肥大细胞TLR2、TLR4在人慢性牙周炎牙龈组织中表达的研究

发布时间：2018-07-17 00:38

【摘要】：背景及目的：牙周病是由菌斑微生物引起的、发生在牙齿周围组织的炎症性疾病。菌斑微生物是牙周病的始动因子，但是单纯的牙周致病菌的致病作用不能解析牙周病的破坏作用，多种免疫细胞及细胞因子参与了牙周病的病理损害。牙周致病菌与宿主之间的相互作用决定了牙周疾病的过程和进展，牙周致病菌一方面直接破坏牙周组织，另一方面通过刺激、调节宿主的免疫炎症反应间接造成牙周组织的病理损害。尽管对牙周病的免疫机制进行了许多研究，但对参与牙周病感染的免疫细胞类型及免疫机制尚未阐明。近年的研究发现，肥大细胞（mast cells, MCs）作为效应和调节性免疫细胞在固有免疫的建立和适应性免疫反应中发挥重要作用。研究显示在慢性牙周炎牙龈组织中观察到MCs，且主要为类胰蛋白酶（tryptase）亚型，提示MCs可能参与了慢性牙周炎病变的发生和发展。在固有免疫防御中，识别病原相关分子模式（pathogen-associated molecule patterns, PAMPs）的Toll样受体（toll-like receptors,TLRs）发挥了重要的作用。Toll样受体2（toll-like receptor2, TLR2）、Toll样受体4（toll-like receptor4, TLR4）作为TLRs家族的重要成员，可识别各种病原微生物表达的PAMPs，介导炎症反应。目前有关肥大细胞TLR2、TLR4在慢性牙周炎中作用的研究尚未见报道。本研究通过观察慢性牙周炎牙龈组织中肥大细胞TLR2、TLR4的表达情况，探讨肥大细胞TLR2、TLR4在牙周感染中的作用及其作用机制。研究结果将对后续以肥大细胞TLR2、TLR4为靶点的慢性牙周炎免疫防治提供理论基础和实验依据。方法：将自愿接受本研究的慢性牙周炎患者按慢性牙周炎分类标准分为4组：1）正常对照组15例；2）轻度慢性牙周炎组15例；3）中度慢性牙周炎组15例；4）重度慢性牙周炎组15例。牙龈标本在10%中性福尔马林固定液中浸泡48h以上，制作颊舌向5μm的牙龈组织连续切片。HE染色，光学显微镜下观察牙龈的组织学变化；采用免疫组织化学染色方法，光学显微镜下观察TLR2、TLR4阳性细胞在牙龈组织中的表达，并计算TLR2、TLR4平均阳性细胞率；采用免疫荧光双染色法，荧光显微镜下观察Tryptase-TLR2、TLR4双阳性MCs在牙龈组织中的表达，并计算Tryptase-TLR2、TLR4双阳性MCs的密度，，即单位面积免疫荧光阳性细胞数（cells/mm2）。用统计学软件SPSS13.0分析处理，多样本间的比较采用完全随机设计的单因素方差分析，用Levene法进行方差齐性检验；方差齐性时，多个样本均数间的两两比较采取Bonferroni检验；若方差不齐时采用Tamhane’s T2法；各组间肥大细胞TLR2、TLR4的密度与慢性牙周炎炎症程度之间作Pearson相关分析，P0.05时认为差异有统计学意义。结果： 1、组织学观察结果慢性牙周炎组牙龈组织中炎症细胞比正常对照组明显增多；慢性牙周炎组的炎症程度评分明显高于正常对照组（P0.01）；中度慢性牙周炎组的炎症程度评分明显高于轻度慢性牙周炎组（P0.01）；重度慢性牙周炎组的炎症程度评分明显高于轻、中度慢性牙周炎组（P0.01）。 2、免疫组织化学染色结果慢性牙周炎组TLR2、TLR4平均阳性细胞率均比正常对照组明显升高（P0.01）；中度慢性牙周炎组的TLR2、TLR4平均阳性细胞率均显著高于轻度慢性牙周炎组（P0.01）；重度慢性牙周炎组的TLR2、TLR4平均阳性细胞率也显著高于轻、中度慢性牙周炎组（P0.01）。 3、免疫荧光双染色结果慢性牙周炎组Tryptase-TLR2、TLR4双阳性MCs的密度比正常对照组明显升高（P0.01）；中度慢性牙周炎组的Tryptase-TLR2、TLR4双阳性MCs的密度显著高于轻度慢性牙周炎组（P0.01）；重度慢性牙周炎组的Tryptase-TLR2、TLR4双阳性MCs的密度也显著高于轻、中度慢性牙周炎组（P0.01）。 4、免疫阳性细胞与慢性牙周炎炎症程度相关性分析结果经Pearson相关分析，各组TLR2、TLR4平均阳性细胞率、Tryptase-TLR2、TLR4双阳性MCs密度均与慢性牙周炎的炎症程度存在正相关关系。结论： 1、慢性牙周炎牙龈组织中TLR2、TLR4平均阳性细胞率随着慢性牙周炎的炎症程度加重而增加。 2、慢性牙周炎牙龈组织中Tryptase-TLR2、TLR4双阳性MCs的密度随着慢性牙周炎的炎症程度加重而增加。肥大细胞TLR2、TLR4可能参与慢性牙周炎的炎症反应，在慢性牙周炎的发病和疾病进程中起着关键作用。
[Abstract]:Background and Purpose :

