摘要
目的探究Indian Hedgehog(IHH)信号通路对软骨内成骨过程中软骨细胞成熟以及转分化的影响。方法取10日龄野生型小鼠的胫骨组织,采用原位杂交和免疫组织化学染色检测生长板区域IHH信号通路相关分子Ihh、Ptch1和Gli1的表达水平。构建肥大软骨细胞特异性Ihh基因敲除小鼠(Col10a1^(Cre/+);Ihh^(null/C)),并采用影像学检查和阿利新蓝染色评估该小鼠的骨骼发育状况。构建肥大软骨细胞IHH信号通路持续激活小鼠(Col10a1^(Cre/+);R26Smo^(M2/M2)和Col10a1^(Cre/+);Ptch1^(LacZ/C)),采用HE染色、原位杂交和TUNEL染色分别对受精15.5天胎鼠胫骨组织形态结构、Ihh(肥大软骨细胞分子标志物)和Col1a1(成骨细胞分子标志物)以及肥大软骨细胞凋亡水平进行检测;另外应用HE染色对10日龄小鼠的胫骨组织进行组织学分析。结果肥大软骨细胞合成分泌IHH,但不表达Ptch1和Gli1。抑制肥大软骨细胞合成IHH蛋白会导致出生后小鼠出现侏儒症;X线检查结果显示小鼠出现严重的骨骼发育不良,包括胸廓狭小、球形头骨以及椎骨发育异常等表现。持续启动IHH信号通路时,胚胎早期软骨细胞成熟分化过程虽未见异常,但是出生后小鼠的骨小梁、骨内膜以及皮质骨等结构均出现一定的异常表现。结论IHH信号通路虽然不参与肥大软骨细胞的终末分化过程,但在软骨细胞转分化的过程中起到了重要的调控作用。
Objective To explore the effect of Indian Hedgehog (IHH) signaling on the maturation andtransdifferentiation of chondrocytes in endochondral ossification.Methods We utilized in situ hybridizationand immunohistochemistry to detect the expression of IHH signaling-related molecules (Ihh,Ptch1 and Gli1) ontibia sec;tions of wild type mice at P10.Next,we ablated Ihh gene in hypertrophic chondrocytes to establishCol10a1;Ihh;mice.We evaluated the;s;k;eletal development of the mice via X-ray and Alcian bluestaining.Finally,we also established Col10a1;R26Smo;mice and Col10a1;Ptch1;mice in whichIHH signaling was consistently activated in hypertrophic chondrocytes.We used HE staining,in situ hybridization and TUNEL assay to detect bone structure,the molecular markers of hypertrophic chondrocytesand osteoblasts and the situation of cell apoptosis on tibia sections of mice at E15.5.In addition,histologicalanalysis on the tibias of these mice were performed by HE staining.Results Hypertrophic chondrocytessynthesized and secreted IHH,but they did not express Ptch1 and Gli1.Inhibiting the synthesis of IHH inhypertrophic chondrocytes led to dwarfism at postnatal stage.X-ray analysis revealed abnormal skeletaldevelopment,including small rib cage,spherical skull and abnormal vertebra.Although constitutive activationof IHH signaling did not impinge on the maturation of chondrocytes in early embryo,there were abnormalmanifestations in primary spongiosa,endosteum and cortical bone at postnatal stage.Conclusion IHHsignaling pathway does not regulate the terminal differentiation of hypertrophic chondrocytes,but it plays acritical role in cartilage-to-bone transition.
作者
王欢博
贺婷
郑超
卢玮光
范静
颉强
杨柳
WANG Huan-bo;HE Ting;ZHENG Chao;LU Wei-guang;FAN Jing;JIE Qiang;YANG Liu(Institute of Orthopaedic Surgery,Xijing Hospital,Air Force Medical University,Xi'an 710032,China;Medical Research Institute,Northwestern Polytechnical University,Xi'an 710072,China;Pediatric Orthopaedics Hospital,Honghui Hospital Affiliated to Medical College of Xi’an Jiaotong University,Xi'an 710054,China)
出处
《骨科》
CAS
2021年第6期485-492,共8页
ORTHOPAEDICS
基金
国家自然科学基金资助项目(81772377、82002261)
陕西省创新能力支撑项目计划(S2020-ZC-0036)。
