【摘要】：背景及目的： 牙周病是由菌斑微生物引起的、發(fā)生在牙齒周圍組織的炎癥性疾病。菌斑微生物是牙周病的始動因子，但是單純的牙周致病菌的致病作用不能解析牙周病的破壞作用，多種免疫細(xì)胞及細(xì)胞因子參與了牙周病的病理損害。牙周致病菌與宿主之間的相互作用決定了牙周疾病的過程和進展，牙周致病菌一方面直接破壞牙周組織，另一方面通過刺激、調(diào)節(jié)宿主的免疫炎癥反應(yīng)間接造成牙周組織的病理損害。盡管對牙周病的免疫機制進行了許多研究，但對參與牙周病感染的免疫細(xì)胞類型及免疫機制尚未闡明。 近年的研究發(fā)現(xiàn)，肥大細(xì)胞（mast cells, MCs）作為效應(yīng)和調(diào)節(jié)性免疫細(xì)胞在固有免疫的建立和適應(yīng)性免疫反應(yīng)中發(fā)揮重要作用。研究顯示在慢性牙周炎牙齦組織中觀察到MCs，且主要為類胰蛋白酶（tryptase）亞型，提示MCs可能參與了慢性牙周炎病變的發(fā)生和發(fā)展。在固有免疫防御中，識別病原相關(guān)分子模式（pathogen-associated molecule patterns, PAMPs）的Toll樣受體（toll-like receptors,TLRs）發(fā)揮了重要的作用。Toll樣受體2（toll-like receptor2, TLR2）、Toll樣受體4（toll-like receptor4, TLR4）作為TLRs家族的重要成員，可識別各種病原微生物表達的PAMPs，介導(dǎo)炎癥反應(yīng)。目前有關(guān)肥大細(xì)胞TLR2、TLR4在慢性牙周炎中作用的研究尚未見報道。本研究通過觀察慢性牙周炎牙齦組織中肥大細(xì)胞TLR2、TLR4的表達情況，探討肥大細(xì)胞TLR2、TLR4在牙周感染中的作用及其作用機制。研究結(jié)果將對后續(xù)以肥大細(xì)胞TLR2、TLR4為靶點的慢性牙周炎免疫防治提供理論基礎(chǔ)和實驗依據(jù)。 方法： 將自愿接受本研究的慢性牙周炎患者按慢性牙周炎分類標(biāo)準(zhǔn)分為4組：1）正常對照組15例；2）輕度慢性牙周炎組15例；3）中度慢性牙周炎組15例；4）重度慢性牙周炎組15例。牙齦標(biāo)本在10%中性福爾馬林固定液中浸泡48h以上，制作頰舌向5μm的牙齦組織連續(xù)切片。HE染色，光學(xué)顯微鏡下觀察牙齦的組織學(xué)變化；采用免疫組織化學(xué)染色方法，光學(xué)顯微鏡下觀察TLR2、TLR4陽性細(xì)胞在牙齦組織中的表達，并計算TLR2、TLR4平均陽性細(xì)胞率；采用免疫熒光雙染色法，熒光顯微鏡下觀察Tryptase-TLR2、TLR4雙陽性MCs在牙齦組織中的表達，并計算Tryptase-TLR2、TLR4雙陽性MCs的密度，，即單位面積免疫熒光陽性細(xì)胞數(shù)（cells/mm2）。用統(tǒng)計學(xué)軟件SPSS13.0分析處理，多樣本間的比較采用完全隨機設(shè)計的單因素方差分析，用Levene法進行方差齊性檢驗；方差齊性時，多個樣本均數(shù)間的兩兩比較采取Bonferroni檢驗；若方差不齊時采用Tamhane’s T2法；各組間肥大細(xì)胞TLR2、TLR4的密度與慢性牙周炎炎癥程度之間作Pearson相關(guān)分析，P0.05時認(rèn)為差異有統(tǒng)計學(xué)意義。 結(jié)果： 1、組織學(xué)觀察結(jié)果 慢性牙周炎組牙齦組織中炎癥細(xì)胞比正常對照組明顯增多；慢性牙周炎組的炎癥程度評分明顯高于正常對照組（P0.01）；中度慢性牙周炎組的炎癥程度評分明顯高于輕度慢性牙周炎組（P0.01）；重度慢性牙周炎組的炎癥程度評分明顯高于輕、中度慢性牙周炎組（P0.01）。 2、免疫組織化學(xué)染色結(jié)果 慢性牙周炎組TLR2、TLR4平均陽性細(xì)胞率均比正常對照組明顯升高（P0.01）；中度慢性牙周炎組的TLR2、TLR4平均陽性細(xì)胞率均顯著高于輕度慢性牙周炎組（P0.01）；重度慢性牙周炎組的TLR2、TLR4平均陽性細(xì)胞率也顯著高于輕、中度慢性牙周炎組（P0.01）。 3、免疫熒光雙染色結(jié)果 慢性牙周炎組Tryptase-TLR2、TLR4雙陽性MCs的密度比正常對照組明顯升高（P0.01）；中度慢性牙周炎組的Tryptase-TLR2、TLR4雙陽性MCs的密度顯著高于輕度慢性牙周炎組（P0.01）；重度慢性牙周炎組的Tryptase-TLR2、TLR4雙陽性MCs的密度也顯著高于輕、中度慢性牙周炎組（P0.01）。 4、免疫陽性細(xì)胞與慢性牙周炎炎癥程度相關(guān)性分析結(jié)果 經(jīng)Pearson相關(guān)分析，各組TLR2、TLR4平均陽性細(xì)胞率、Tryptase-TLR2、TLR4雙陽性MCs密度均與慢性牙周炎的炎癥程度存在正相關(guān)關(guān)系。 結(jié)論： 1、慢性牙周炎牙齦組織中TLR2、TLR4平均陽性細(xì)胞率隨著慢性牙周炎的炎癥程度加重而增加。 2、慢性牙周炎牙齦組織中Tryptase-TLR2、TLR4雙陽性MCs的密度隨著慢性牙周炎的炎癥程度加重而增加。 肥大細(xì)胞TLR2、TLR4可能參與慢性牙周炎的炎癥反應(yīng)，在慢性牙周炎的發(fā)病和疾病進程中起著關(guān)鍵作用。
[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 .
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R781.42

【參考文獻】

相關(guān)期刊論文 前1條

1 謝敏;黃世光;宋寧;張增方;鄧國珍;;Th1極化反應(yīng)與牙周炎病理改變的實驗研究[J];實用口腔醫(yī)學(xué)雜志;2010年04期

本文編號：2128251