TGF-βⅡ型受体小分子抑制剂ITD-1对骨髓基质细胞成骨分化和血管形成的作用  

Effects of ITD-1, a small-molecule inhibitor of TGF-β type Ⅱ receptor, on osteogenic differentiation and angiogenesis of bone marrow stromal cells

作　　者：曾继涛 涂小林[2] 刘宏[1] ZENG Jitao;TU Xiaolin;LIU Hong(Center of Reproduction Medicine and Infertility,the First Affiliated Hospital of Chongqing Medical University,Chongqing,400016;Laboratory of Bone Development and Regeneration,Institute of Life Sciences,Chongqing Medical University,Chongqing,400016,China)

机构地区：[1]重庆医科大学附属第一医院生殖健康与不孕症中心,重庆400016 [2]重庆医科大学:生命科学研究院骨发育与再生实验室,重庆400016

出　　处：《第三军医大学学报》2020年第4期384-391,共8页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81672118)~~

摘　　要：目的探讨TGF-βⅡ型受体(transforming growth factor beta receptorⅡ,TGFBR2)的小分子抑制剂ITD-1对小鼠骨髓基质细胞(bone marrow stromal cells,BMSCs)成骨分化和血管形成的影响。方法将C57/BL6小鼠BMSCs分为3组:不经药物处理为空白组;1‰DMSO处理为DMSO组;10μmol/L ITD-1处理为ITD-1组。采用CCK-8检测细胞增殖变化;流式细胞仪检测凋亡细胞比例;实时荧光定量PCR(qPCR)测定成骨标志物碱性磷酸酶(ALP)、ostrix(OSX)、Ⅰ型胶原(collagen1,COL1)和骨钙蛋白(osteocalcin,OCN)及成血管相关标志物血管内皮生长因子(vascular endothelial growth factor,VEGF)、血管生成素1(angiopoietin1,ANGPT1)mRNA的表达;碱性磷酸酶染色检测ALP表达情况;茜素红染色检测成骨晚期矿化水平;免疫荧光染色检测成骨标志物OPN及成血管标志物VEGF的表达;小鼠组织培养实验检测体外培养小鼠肱骨骨密度。结果碱性磷酸酶染色显示ITD-1组染色变浅,茜素红染色结果提示ITD-1组钙盐结节显著减少;与DMSO组比较,ITD-1组BMSCs增殖水平显著降低;流式细胞仪检测显示10μmol/L的ITD-1不导致细胞凋亡;与DMSO组比较,ITD-1组成骨标志物ALP、OSX、COL1及OCN的mRNA相对表达量均显著降低(P<0.01),但成血管标志物VEGF和ANGPT1的mRNA相对表达量显著升高(P<0.05),OPN表达降低而VEGF表达升高(P<0.05),肱骨骨密度也显著降低(P<0.05)。结论TGFBR2的小分子抑制剂ITD-1可抑制骨髓基质细胞增殖,在不影响细胞凋亡的情况下对成骨分化具有显著抑制作用,但显著增强血管形成。ObjectiveTo investigate the effects of ITD-1,a small-molecule inhibitor of transforming growth factor-β(TGF-β)typeⅡreceptor(TGFBR2),on osteogenic differentiation and angiogenesis of bone marrow stromal cells(BMSCs).MethodsBMSCs from C57/BL6 mice were treated with 10μmol/L ITD-1 or 0.1%DMSO,and the changes in the cell proliferation and apoptosis were examined using cell counting kit-8(CCK-8)and flow cytometry,respectively.Real-time quantitative PCR(qPCR)was performed to detect the mRNA expression of the osteogenic markers including alkaline phosphatase(ALP),ostrix(OSX),collagen typeⅠ(COL1)and osteocalcin(OCN)and the angiogenesis-related markers including vascular endothelial growth factor(VEGF)and angiopoietin 1(ANGPT1).ALP staining and alizarin red staining were used to assess the activity of ALP and matrix mineralization in the cells,respectively;immunofluorescence assay was used to detect the expression of the osteogenic marker OPN and the angiogenic marker VEGF.Mouse tissue culture experiment was conducted to test the bone mineral density of mouse humerus cultured in the presence of ITD-1.ResultsThe intensity of ALP staining was obviously lowered in ITD-1-treated mouse BMSCs as compared with DMSO-treated cells.Alizarin red staining revealed significantly reduced matrix mineralization in ITD-1-treated BMSCs.Treatment with 10μmol/L ITD-1 for 12 h significantly suppressed the proliferation of the BMSCs but did not cause obvious cell apoptosis.Treatment with ITD-1 significantly decreased the mRNA expression of the osteogenic markers ALP,OSX,COL1 and OCN(P<0.05)but increased the mRNA expression of the angiogenesis-related markers VEGF and ANGPT1(P<0.05).Immunofluorescence assay also confirmed the decrease of OPN and increase of VEGF expression.The tissue culture experiment showed that culture in the presence of ITD-1 significantly reduced bone mineral density of mouse humerus.ConclusionITD-1 inhibits the proliferation and osteogenic differentiation and enhances angiogenesis of mouse BMSCs without affecting

关 键 词：骨髓基质细胞 转化生长因子&beta Ⅱ型受体 成骨分化 血管生成 小分子抑制剂 

分 类 号：R322.71[医药卫生—人体解剖和组织胚胎学] R329.28[医药卫生—基础医学]