【摘要】：第一部分口腔扁平苔蘚與NF κB相關(guān)細(xì)胞因子和幽門(mén)螺桿菌關(guān)系的研究目的:口腔扁平苔蘚(Oral lichen planus OLP)是一種與自身免疫相關(guān)常見(jiàn)口腔疾病,目前為止OLP病因機(jī)制尚未完全明確。近年來(lái)核轉(zhuǎn)錄因子-κB(nuclearfactor-KB NF-κB)引導(dǎo)的炎癥通路及其介導(dǎo)的炎癥介質(zhì)的紊亂一直成為研究的熱點(diǎn)。NF-κB可能通過(guò)引起細(xì)胞因子網(wǎng)絡(luò)紊亂,進(jìn)一步介導(dǎo)炎癥反應(yīng)參與口腔扁平苔蘚疾病的發(fā)生。楊鳳英等采用基因芯片技術(shù)篩選出與OLP關(guān)系最密切的是幽門(mén)螺桿菌感染上皮細(xì)胞信號(hào)傳導(dǎo)通路ECS-HP(Epithelial cell signaling in Helicobacter pylori infection)[1],口腔中幽門(mén)螺桿菌(Helicobacter pylori HP)感染與口腔扁平苔蘚疾病的發(fā)生存在密切關(guān)系,HP也與慢性胃炎、消化性潰瘍、胃黏膜相關(guān)淋巴組織淋巴瘤、胃癌的發(fā)病密切相關(guān)。HP感染胃壁粘膜后可以激活NF-κB通路并介導(dǎo)炎癥反應(yīng)�？谇徽衬づc胃壁粘膜均來(lái)自于外胚層,口腔黏膜感染HP后引起的炎癥機(jī)制可能與胃壁粘膜一致。口腔中HP感染激活NF-κB炎癥通路在OLP發(fā)病中的作用有待進(jìn)一步研究。本研究采用酶聯(lián)免疫吸附實(shí)驗(yàn)(ELISA),檢測(cè)口腔扁平苔蘚患者與健康者血清、唾液中IL-8、RANTES的蛋白水平變化,通過(guò)觀察血清和唾液中IL-8、RANTES等炎癥因子的變化水平以及感染幽門(mén)螺桿菌OLP患者血清和唾液中細(xì)胞因子變化水平,初步探討NF-κB炎癥通路在口腔扁平苔蘚發(fā)病中的作用機(jī)制以及OLP與HP的關(guān)系。方法:1采用ELISA方法對(duì)口腔扁平苔蘚患者和健康者血清、唾液中IL-8、RANTES的含量進(jìn)行測(cè)定,分析IL-8、RANTES在血清、唾液中表達(dá)。病例納入標(biāo)準(zhǔn):口腔扁平苔蘚患者30例(糜爛型14例,普通型16例)和健康志愿者30例,分別收集口腔扁平苔蘚患者和健康志愿者的唾液、血清樣本。2采用ELISA方法檢測(cè)HP感染陽(yáng)性及HP陰性的口腔扁平苔蘚患者血清、唾液中IL-8、RANTES的表達(dá),分析HP感染與NF-κB通路在口腔扁平苔蘚發(fā)病中的關(guān)系及其關(guān)鍵因子的表達(dá)。3幽門(mén)螺桿菌感染檢測(cè):快速尿素酶試驗(yàn)法檢測(cè)口腔扁平苔蘚患者牙菌斑中的幽門(mén)螺桿菌,探討OLP患者和HP感染的相關(guān)性。結(jié)果:1口腔扁平苔蘚患者與對(duì)照組相比血清和唾液中IL-8、RANTES顯著增高,P0.05,差別有統(tǒng)計(jì)學(xué)意義;2口腔扁平苔蘚血清中糜爛型與普通型IL-8、RANTES細(xì)胞因子表達(dá)水平差別無(wú)統(tǒng)計(jì)學(xué)意義,P0.05,口腔扁平苔蘚唾液中糜爛型IL-8含量較普通型扁平苔蘚顯著升高,P0.05;3將口腔扁平苔蘚分為HP感染陽(yáng)性和HP感染陰性,血清中HP感染陽(yáng)性較HP感染陰性者IL-8、RANTES表達(dá)水平顯著增高,P0.05。唾液中HP感染陽(yáng)性較HP感染陰性者IL-8、RANTES表達(dá)水平差別無(wú)統(tǒng)計(jì)學(xué)意義,P0.05。結(jié)論:1口腔扁平苔蘚患者血清和唾液中IL-8、RANTES細(xì)胞因子水平與對(duì)照組相比均有不同程度的升高,提示口腔扁平苔蘚的發(fā)病可能與NF κB通路介導(dǎo)的炎癥反應(yīng)相關(guān),阻斷NF κB炎癥通路的激活可能成為治療口腔扁平苔蘚新的靶點(diǎn),為口腔扁平苔蘚的治療提供新思路與方法。2血清中HP感染陽(yáng)性較HP感染陰性者IL-8、RANTES表達(dá)水平顯著增高,提示口腔扁平苔蘚的發(fā)病可能與口腔中HP感染有一定相關(guān)性,并進(jìn)一步介導(dǎo)免疫炎癥反應(yīng)。第二部分藥物對(duì)牙齦成纖維細(xì)胞增殖作用的影響目的:目前藥物性牙齦增生的機(jī)制尚未完全明確。一些研究表明牙齦炎、牙周炎菌斑的刺激可能有助于牙齦增生發(fā)展。本課題利用健康人牙齦組織原代培養(yǎng)牙齦成纖維細(xì)胞,分別應(yīng)用硝苯地平、IL-1以及兩種藥物共同干預(yù)牙齦成纖維細(xì)胞,觀察不同藥物對(duì)牙齦成纖維細(xì)胞的增殖活性的影響,進(jìn)一步探討藥物性牙齦增生機(jī)制以及藥物性牙齦增生與牙齦炎的關(guān)系,為臨床上藥物性牙齦增生治療提供新的思路。