periodontal disease is an inflammatory disease caused by plaque microorganisms , which occurs around the teeth . The plaque microorganism is the initial factor of periodontal disease , but only the pathogenic role of periodontal disease can not resolve the pathological damage of periodontal disease . The interaction between the pathogenic bacteria and the host determines the pathological damage of periodontal disease .

Toll - like receptor2 ( TLR2 ) and Toll - like receptor4 ( TLRs ) play an important role in the pathogenesis and development of chronic periodontitis . Toll like receptor 2 ( toll - like receptor2 , TLR2 ) and Toll - like receptor4 ( TLRs ) play an important role in the pathogenesis of chronic periodontitis .

Method :

Patients with chronic periodontitis who voluntarily accepted the study were divided into 4 groups according to the classification standard of chronic periodontitis : 1 ) normal control group ( n = 15 ) ;
2 ) 15 patients with mild chronic periodontitis ;
3 ) 15 patients with moderate chronic periodontitis ;
4 ) In 15 patients with severe chronic periodontitis , gingival specimens were soaked in 10 % neutral formalin for more than 48 hours , and the buccal tongues were sectioned into 5 渭m gingival tissue . HE staining and optical microscope were used to observe the histological changes of the gums .
Immunohistochemical staining was used to observe the expression of TLR2 and TLR2 in gingival tissues and to calculate the average positive cell rate of TLR2 and TLR2 .
Using immunofluorescence double staining method , the expression of Tryptase - TLR2 was observed under the fluorescence microscope , and the density of the two positive cells of Tryptase - TLR2 was calculated . The number of cells per mm2 was calculated by SPSS 13.0 , and the variance analysis was performed with Levene method .
When the variance is homogeneous , the two comparisons between the number of samples take the Bondevii test ;
Tamhane ' s T2 method is adopted if the variance is not aligned ;
There was a Pearson correlation between the density of TLR2 and TLR2 and the degree of inflammation of chronic periodontitis in each group , and the difference was considered statistically significant at P0.05 .

Results :

1 . Histological Observations

The inflammatory cells in the gingival tissues of the chronic periodontitis group were significantly higher than those in the normal control group .
The degree of inflammation in the chronic periodontitis group was significantly higher than that in the control group ( P0.01 ) .
The degree of inflammation in patients with moderate chronic periodontitis was significantly higher than that in mild chronic periodontitis group ( P0.01 ) .
The scores of inflammation in patients with severe chronic periodontitis were significantly higher than those in mild and moderate chronic periodontitis ( P0.01 ) .

2 . Results of immunohistochemistry staining

Compared with the control group , the average positive rate of TLR2 and TLR2 in the chronic periodontitis group was significantly higher than that in the normal control group ( P0.01 ) .
Compared with mild chronic periodontitis group , the average positive cell rate of TLR2 and TLR2 in moderate chronic periodontitis group was significantly higher than that in mild chronic periodontitis group ( P0.01 ) .
In severe chronic periodontitis group , the average positive rate of TLR2 was higher than that in mild and moderate chronic periodontitis group ( P0.01 ) .

3 . Immunofluorescence double staining results

Compared with the control group , the density of Tryptase - TLR2 was significantly higher in the chronic periodontitis group than in the control group ( P0.01 ) .
In moderate chronic periodontitis group , the density was significantly higher than that in mild chronic periodontitis group ( P0.01 ) .
In the severe chronic periodontitis group , the density of the two positive clones was significantly higher than that in the mild and moderate chronic periodontitis group ( P0.01 ) .

4 . Analysis of the correlation between immune - positive cells and the degree of inflammation in chronic periodontitis

Pearson correlation analysis showed that the average positive cell rate of TLR2 , TLR2 and TLR2 in each group had positive correlation with the degree of inflammation in chronic periodontitis .

Conclusion :

1 . In the gingival tissues of chronic periodontitis , the average positive cell rate of TLR2 was increased with the degree of inflammation of chronic periodontitis .

2 . Tryptase - TLR2 in the gingival tissues of chronic periodontitis increased with the severity of chronic periodontitis .

Mast cell TLR2 , Toll - like receptor can participate in the inflammatory reaction of chronic periodontitis and play a key role in the pathogenesis of chronic periodontitis and disease progression .
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R781.42

【参考文献】