PGP9.5在咬合创伤大鼠颞下颌关节骨关节炎中作用的初步研究
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摘要
目的：建立颞下颌关节骨关节炎大鼠模型,观察并评价大鼠颞下颌关节髁突软骨的组织学改变,检测PGP9.5在关节囊神经及髁突软骨中的表达及变化,并研究组织学改变与PGP9.5表达量之间的相关性,探讨PGP9.5在大鼠颞下颌关节骨关节炎中的作用,为临床上颞下颌关节紊乱病中骨关节炎的治疗提供一定的理论基础。
方法：8周龄雌性Wistar大鼠60只(山东大学动物实验中心提供),体重200-250g,随机分为5个实验组(n=10)与1个对照组(n=10),通过在大鼠右上第一磨牙(?)面粘结正畸方丝造成咬合创伤,建立颞下颌关节骨关节炎模型。于建模后1、3、7、14、28d分批处死动物,取双侧颞下颌关节,制作石蜡切片,HE染色观察并评价髁突软骨的组织学改变；免疫组化染色检测PGP9.5在关节囊神经及髁突软骨中的表达；应用实时荧光定量PCR法检测关节囊神经及髁突软骨中PGP9.5mRNA的相对表达量。比较各实验组与对照组以及创伤侧与非创伤侧的各项指标之间的差别,并分析髁突组织学改变与PGP9.5表达量之间的相关性。
结果：
1)髁突组织学改变：HE染色显示各实验组大鼠髁突软骨发生不同程度的病理性改变,实验组Colombo关节软骨病理积分在6.32至18.35之间,颞下颌关节骨关节炎模型建立成功；实验组大鼠双侧Colombo关节软骨病理积分在1、3、7d逐渐升高,7d达到峰值,14、28d逐渐降低,其中3、7、14d创伤侧高于对照侧(p<0.05)；3、7、14、28d实验组高于对照组(p<0.05),
2)PGP9.5免疫组化染色平均光密度(optical density, OD)值：实验组大鼠双侧关节囊神经PGP9.5平均OD值在1、3、7d逐渐升高,7d达到峰值,14、28d逐渐降低,其中3、7d创伤侧高于对照侧(p<0.05)；1、3、7d实验组高于对照组(p<0.05)。实验组大鼠双侧髁突软骨PGP9.5平均OD值在1、3、7d逐渐升高,7d达到峰值,14、28d逐渐降低,其中7、14d创伤侧高于对照侧(p<0.05)；3、7、14d实验组高于对照组(p<0.05)。
3) PGP9.5mRNA相对表达量：实验组大鼠双侧关节囊神经PGP9.5mRNA相对表达量在1、3d逐渐升高,7、14、28d逐渐降低,其中3、7d创伤侧高于对照侧(p<0.05)；1、3、7d实验组高于对照组(p<0.05)。实验组大鼠双侧髁突软骨PGP9.5mRNA相对表达量在3d时出现明显升高,7、14、28d逐渐降低,其中1、3、7d创伤侧高于对照侧(p<0.05)：1、3、7、14d实验组高于对照组(p<0.05)。
4)相关性分析：髁突软骨PGP9.5平均OD值与Colombo关节软骨病理积分呈显著正相关(r=0.817,p<0.05)。
结论：通过在大鼠右上第一磨牙(?)面粘结正畸方丝造成咬合创伤,可建立颞下颌关节骨关节炎模型。咬合创伤可引起大鼠颞下颌关节关节囊神经及髁突软骨中PGP9.5蛋白及mRNA的表达增高,且髁突软骨中PGP9.5表达量与Colombo关节软骨病理积分呈显著正相关,本研究为颞下颌关节紊乱病中骨关节炎的发病机制研究和治疗提供了新思路。
Objectives:To establish TMJ OA (tempromandibular join osteoarthritis) rat models, evaluate the histological changes of condylar cartilage, and investigate the expression of PGP9.5in the nerves of joint capsules and condylar cartilage. Explore the correlation between histological changes and PGP9.5expression, and discuss the roles PGP9.5plays in TMJ OA, in order to provide a theoretical basis for the treatment of TMJ OA.
Methods:A total of608-week-old female Wistar rats (provided by the Experimental Animal Center of Shandong University), weighing200-250g, were randomly divided into one control group (n=10) and five experimental groups (n=10). TMJ OA rat models were established by bonding an orthodontic square wire on the occlusal surface of the right first maxillary molar to create occlusal trauma. The TMJs were prepared for HE, immunohistochemical staining and real-time PCR analysis at1,3,7,14and28days after teeth operation. Compare the histological changes and PGP9.5expression between the experimental groups and the control group, and between the traumatic sides and the non-traumatic sides of experimental groups; analyze the correlation between histological changes and PGP9.5expression.
