DDH髋臼顶壁软骨骨化发育异常的分子生物学机制

Developmental dislocation of the hip (DDH) is a kind of complicated malformation of three dimensional morphology in the immature hips. To date, the etiology of DDH remains unclear. In our previous study, the acetabulum of patients with DDH was analyzed by 3D-CT and MRI. We reported for the first time that the chondrocyte of acetabular roof on the dysplasia hips and uninvolved hips present abnormal ossification. The characteristic of morphology of the chondrocyte of acetabular roof in Li's study that the hind legs of animals were keeps on the position of extension in order to produce the animal model of DDH is similar to our previous observation of MRI on DDH. Therefore, in the present study, the authors intend to employ MRI successively observing the change of the chondrocyte of acetabular roof on the dogs that the hind legs were keeps on the position of extension and produce the animal model of acetabular dysplasia, the subluxation of hips and dislocation of the hips. Meanwhile, genic differential expression was compared between the group of acetabular dysplasia, the group of subluxation of hips, the group of dislocation of hips and the group of normal control to reveal the responsible mechanism of molecular biology leading to abnormal ossification of the chondrocyte of acetabular roof. Studying those issues are vital for comprehending the etiology of DDH and improving the curative effect of DDH.

发育性髋臼发育不良（DDH）是小儿骨科最常见、最复杂的三维形态学畸形之一，目前病因不清。前期我们使用3D-CT拆分技术和MRI技术对单髋脱位DDH患者髋臼各骨的病理形态学进行了研究，首次报道了髋臼发育不良髋和未受累髋的髋臼顶壁软骨均存在软骨骨化异常。Li等模拟伸直襁褓体位所建立髋臼发育不良动物模型的髋臼顶壁软骨病变与我们的DDH患儿MRI观察结果相似。因此，本次课题拟对犬进行伸直体位襁褓，结合MRI技术连续动态观察髋臼软骨变化，建立髋臼发育不良、髋关节半脱位和髋关节脱位动物模型，并对其生物力学进行三维有限元分析。对髋臼发育不良、髋关节半脱位、髋关节脱位和正常对照犬的髋臼软骨进行基因差异表达分析，揭示其髋臼顶壁软骨骨化发育异常、DDH发生发展的分子生物学机制和基因信号通路。上述问题的揭示，对于DDH病因学的研究和实验性干预DDH的治疗均具有重要意义。

发育性髋臼发育不良（DDH）是小儿骨科最常见的先天性畸形，目前病因不清。流行病学研究证实伸直襁褓体位是DDH高危因素，但具体发病机制尚不明确。前期我们使用3D-CT拆分技术和MRI技术对单髋脱位DDH患者髋臼各骨的病理形态学进行了研究，首次报道了髋臼发育不良髋和未受累髋的髋臼顶壁软骨均存在软骨骨化异常。而且通过伸直襁褓体位所建立髋臼发育不良动物模型的髋臼顶壁软骨病变与我们的DDH患儿MRI观察结果相似。因此课题组推测，伸直襁褓体位导致调控髋臼顶壁软骨骨化发育的信号通路出现异常，引起髋臼顶壁软骨骨化延迟，最终出现髋臼发育不良。.课题组对兔进行单侧下肢进行石膏固定，模拟伸直襁褓，成功建立髋臼发育不良，半脱位和全脱位动物模型。X线、MRI和组织大体切片证实髋臼顶壁出现软骨骨化延迟和髋臼发育不良。番红O-快绿染色和电镜检测发现病变组软骨细胞亚细胞器等出现形态学异常。缺氧探针（Hypoxyprobe-1）观察到病变组织出现缺氧表现，5-溴脱氧尿嘧啶核苷（Brdu）和TUNEL检测证实病变组软骨细胞增殖下降和凋亡增加。X型胶原及Runx2表达检测结果表明病变组软骨细胞出现骨化异常。.为进一步明确其具体分子生物学机制，课题组对12个标本进行miRNA高通量测序，通过对预测到miRNA的靶基因进行KEGG通路富集分析，以鉴定骺板软骨细胞响应造模后的调控通路。对比模型组和对照组，共有包含147条差异基因，被注释到95条信号通路中。选择其中4个与软骨发育密切相关的miRNA进行验证，结果证实miR-129和miR-133低表达。荧光素酶、原位杂交、QPCR、过表达和沉默技术，证实低表达miR-133靶向升高SOX9表达，引起PTHrP高表达，导致软骨骨化延迟；低表达miR-129靶向升高SMAD3，下调IHH引起软骨骨化延迟。慢病毒体内转染分别沉默miR-133和miR-129表达，兔髋臼顶壁软骨出现骨化异常，体内实验证实了体外实验结果。上述问题的揭示，对于DDH病因学的研究和实验性干预DDH的治疗均具有重要意义，并对DDH的治疗和预防提供了新思路。