方法:1牙齦成纖維細(xì)胞原代培養(yǎng):按照原代細(xì)胞組織塊培養(yǎng)方法,將牙齦組織塊接種于25m L細(xì)胞培養(yǎng)瓶中,觀察原代細(xì)胞游出狀態(tài)。細(xì)胞爬滿(mǎn)瓶底約80%時(shí)進(jìn)行傳代。使用0.25%胰酶消化傳代培養(yǎng),首次傳代按1:1的比例,再次傳代以1:3的比例進(jìn)行。取第5代牙齦成纖維細(xì)胞用于實(shí)驗(yàn)。其余細(xì)胞凍存?zhèn)溆谩?牙齦成纖維細(xì)胞鑒定:倒置顯微鏡下觀察原代細(xì)胞形態(tài)學(xué)變化;光鏡下觀察原代細(xì)胞HE染色及免疫組化結(jié)果(抗波形蛋白、抗角蛋白)。3細(xì)胞增殖實(shí)驗(yàn):采用甲基噻唑基四唑(methyl thiazolyltetrazolium,MTT)法測(cè)定不同條件培養(yǎng)液下細(xì)胞數(shù)量變化(A組為空白對(duì)照組,不加任何藥物;B組為硝苯地平組,又分為3個(gè)濃度亞組,B1組加入硝苯地平1200μg/L,B2組加入硝苯地平360μg/L,B3組加入硝苯地平108μg/L;C組為IL-1組,加入IL-1β10ng/ml;D組為硝苯地平+IL-1組,分為3個(gè)亞組,D1組=B1組+C組,D2組=B2組+C組,D3組=B3組+C組),在酶聯(lián)免疫儀上測(cè)定490 nm處各孔吸光度值(OD值)。本實(shí)驗(yàn)重復(fù)3次。利用SPSS21.0軟件分析數(shù)據(jù)。結(jié)果:1原代培養(yǎng)細(xì)胞從組織塊游離出的時(shí)間約為10天,倒置顯微鏡鏡下觀察可見(jiàn)散在的貼壁伸展細(xì)胞,細(xì)胞形態(tài)呈長(zhǎng)梭形,第11天組織塊周?chē)?xì)胞明顯增多,組織塊周?chē)纬杉?xì)胞生長(zhǎng)暈形態(tài)。原代第12天,可見(jiàn)細(xì)胞呈長(zhǎng)梭形,胞核圓形或卵圓形,細(xì)胞數(shù)量明顯增多。約12-14天細(xì)胞逐漸融合;2牙齦成纖維細(xì)胞鑒定:經(jīng)傳代培養(yǎng)后牙齦成纖維細(xì)胞形態(tài)逐漸向典型的成纖維樣細(xì)胞轉(zhuǎn)化,細(xì)胞生長(zhǎng)呈漩禍狀或放射走行,有極性且排列緊密,突起變短,呈長(zhǎng)梭形、紡綞形、不規(guī)則三角形態(tài)(見(jiàn)Fig.1)。HE染色示細(xì)胞呈梭形或不規(guī)則三角形態(tài),核圓或卵圓,胞核內(nèi)可見(jiàn)分裂象,胞漿粉紅,胞核藍(lán)染(見(jiàn)Fig.2)。光鏡下觀察細(xì)胞免疫組織化學(xué)染色,波形蛋白表達(dá)為強(qiáng)陽(yáng)性,呈棕黃著色,定位于細(xì)胞的胞質(zhì)中,陽(yáng)性顆粒在胞質(zhì)內(nèi)分布均勻,細(xì)胞核內(nèi)染色陰性,細(xì)胞外未見(jiàn)陽(yáng)性表達(dá),細(xì)胞核經(jīng)蘇木精復(fù)染為藍(lán)色,說(shuō)明細(xì)胞來(lái)源于外胚間充質(zhì)(見(jiàn)Fig.3),對(duì)照組細(xì)胞染色陰性。角蛋白表達(dá)為陰性(見(jiàn)Fig.4);3細(xì)胞增殖實(shí)驗(yàn):不同濃度培養(yǎng)液作用后細(xì)胞增殖結(jié)果:培養(yǎng)0h、24h、48h、72h、96h各組OD值變化見(jiàn)Table 1。結(jié)論:1原代培養(yǎng)的細(xì)胞抗波形蛋白表達(dá)陽(yáng)性、抗角蛋白表達(dá)陰性以及形態(tài)學(xué)上觀察結(jié)果符合牙齦成纖維細(xì)胞,保留此細(xì)胞株,以便用于今后實(shí)驗(yàn)。2三種不同濃度硝苯地平都在一定程度上促進(jìn)牙齦成纖維細(xì)胞細(xì)胞增殖,當(dāng)硝苯地平濃度為1200μg/L時(shí)細(xì)胞增殖作用最明顯,說(shuō)明硝苯地平局部藥物濃度可能在牙齦增生方面有著重要作用;3 IL-1濃度為10ng/ml對(duì)牙齦成纖維細(xì)胞的增殖活性產(chǎn)生抑制。硝苯地平與IL-1共同作用于牙齦成纖維細(xì)胞時(shí),對(duì)牙齦成纖維細(xì)胞增殖作用不明顯,有可能是牙齦炎癥對(duì)硝苯地平引起的牙齦增生效果不明顯。
