基于TGF-β1/Smads信号通路探讨肩袖损伤肌腱止点处异常骨重塑的机制研究

Mechanism of rotator cuff injury at the tendon insertion in abnormal bone remodeling based on the TGF-β1/Smads signaling pathway

目的通过组织学方法探讨肩袖损伤的病理机制,并明确转化生长因子-β1(TGF-β1)/Smads信号通路是否参与肩袖损伤肌腱止点处异常骨重塑。方法选取SPF级雄性SD大鼠40只,鼠龄12周,体质量(450±30)g。随机数字表法分为正常组、模型组术后14、30、60d组,各10只。正常组不做任何操作,模型组离断大鼠右肩冈上肌肌腱止点构建肩袖损伤模型,分别于术后14、30、60d人道处死大鼠,取右肩关节。通过HE染色观察透明软骨和钙化软骨的厚度,潮线是否改变;通过番红-固绿(SO-FG)染色观察关节软骨组织的破坏情况;通过抗酒石酸酸性磷酸酶(TRAP)染色检测软骨下骨破骨情况;通过免疫组织化学法检测分析软骨下骨中TGF-β1、磷酸化的Smad2/3(p-Smad2/3)、成骨转录因子(Osterix)及血小板内皮细胞黏附因子(CD31)的表达量。结果HE染色显示,与正常组比较,模型组术后30、60d透明软骨厚度减小,钙化软骨厚度增加,潮线上移(P<0.05)。SO-FG染色显示,与正常组比较,模型组术后14、30、60d软骨细胞排列紊乱,番红着色逐渐较浅。TRAP染色显示,与正常组比较,模型组术后14、30d软骨下骨破骨细胞表达更加活跃(P<0.05);模型组术后60d破骨细胞表达量与正常组比较,差异无统计学意义(P>0.05)。免疫组织化学结果显示,TGF-β1、p-Smad2/3在模型组术后14、30d阳性细胞表达量水平均显著高于正常组(P<0.05);CD31、Osterix在模型组术后14、30、60d阳性细胞表达量均显著高于正常组(P<0.05)。结论肩袖止点处发生异常骨重塑可能参与了肩袖损伤的病理机制,且TGF-β1/Smads信号通路参与肩袖损伤肌腱止点处异常的骨重塑。

Objective To explore the pathological mechanism of rotator cuff injury through histological methods and to determine whether the TGF-β1/Smads signaling pathway is involved in the abnormal bone remodeling at the tendon insertion of the rotator cuff injury.Methods A total of 40 SPF male Sprague Dawley(SD)rats were selected,12 weeks old,with body weight of(450±30)g,were randomly divided into normal group and model group 14,30,60 days after operation,with 10 rats in each group.The normal group did not receive any operation,and the rotator cuff injury model was established by transecting the right supraspinatus tendon insertion in the model group.Each group of rats was euthanized at 14,30,and 60 days after surgery,and the right shoulder joint was taken.Hematoxylin-eosin staining(HE)was applied to analyze the thickness of hyaline cartilage and calcified cartilage,and whether the tide line changes.The destruction of articular cartilage tissue were observed by safranin O-fast green staining(SO-FG).Osteoclast expression in the subchondral bone was detected by tartrate-resistant acid phosphatase staining(TRAP).The expression levels of transforming growth factorβ1(TGF-β1),phosphorylated Smad2/3(p-Smad2/3),osteogenic transcription factor(Osterix),and platelet-endothelial cell adhesion factor(CD31)in subchondral bones were detected by immunohistochemistry.Results HE staining showed that,compared with the normal group,the thickness of hyaline cartilage decreased,the thickness of calcified cartilage increased,and the tide line shifted in the model group 30 and 60 days after peration(P<0.05).SO-FG staining showed that,compared with the normal group,the arrangement of chondrocytes in each model group at 14,30 and 60 days after operation were disordered,and the saffron staining was lighter.TRAP staining results showed that,compared with the normal group,the expression of osteoclast cells of subchondral bone was more positive at 14 and 30 days after operation(P<0.05),and there was no significant difference in osteoclast expression between the model group at 60 days after operation and the normal group(P>0.05).Immunohistochemistry results showed that the expression levels of TGF-β1 and p-Smad2/3 were significantly higher at 14 and 30 days after operation than in the normal group(P<0.05),the expression levels of Osterix and CD31 in each model group at 14,30 and 60 days after operation were significantly higher than in the normal group,and the differences were statistically significant(P<0.05).Conclusion Abnormal bone remodeling at the tendon insertion of the rotator cuff may be involved in the pathological mechanism of rotator cuff injury,and the TGF-β1/Smads signaling pathway is involved in the abnormal bone remodeling at the tendon insertion of the rotator cuff injury.