精神心理因素与颞下颌关节紊乱病的关联研究
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摘要
目的：第一,通过对乌鲁木齐青少年TMD流行病学的调查,综合分析TMD症状和体征的发生率,以及与精神心理问题之间可能的关联。第二,通过建立大鼠心理应激动物模型,观察颞下颌关节组织结构变化,并经免疫组化分析一些细胞因子在关节组织内的表达情况；研究心理应激状态下大鼠咬肌组织功能活动性变化,以及COMT基因在咬肌组织的表达,初步探讨心理应激对颞下颌关节紊乱病的影响及可能的机制。方法：第一部分：采用随机整群抽样调查乌鲁木齐市水磨沟区3所学校的30个班的7-9年级学生1408名,12～15岁,进行精神状态的相关问卷调查和颞下颌关节系统的症状和体征检查,计算TMD症状与体征发生率及与精神心理状态之间的关联程度。第二部分：①wister雄性大鼠48只,随机分成4组,每组12只。心理应激组分为3周组、6周组、和恢复6周组,另一组为不加刺激的空白对照。在同等喂养环境和条件下,应激组给予电击足部、气候箱温度刺激、食物诱惑、和铁筒制动四种方式交替进行刺激,期间测量各组大鼠平均每日进食进水量和体重变化及场旷实验得分,并于刺激3周、6周和恢复6周的时间点分别处死,解剖取完整咬肌和颞下颌关节组织备用。咬肌组织左右侧分别随机放入到-20℃和-80℃冰箱中保存备用。②对取得的颞下颌关节组织进行固定、切片,光镜下观察关节盘、关节软骨、关节骨质和滑膜四种组织结构。③切片行免疫组化分析大鼠颞下颌关节髁突软骨细胞MMP-3、TIMP-3、VEGF、TGF-β1的表达变化。第三部分：①利用低温离心机和全自动生化仪测试各组咬肌组织的肌酸肌酶(CK)、C反应蛋白(CRP)、和乳酸脱氢酶(LDH)含量,并进行比较。②对-80℃冰箱保存之咬肌做儿茶酚胺氧位甲基转移酶(COMT)荧光定量PCR及凝胶电泳。结果：第一部分：1408名初中生中,有一种以上的TMD体征者156名(11.8%),有1种以上TMD症状者有298名(21.16%)。TMD的3个主要症状和体征(张口困难、弹响与杂音、肌肉及关节区疼痛)以及口腔异常功能(紧咬牙和夜磨牙)都与精神心理状态密切相关,P<0.001。第二部分:①平均体重、平均摄食摄水量和场旷实验的行为学改变分析各组大鼠应激状态,应激的三个组与对照组比较有明显差异,应激组之间比较6周组比3周组和恢得组的评分都明显高,差异均有统计学意义。②实验3周组和6周组大鼠髁突表面胶原纤维排列紊乱,局部粗糙,增殖带和透明软骨带细胞层数减少,软骨细胞层数减少到3-5层以下,髁突软骨出现裂隙,骨小梁排列紊乱,关节盘变薄等,且6周组比3周组变化明显,差异均有统计学意义。恢复组与3周组表现相似,滑膜水肿等表现有缓解。③免疫组化结果显示各细胞因子在关节组织中均可见表达。MMP-3表达阳性率,实验6周组明显大于对照组,P<0.01；实验3周组明显大于对照组,P<0.05,其余各组间比较无差异；恢复组TIMP-3表达阳性率明显大于对照组,P<0.05；恢复组VEGF表达阳性率明显大于实验3周组和对照组,P<0.05;TGF-β1表达阳性率实验6周组明显大于对照组,P<0.05；第三部分：①CK和LDH测定,实验3周组、6周组、恢复组均明显高于对照组,恢复组明显高于实验3周组和6周组,差异有统计学意义(P<0.05)。CRP测试恢复组明显高于实验3周组、实验6周组和对照组,差异有统计学意义(P<0.05)其它各组间无明显差异(P>0.05)。②OMT基因荧光定量PCR四组数据总体比较有统计学意义,Hc=8.355,P=0.039,大鼠咬肌组织中COMT表达水平由低到高依次为实验6周组、3周组、恢复组和对照组,但均无统计学意义。结论：①儿童青少年的TMD患病率比较高,而且其症状体征与一些精神心理因素有关联,需引起重视。②长期的足底电击、制动、气候箱和食物诱惑刺激可以明显改变大鼠的行为,精神状态极为紧张恐惧,体重、摄食摄水量和场旷试验综合评价证明动物处于心理应激状态或大鼠心理应激模型构建成功。心理应激下大鼠颞下颌关节组织结构出现了病理变化,主要为髁状突软骨结构紊乱,层数变少,并有断裂,病变程度与刺激时间成正比,刺激去除后关节组织病变可轻度恢复并伴有部分修复现象。大鼠颞下颌关节组织结构病变程度重者髁突软骨细胞MMP-3表达阳性率高,MMP-3高表达与颞下颌关节组织结构破坏有关,恢复组TIMP-3高表达说明其在软骨组织修复中起了重要作用；恢复组VEGF在关节软骨表达明显增高说明了刺激去除之后组织有血管生成修复现象。TGF的表达与组织破坏程度成正比,可能在心理应激状态之下发生较多骨吸收的作用,相对成骨的作用较少,而且各因子之间可能有着相互调节的作用。③大鼠在心理应激的刺激之后,咬肌中CK、LDH含量较对照组明显升高,大鼠咬肌肌肉的活动性功能改变,并且肌肉组织也发生了相应的病理性变化,存在炎症性改变以及可能的能量代谢障碍等。长时间刺激使咬肌组织内COMT含量减少,CA分泌增加,引起咬肌痉挛收缩,肌肉血液循环障碍和缺血,继而发生功能障碍。
Objective:The first, by an epidemiological investigation of TMD in the Urumqi adolescent, to achieve a comprehensive analysis of TMD signs and symptoms and relationship associated with the mental problems, the second, by establishing rat model of psychological stress, to explore the function and pathological changes of masseter muscle tissue and temporomandibular joint tissue, and by immunohistochemical method, to analyze the expression of some cytokines and their effect on TMD, then by testing COMT gene with the fluorescent quantitative PCR techonology, to discuss preliminarily the effect of psychological stress on TMD and possibly infuencing mechanisms. Methods: part1:Epidemiology:1408students in Urumqi city, between7th to9th grades, age from12～15, were investigated using random cluster sampling survey, including mental status questionnaire and temporomandibular joint symptoms and signs check, and the incidence of TMD and the relationship associated with mental stress were analyzed. Part2:①48male wister'rats, were randomly divided into4groups with12rats in each group, of which psychological stress group was divided into3-week-group,6-week-group, and a recovery group of6weeks, while another group is control group. Under the same feeding conditions, stressed groups were stimulated with electric shock in feet, temperature conditioning of climate box, food temptations, and body braking of iron drum, and four ways were chosed alternately and randomly. During the different time of3th week,6th week, and recovered6week, measured the consume quantity of water and food, weight change and rats' scores in opened field experiment, and then killed the rats and got anatomically complete masseter muscle and temporomandibular joint tissue for reserve.②The temporomandibular joint were fixed, sliced, and observed under light microscope.