大鼠早期骨关节炎软骨下骨结构与骨改建相关基因表达研究

Research on the subchondral bone architecture and bone remodeling related genes change in the initial stage of osteoarthritis in rat

目的考察创伤性关节炎早期软骨下骨的微结构变化和基因表达变化,探索软骨下骨的骨重建特点及其在关节软骨退变中的作用。方法选择13只SD大鼠,利用内侧半月板撕裂(MMT)模型模拟创伤性骨关节炎,右侧膝关节行MMT手术,左侧行假手术,术后3周处死大鼠并取膝关节组织标本4%PFA固定。取10只SD大鼠的造模侧及对照侧胫骨关节,利用microCT扫描并重建分析软骨下骨的微结构变化。标本脱钙后石蜡包埋切片,番红O固绿染色,普通光学显微镜观察摄片。另取3只SD大鼠,提取软骨下骨的组织RNA,RT-PCR检测两组之间骨形成标志基因(ALP、RUNX2、OCN)与骨吸收相关基因(TRAP、CTSK、MMP9)mRNA水平的表达变化。结果 MMT术后3周,胫骨关节micro-CT扫描显示模型组软骨下骨的小梁骨结构紊乱,软骨下骨小梁骨的骨体积分数(BV/TV)、骨小梁连接密度(Conn.D)、骨小梁厚度(Tb.Th)降低(P<0.05),骨小梁间隔(Tb.Sp)增大(P<0.05)。组织病理结果显示,模型组关节软骨未发生明显结构变化、软骨下骨骨小梁结构稀疏。与对照组比较,模型组骨形成标记基因mRNA表达水平降低(P<0.05),骨吸收相关基因mRNA表达水平升高(P<0.05)。结论大鼠膝关节内侧MMT诱导的创伤性骨关节模型早期,软骨下骨体积分数降低、骨小梁厚度变薄,成骨细胞的标志基因表达下降,破骨细胞的功能基因表达增加。

Objective To investigate the changes of microarchitecture and gene expression of subchondral bone in the initial stage of traumatic arthritis,to explore the characteristics of subchondral bone remodeling and its role in the articular cartilage degeneration. Methods The medial meniscal tear （MMT） was performed on the right knees of 13 SD rats to simulate the traumatic osteoarthritis, while sham operation on the control group. Three weeks later, all the rats were executed and dissected, with proximal tibiae being kept and distributed into the two groups, 10 respectively. Micro-computed tomography （micro-CT） was adopted to reconstruct and analyze the subchondral bone. After being fixed by 4% paraformaldehyde, all the samples were decalcified until six weeks passed, followed by paraffin-sectioning, safranin O and fast green staining, ar/d examining and photographing under an ordinary optical microscope. The RNA of another 3 SD rats＇ subchondral bone was extracted,and a real-time PCR test was carried out to illuminate the expression variation of bone-formation marker genes （ALP, RUNX2, and OCN）, and bone-resorption marker genes （TRAP,CTSK and MMP9） ,between the two groups. Results Three weeks after MMT surgery,subchondral bone disorders were observed among the experimental samples through micro-CT scanning. There was lesser BV/TV, Conn. D and Tb. Th（P〈0.05） and more Tb. Sp（P〈0.05） in the experimental group compared with the control group. In the pathological section, arthritic degeneration was not spotted in both groups, but trabeculae of the experimental group were found to be sparse. Compared with control group, the level of mRNA expression of the bone-formation marker genes of the experimental group was decreased（P〈0.05）, while bone-resorption related genes increased（P〈0.05）. Conclusion The model of initial traumatic osteoarthritis induced by MMT in rats＇ knees showed an active bone remodeling,more bone absorbing than bone formation,lowered bone volume, and microarchitecture changing of the subchondral bone.