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[1]王玉杭,王煦程,王迪,等.基于“髓系骨病”理論探索Osterix陽性骨髓間充質(zhì)干細(xì)胞維持骨穩(wěn)態(tài)的作用[J].中醫(yī)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”理論探索Osterix陽性骨髓間充質(zhì)干細(xì)胞維持骨穩(wěn)態(tài)的作用()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第35卷
期數(shù):
2023年01期
頁碼:
10-16
欄目:
基礎(chǔ)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; 楊婧1; 曾慶賀1; 袁文華4; 童培建4; 金紅婷1
(1.浙江中醫(yī)藥大學(xué)第一臨床醫(yī)學(xué)院,浙江 杭州 310053; 2.浙江中醫(yī)藥大學(xué)第三臨床醫(yī)學(xué)院,浙江 杭州 310053; 3.浙江中醫(yī)藥大學(xué)醫(yī)學(xué)技術(shù)學(xué)院,浙江 杭州 310053; 4.浙江省中醫(yī)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
關(guān)鍵詞:
髓;  骨髓;  間充質(zhì)干細(xì)胞;  骨生成;  Osterix;  動(dòng)物實(shí)驗(yàn)
Keywords:
marrow;  bone marrow;  mesenchymal stem cells;  osteogenesis;  Osterix;  animal experimentation
摘要:
目的:探索Osterix陽性骨髓間充質(zhì)干細(xì)胞維持骨穩(wěn)態(tài)的作用。方法:Osterix-CreERT2小鼠(Osterix基因靶標(biāo)小鼠)與Rosa26-tdTomato小鼠(番茄紅熒光報(bào)告小鼠)交配獲得子代Osterix-CreERT2/Rosa26-tdTomato小鼠,Osterix-CreERT2小鼠與Rosa26-DTA小鼠(白喉毒素A片段報(bào)告小鼠)交配獲得子代Rosa26-DTA小鼠和Osterix-CreERT2/Rosa26-DTA小鼠。選取3只子代Osterix-CreERT2/Rosa26-tdTomato小鼠納入示蹤組,6只子代Rosa26-DTA小鼠納入空白組,6只子代Osterix-CreERT2/Rosa26-DTA小鼠納入Osterix陽性細(xì)胞剔除組。各組小鼠均于1月齡時(shí)按0.1 mg·g-1體質(zhì)量腹腔注射他莫昔芬,每天1次,連續(xù)注射5 d,以誘導(dǎo)白喉毒素表達(dá),特異性剔除Osterix陽性細(xì)胞。他莫昔芬干預(yù)結(jié)束后2周,脫頸處死示蹤組小鼠,切取左側(cè)股骨,通過免疫熒光染色觀察Osterix陽性細(xì)胞在小鼠股骨中的分布情況。他莫昔芬干預(yù)結(jié)束后6個(gè)月,脫頸處死Osterix陽性細(xì)胞剔除組和空白組小鼠,切取左側(cè)股骨,以Micro-CT觀察小鼠股骨骨微結(jié)構(gòu)、阿爾新藍(lán)-蘇木精染色觀察小鼠股骨組織形態(tài)、免疫組織化學(xué)染色測(cè)定小鼠股骨中堿性磷酸酶(alkaline phosphatase,ALP)和脂肪酸結(jié)合蛋白4(fatty acid-binding protein,FABP4)表達(dá)量。結(jié)果:①Osterix陽性細(xì)胞在小鼠股骨中的分布情況。Osterix陽性細(xì)胞中tdTomato蛋白的紅色熒光與DAPI染色的細(xì)胞核藍(lán)色熒光分布高度重合,均位于髓腔; Osterix陽性細(xì)胞中tdTomato蛋白的紅色熒光與CD73染色的骨髓間充質(zhì)干細(xì)胞細(xì)胞核綠色熒光分布高度重合,均位于髓腔。②小鼠股骨骨微結(jié)構(gòu)觀察結(jié)果。Osterix陽性細(xì)胞剔除組的骨松質(zhì)骨密度和骨松質(zhì)骨小梁厚度均低于空白組[(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],骨松質(zhì)骨小梁分離度高于空白組[(0.332±0.012)mm,(0.381±0.004)mm,t=-2.159,P=0.009]; 2組的骨松質(zhì)骨體積分?jǐn)?shù)、骨松質(zhì)骨小梁數(shù)量比較,組間差異均無統(tǒng)計(jì)學(xué)意義[(6.902±2.216)%,(2.531±0.399)%,t=1.848,P=0.129;(0.950±0.266)個(gè)·mm-1,(0.418±0.037)個(gè)·mm-1,t=1.626,P=0.122]。Osterix陽性細(xì)胞剔除組的骨皮質(zhì)骨密度、骨皮質(zhì)骨體積分?jǐn)?shù)、骨皮質(zhì)骨小梁厚度均低于空白組[(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組的骨皮質(zhì)骨小梁分離度、骨皮質(zhì)骨小梁數(shù)量比較,組間差異均無統(tǒng)計(jì)學(xué)意義[(0.295±0.001)mm,(0.296±0.003)mm,t=-0.372,P=0.072;(1.124±0.221)個(gè)·mm-1,(1.146±0.042)個(gè)·mm-1,t=1.525,P=0.171]。③小鼠股骨組織形態(tài)觀察結(jié)果。與空白組相比,Osterix陽性細(xì)胞剔除組小鼠股骨生長(zhǎng)板軟骨-骨連接處有大量脂滴生成,脂肪空泡堆積。Osterix陽性細(xì)胞剔除組的脂滴面積大于空白組[(203.514±0.957)%,(241.061±5.805)%,t=-6.381,P=0.003]。④小鼠股骨中ALP和FABP4表達(dá)量測(cè)定結(jié)果。Osterix陽性細(xì)胞剔除組股骨ALP表達(dá)量低于空白組[(1.143±0.122)%,(0.550±0.641)%,t=4.305,P=0.013],FABP4表達(dá)量高于空白組[(10.419±1.113)%,(15.670±1.405)%,t=-2.930,P=0.043]。結(jié)論:Osterix陽性骨髓間充質(zhì)干細(xì)胞發(fā)揮著維持骨穩(wěn)態(tài)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 本文編輯:李曉樂)

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備注/Memo

備注/Memo:
基金項(xiàng)目:浙江省中醫(yī)藥科技計(jì)劃項(xiàng)目(2021ZZ014) 通訊作者:金紅婷 E-mail:[email protected]
更新日期/Last Update: 1900-01-01