③The expressions of MMP-3, TIMP-3, VEGF, and TGF-β1were observed and analyzed in condylar cartilage cells of rats'temporomandibular joint by immunohistochemical technology. Part3:①The serum creatine kinase (CK), C reactive protein (CRP), and lactate dehydrogenase (LDH) in each group were tested and analyzed under the low-temperature centrifuge and the automatic biochemical analyzer.②COMT fluorescence quantitative PCR and gel electrophoresis were done with the masseter muscle reserved in refrigerator of-80℃. Results:part1:Of1408junior high school students, people with more than one TMD signs was156(11.8%), and people with more than1TMD symptoms was298(21.16%). There was a close relation with the three chief TMD signs (trismus, snapping and murmur, muscle and joint pain), as well as abnormal oral function (clenching and bruxism) and mental state, P<0.001.part2:①According to the the average weight, average feeding intake of water and the scores of field open experiment behavioral changes of rats, the three stressed groups were different from the control group, with a statistics significance. The score of6th week group stressed was significantly higher than that of the3th week group, and the difference was statistically significant.②In the3rd week and the6th week group stressed psychologically, the collagen fibrils on the surface of condylar arranged in disorder, with local roughness, and the number of cartilage cell layers decreased in the area of proliferation and hyaline cartilage to less than3-5layers. The TMJ showed cartilage fracture, bone trabecular derangement, and squeezed disc, of which the6th group changed more significantly than the3th group (P<0.05), while the recorvered group was similar to the3th group with relieved synovial edema.③Immunohistochemistry showed various cytokines in joint tissues were visible expression. The expression rate of MMP-3of stressed6th group was significantly higher than that of control group (P<0.01), and the3th group was also higher than the control group (P<0.05), but between the other groups hadn't significant difference; the expression of TIMP-3of the recovered group was significantly higher than the control group, P<0.05; the expression of VEGF in the recovered group was significantly greater than that in the3th stressed group and control group, P<0.05; TGF-β1of the6th group expressed more significantly than in the control group, P<0.05. part3:①Values of CK and LDH in the3rd,6th week group stressed psychologically and the recorvered group were significantly higher than those in control group and there was statistical significance (P<0.05). CRP of the recovered group was significantly higher than other groups (P<0.05), and the others showed no significant differences (P>0.05).②The gene fluorescence quantitative PCR of COMT showed statistical significance in four groups,Hc=8.355, P=0.039, the COMT expression levels of rat masseter muscle followed by6th week,3th week, the recovered group and the control group from low to high. Conclusion:Part1:The prevalence of TMD in children and adolescents is relatively high, the symptoms and signs were related to some psychological factors, and this situation should be concerned. Part2:the rats' behavior can be changed under longtime stimulation by foot electric shock, body brake, climate conditioner, and food temptation, and the rats can display extremely nervous and fear. Due to the comprehensive valuation of body weight, food and water intake, and open field testing, it can be concluded that the the stressed animal models was achieved, or the rats can be considered in such a state of psychological stress. Temporomandibular joint structure showed some pathological changes, especially in cartilage layer of mandibular condyle, with structure disorders, decreased number of cartilage layer, fracture, and these pathological changes was proportional to the stimulation time. After removal of stress stimulation, the lesions can be recovered or restored partially. In severe case of pathological changes, the positive rate of MMP-3was high