Results:
1) Histological changes of condylar cartilage:OA-like lesions were induced in the experimental group rats, with the Colombo histological scores ranging from6.32to18.35in different groups, which indicated that the TMJ OA models were established successfully. The Colombo histological scores of the experimental groups gradually rose on the1st,3rd and7th days, and reached the peak on the7th day before decreasing by the14th and28th days. The scores of experimental groups were significantly higher than those of the control group on the1st,3rd,7th and14th days (p<0.05). The scores of traumatic sides were significantly higher than those of the non-traumatic sides on the3rd,7th and14th days (p<0.05).
2) PGP9.5mean OD value:The PGP9.5mean OD value of the nerves of joint capsules gradually rose on the1st,3rd and7th days, reaching the peak on the7th day, and then decreasing by the14th and28th days. The mean OD of experimental groups were significantly higher than those of the control group on the1st,3rd and7th days (p<0.05). The mean OD of traumatic sides were significantly higher than those of the non-traumatic sides on the3rd and7th days (p<0.05). The PGP9.5mean OD value of condylar cartilage gradually rose on the1st,3rd and7th days, reaching the peak on the7th day, and then decreasing by the14th and28th days. The mean OD of experimental groups were significantly higher than those of the control group on the3rd,7th and14th days (p<0.05). The mean OD of traumatic sides were significantly higher than those of the non-traumatic sides on the7th and14th days (p<0.05).
3) The expression of PGP9.5mRNA:The expression of PGP9.5mRNA of the nerves of joint capsules gradually rose on the1st and3rd days, and then decreasing by the7th,14th and28th days (p<0.05). The mRNA expression of experimental groups were significantly higher than those of the control group on the1st,3rd and7th days (p<0.05). The mRNA expression of traumatic sides were significantly higher than those of the non-traumatic sides on the3rd and7th days (p<0.05). The expression of PGP9.5mRNA of condylar cartilage gradually rose on the1st and3rd days, and then decreasing by the7th,14th and28th days (p<0.05). The mRNA expression of experimental groups were significantly higher than those of the control group on the7th,14th and28th days (p<0.05). The mRNA expression of traumatic sides were significantly higher than those of the non-traumatic sides on the1st,3rd, and7th days (p<0.05).
4) Correlation analysis:A positive correlation exists between PGP9.5mean OD and Colombo histological scores of condylar cartilage (r=0.817,p<0.05).
Conclusions:Experimentally created traumatic occlusion can induce obvious TMJ OA-like lesions. Traumatic occlusion was found to raise the expression of PGP9.5and PGP9.5mRNA in the nerves of joint capsules and condylar cartilage. In addition, a positive correlation exists between PGP9.5expression and Colombo histological scores of condylar cartilage. Therefore, new ideas for etiological mechanism study and clinical treatment of TMJ OA were provided.
方法：8周龄雌性Wistar大鼠60只(山东大学动物实验中心提供),体重200-250g,随机分为5个实验组(n=10)与1个对照组(n=10),通过在大鼠右上第一磨牙(?)面粘结正畸方丝造成咬合创伤,建立颞下颌关节骨关节炎模型。于建模后1、3、7、14、28d分批处死动物,取双侧颞下颌关节,制作石蜡切片,HE染色观察并评价髁突软骨的组织学改变；免疫组化染色检测PGP9.5在关节囊神经及髁突软骨中的表达；应用实时荧光定量PCR法检测关节囊神经及髁突软骨中PGP9.5mRNA的相对表达量。比较各实验组与对照组以及创伤侧与非创伤侧的各项指标之间的差别,并分析髁突组织学改变与PGP9.5表达量之间的相关性。
结果：
1)髁突组织学改变：HE染色显示各实验组大鼠髁突软骨发生不同程度的病理性改变,实验组Colombo关节软骨病理积分在6.32至18.35之间,颞下颌关节骨关节炎模型建立成功；实验组大鼠双侧Colombo关节软骨病理积分在1、3、7d逐渐升高,7d达到峰值,14、28d逐渐降低,其中3、7、14d创伤侧高于对照侧(p<0.05)；3、7、14、28d实验组高于对照组(p<0.05),
2)PGP9.5免疫组化染色平均光密度(optical density, OD)值：实验组大鼠双侧关节囊神经PGP9.5平均OD值在1、3、7d逐渐升高,7d达到峰值,14、28d逐渐降低,其中3、7d创伤侧高于对照侧(p<0.05)；1、3、7d实验组高于对照组(p<0.05)。实验组大鼠双侧髁突软骨PGP9.5平均OD值在1、3、7d逐渐升高,7d达到峰值,14、28d逐渐降低,其中7、14d创伤侧高于对照侧(p<0.05)；3、7、14d实验组高于对照组(p<0.05)。
3) PGP9.5mRNA相对表达量：实验组大鼠双侧关节囊神经PGP9.5mRNA相对表达量在1、3d逐渐升高,7、14、28d逐渐降低,其中3、7d创伤侧高于对照侧(p<0.05)；1、3、7d实验组高于对照组(p<0.05)。实验组大鼠双侧髁突软骨PGP9.5mRNA相对表达量在3d时出现明显升高,7、14、28d逐渐降低,其中1、3、7d创伤侧高于对照侧(p<0.05)：1、3、7、14d实验组高于对照组(p<0.05)。
4)相关性分析：髁突软骨PGP9.5平均OD值与Colombo关节软骨病理积分呈显著正相关(r=0.817,p<0.05)。
结论：通过在大鼠右上第一磨牙(?)面粘结正畸方丝造成咬合创伤,可建立颞下颌关节骨关节炎模型。咬合创伤可引起大鼠颞下颌关节关节囊神经及髁突软骨中PGP9.5蛋白及mRNA的表达增高,且髁突软骨中PGP9.5表达量与Colombo关节软骨病理积分呈显著正相关,本研究为颞下颌关节紊乱病中骨关节炎的发病机制研究和治疗提供了新思路。
Objectives:To establish TMJ OA (tempromandibular join osteoarthritis) rat models, evaluate the histological changes of condylar cartilage, and investigate the expression of PGP9.5in the nerves of joint capsules and condylar cartilage. Explore the correlation between histological changes and PGP9.5expression, and discuss the roles PGP9.5plays in TMJ OA, in order to provide a theoretical basis for the treatment of TMJ OA.
