266)个·mm-1,(0.418±0.037)个·mm-1,t=1.626,P=0.122]。Osterix阳性细胞剔除组的骨皮质骨密度、骨皮质骨体积分数、骨皮质骨小梁厚度均低于空白组[(40.127±1.718)g·mm-3,(24.990±3.099)g·mm-3,t=4.522,P=0.003;(19.482±0.803)%,(13.444±1.604)%,t=3.600,P=0.009;(0.161±0.006)mm,(0.117±0.010)mm,t=3.818,P=0.007]; 2组的骨皮质骨小梁分离度、骨皮质骨小梁数量比较,组间差异均无统计学意义[(0.295±0.001)mm,(0.296±0.003)mm,t=-0.372,P=0.072;(1.124±0.221)个·mm-1,(1.146±0.042)个·mm-1,t=1.525,P=0.171]。③小鼠股骨组织形态观察结果。与空白组相比,Osterix阳性细胞剔除组小鼠股骨生长板软骨-骨连接处有大量脂滴生成,脂肪空泡堆积。Osterix阳性细胞剔除组的脂滴面积大于空白组[(203.514±0.957)%,(241.061±5.805)%,t=-6.381,P=0.003]。④小鼠股骨中ALP和FABP4表达量测定结果。Osterix阳性细胞剔除组股骨ALP表达量低于空白组[(1.143±0.122)%,(0.550±0.641)%,t=4.305,P=0.013],FABP4表达量高于空白组[(10.419±1.113)%,(15.670±1.405)%,t=-2.930,P=0.043]。结论:Osterix阳性骨髓间充质干细胞发挥着维持骨稳态的作用。
Abstract:
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-MSCs,both of which were located in the medullary cavity.②Microstructure of the femur in mice.The cancellous bone density and trabecular thickness of the Osterix positive cells died group were lower than those of the blank group(36.077±3.449 vs 25.240±1.077 g/mm(3),t=2.831,P=0.031; 0.070±0.004 vs 0.055±0.003 mm,t=2.839,P=0.014),and the degree of separation of cancellous bone trabeculae was higher than that of the blank group(0.332±0.012 vs 0.381±0.004 mm,t=-2.159,P=0.009).There was no significant difference between the two groups in the volume fraction of cancellous bone and the number of cancellous bone trabeculae(6.902±2.216 vs 2.531±0.399%,t=1.848,P=0.129; 0.950±0.266 vs 0.418±0.037 pieces/mm,t=1.626,P=0.122).The cortical bone density,cortical bone volume fraction,and cortical bone trabecular thickness of the Osterix positive cells died group were lower than those of the blank group(40.127±1.718 vs 24.990±3.099 g/mm(3),t=4.522,P=0.003; 19.482±0.803 vs 13.444±1.604%,t=3.600,P=0.009; 0.161±0.006 vs 0.117±0.010 mm,t=3.818,P=0.007).There was no significant difference between the two groups in the degree of separation and number of cortical bone trabeculae(0.295±0.001 vs 0.296±0.003 mm,t=-0.372,P=0.072; 1.124±0.221 vs 1.146±0.042 pieces/mm,t=1.525,P=0.171).③Morphology of the femur in mice.Compared with the blank group,the Osterix positive cells died group showed a large number of lipid droplets and fat vacuoles accumulating at the cartilage-bone junction of the femoral growth plate.The lipid droplet area of the Osterix positive cells died group was larger than that of the blank group(203.514±0.957 vs 241.061±5.805%,t=-6.381,P=0.003).④ALP and FABP4 expression in the femur of mice.The expression of ALP in the femur of the Osterix positive cells died group was lower than that of the blank group(1.143±0.122 vs 0.550±0.641%,t=4.305,P=0.013),and the expression of FABP4 was higher than that of the blank group(10.419±1.113 vs 15.670±1.405%,t=-2.930,P=0.043).Conclusion:Osterix-expressing BM-MSCs play a role in maintaining bone homeostasis.

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(收稿日期:2022-07-14 本文编辑:李晓乐)

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备注/Memo

备注/Memo:
基金项目:浙江省中医药科技计划项目(2021ZZ014) 通讯作者:金红婷 E-mail:hongtingjin@163.com
更新日期/Last Update: 1900-01-01