and the over expression could be related with the damage of TMJ structure tissue. More expression of TIMP-1shows that it plays an important role in the recovery group. The expression of VEGF in articular cartilage illustrates the angiogenesis phenomena after the removal of stimulation. The expression of TGF-β1in chondrocyte is proportional to the degree of tissue lesion, and it is possible that more bone resorption and less osteogenesis happens under the psychological stress, moreover among various factors may have interaction. Part3: Under the situation of psychological stress, CK and LDH in rats' masseter muscle increased and muscle activity changed significantly, morever, proving that there are some relative muscle pathological changes such as inflammatory changes and possible energy metabolism disorder. The COMT of masseter muscle will decrease under longtime psychological stress, and CA secrete more so that the masseter shows spasm, over contraction, and dysfunction, due to the muscle blood circulation disorders and ischemia.
Objective:The first, by an epidemiological investigation of TMD in the Urumqi adolescent, to achieve a comprehensive analysis of TMD signs and symptoms and relationship associated with the mental problems, the second, by establishing rat model of psychological stress, to explore the function and pathological changes of masseter muscle tissue and temporomandibular joint tissue, and by immunohistochemical method, to analyze the expression of some cytokines and their effect on TMD, then by testing COMT gene with the fluorescent quantitative PCR techonology, to discuss preliminarily the effect of psychological stress on TMD and possibly infuencing mechanisms. Methods: part1:Epidemiology:1408students in Urumqi city, between7th to9th grades, age from12～15, were investigated using random cluster sampling survey, including mental status questionnaire and temporomandibular joint symptoms and signs check, and the incidence of TMD and the relationship associated with mental stress were analyzed. Part2:①48male wister'rats, were randomly divided into4groups with12rats in each group, of which psychological stress group was divided into3-week-group,6-week-group, and a recovery group of6weeks, while another group is control group. Under the same feeding conditions, stressed groups were stimulated with electric shock in feet, temperature conditioning of climate box, food temptations, and body braking of iron drum, and four ways were chosed alternately and randomly. During the different time of3th week,6th week, and recovered6week, measured the consume quantity of water and food, weight change and rats' scores in opened field experiment, and then killed the rats and got anatomically complete masseter muscle and temporomandibular joint tissue for reserve.②The temporomandibular joint were fixed, sliced, and observed under light microscope.③The expressions of MMP-3, TIMP-3, VEGF, and TGF-β1were observed and analyzed in condylar cartilage cells of rats'temporomandibular joint by immunohistochemical technology. Part3:①The serum creatine kinase (CK), C reactive protein (CRP), and lactate dehydrogenase (LDH) in each group were tested and analyzed under the low-temperature centrifuge and the automatic biochemical analyzer.②COMT fluorescence quantitative PCR and gel electrophoresis were done with the masseter muscle reserved in refrigerator of-80℃. Results:part1:Of1408junior high school students, people with more than one TMD signs was156(11.8%), and people with more than1TMD symptoms was298(21.16%). There was a close relation with the three chief TMD signs (trismus, snapping and murmur, muscle and joint pain), as well as abnormal oral function (clenching and bruxism) and mental state, P<0.001.part2:①According to the the average weight, average feeding intake of water and the scores of field open experiment behavioral changes of rats, the three stressed groups were different from the control group, with a statistics significance. The score of6th week group stressed was significantly higher than that of the3th week group, and the difference was statistically significant.