Methods:A total of608-week-old female Wistar rats (provided by the Experimental Animal Center of Shandong University), weighing200-250g, were randomly divided into one control group (n=10) and five experimental groups (n=10). TMJ OA rat models were established by bonding an orthodontic square wire on the occlusal surface of the right first maxillary molar to create occlusal trauma. The TMJs were prepared for HE, immunohistochemical staining and real-time PCR analysis at1,3,7,14and28days after teeth operation. Compare the histological changes and PGP9.5expression between the experimental groups and the control group, and between the traumatic sides and the non-traumatic sides of experimental groups; analyze the correlation between histological changes and PGP9.5expression.
Results:
1) Histological changes of condylar cartilage:OA-like lesions were induced in the experimental group rats, with the Colombo histological scores ranging from6.32to18.35in different groups, which indicated that the TMJ OA models were established successfully. The Colombo histological scores of the experimental groups gradually rose on the1st,3rd and7th days, and reached the peak on the7th day before decreasing by the14th and28th days. The scores of experimental groups were significantly higher than those of the control group on the1st,3rd,7th and14th days (p<0.05). The scores of traumatic sides were significantly higher than those of the non-traumatic sides on the3rd,7th and14th days (p<0.05).
2) PGP9.5mean OD value:The PGP9.5mean OD value of the nerves of joint capsules gradually rose on the1st,3rd and7th days, reaching the peak on the7th day, and then decreasing by the14th and28th days. The mean OD of experimental groups were significantly higher than those of the control group on the1st,3rd and7th days (p<0.05). The mean OD of traumatic sides were significantly higher than those of the non-traumatic sides on the3rd and7th days (p<0.05). The PGP9.5mean OD value of condylar cartilage gradually rose on the1st,3rd and7th days, reaching the peak on the7th day, and then decreasing by the14th and28th days. The mean OD of experimental groups were significantly higher than those of the control group on the3rd,7th and14th days (p<0.05). The mean OD of traumatic sides were significantly higher than those of the non-traumatic sides on the7th and14th days (p<0.05).
3) The expression of PGP9.5mRNA:The expression of PGP9.5mRNA of the nerves of joint capsules gradually rose on the1st and3rd days, and then decreasing by the7th,14th and28th days (p<0.05). The mRNA expression of experimental groups were significantly higher than those of the control group on the1st,3rd and7th days (p<0.05). The mRNA expression of traumatic sides were significantly higher than those of the non-traumatic sides on the3rd and7th days (p<0.05). The expression of PGP9.5mRNA of condylar cartilage gradually rose on the1st and3rd days, and then decreasing by the7th,14th and28th days (p<0.05). The mRNA expression of experimental groups were significantly higher than those of the control group on the7th,14th and28th days (p<0.05). The mRNA expression of traumatic sides were significantly higher than those of the non-traumatic sides on the1st,3rd, and7th days (p<0.05).
4) Correlation analysis:A positive correlation exists between PGP9.5mean OD and Colombo histological scores of condylar cartilage (r=0.817,p<0.05).
Conclusions:Experimentally created traumatic occlusion can induce obvious TMJ OA-like lesions. Traumatic occlusion was found to raise the expression of PGP9.5and PGP9.5mRNA in the nerves of joint capsules and condylar cartilage. In addition, a positive correlation exists between PGP9.5expression and Colombo histological scores of condylar cartilage. Therefore, new ideas for etiological mechanism study and clinical treatment of TMJ OA were provided.
引文
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