②In the3rd week and the6th week group stressed psychologically, the collagen fibrils on the surface of condylar arranged in disorder, with local roughness, and the number of cartilage cell layers decreased in the area of proliferation and hyaline cartilage to less than3-5layers. The TMJ showed cartilage fracture, bone trabecular derangement, and squeezed disc, of which the6th group changed more significantly than the3th group (P<0.05), while the recorvered group was similar to the3th group with relieved synovial edema.③Immunohistochemistry showed various cytokines in joint tissues were visible expression. The expression rate of MMP-3of stressed6th group was significantly higher than that of control group (P<0.01), and the3th group was also higher than the control group (P<0.05), but between the other groups hadn't significant difference; the expression of TIMP-3of the recovered group was significantly higher than the control group, P<0.05; the expression of VEGF in the recovered group was significantly greater than that in the3th stressed group and control group, P<0.05; TGF-β1of the6th group expressed more significantly than in the control group, P<0.05. part3:①Values of CK and LDH in the3rd,6th week group stressed psychologically and the recorvered group were significantly higher than those in control group and there was statistical significance (P<0.05). CRP of the recovered group was significantly higher than other groups (P<0.05), and the others showed no significant differences (P>0.05).②The gene fluorescence quantitative PCR of COMT showed statistical significance in four groups,Hc=8.355, P=0.039, the COMT expression levels of rat masseter muscle followed by6th week,3th week, the recovered group and the control group from low to high. Conclusion:Part1:The prevalence of TMD in children and adolescents is relatively high, the symptoms and signs were related to some psychological factors, and this situation should be concerned. Part2:the rats' behavior can be changed under longtime stimulation by foot electric shock, body brake, climate conditioner, and food temptation, and the rats can display extremely nervous and fear. Due to the comprehensive valuation of body weight, food and water intake, and open field testing, it can be concluded that the the stressed animal models was achieved, or the rats can be considered in such a state of psychological stress. Temporomandibular joint structure showed some pathological changes, especially in cartilage layer of mandibular condyle, with structure disorders, decreased number of cartilage layer, fracture, and these pathological changes was proportional to the stimulation time. After removal of stress stimulation, the lesions can be recovered or restored partially. In severe case of pathological changes, the positive rate of MMP-3was high and the over expression could be related with the damage of TMJ structure tissue. More expression of TIMP-1shows that it plays an important role in the recovery group. The expression of VEGF in articular cartilage illustrates the angiogenesis phenomena after the removal of stimulation. The expression of TGF-β1in chondrocyte is proportional to the degree of tissue lesion, and it is possible that more bone resorption and less osteogenesis happens under the psychological stress, moreover among various factors may have interaction. Part3: Under the situation of psychological stress, CK and LDH in rats' masseter muscle increased and muscle activity changed significantly, morever, proving that there are some relative muscle pathological changes such as inflammatory changes and possible energy metabolism disorder. The COMT of masseter muscle will decrease under longtime psychological stress, and CA secrete more so that the masseter shows spasm, over contraction, and dysfunction, due to the muscle blood circulation disorders and ischemia.
引文
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