[Abstract]:The relationship between the first part of oral lichen planus and NF-B-associated cytokines and Helicobacter pylori: Oral lichen planus OLP is a common oral disease associated with autoimmune diseases, so far the cause mechanism of OLP has not been completely clear. In recent years, the inflammatory pathway and the disorder of the mediated inflammatory mediators of the nuclear transcription factor-BNF-B have been the focus of the study. NF-B may further mediate inflammatory response to the occurrence of oral lichen planus by causing a disorder of the cytokine network. The infection of Helicobacter pylori (HP) in the oral cavity is closely related to the occurrence of oral lichen planus disease. HP is also closely related to chronic gastritis, digestive tract ulceration, gastric mucosa-associated lymphoid tissue lymphoma, and gastric cancer. The NF-B-B pathway can be activated and the inflammatory response can be mediated by the HP infection of the gastric wall mucosa. The mucosa of the oral mucosa and the stomach wall all come from the ectodermal layer, and the inflammatory mechanism caused by the infection of HP in the oral mucosa may be consistent with the mucosa of the stomach wall. The role of HP infection in the oral cavity in the pathogenesis of OLP is to be further studied. In this study, an enzyme-linked immunosorbent assay (ELISA) was used to detect the changes of the levels of IL-8 and RANTES in the serum and saliva of oral lichen planus, and by observing the levels of IL-8 in serum and saliva, The changes of the inflammatory factors such as RANTES and the level of cytokines in the serum and saliva of the patients with Helicobacter pylori OLP were studied. The mechanism of the inflammatory pathway in the oral lichen planus and the relationship between OLP and HP were discussed. Methods:1 The levels of IL-8 and RANTES in the serum and saliva of oral lichen planus and healthy persons were determined by ELISA, and the expression of IL-8 and RANTES in serum and saliva was analyzed. The cases were included in 30 cases of oral lichen planus (14 cases of erosion type,16 cases of common type) and 30 healthy volunteers. The saliva of oral lichen planus and healthy volunteers were collected separately. The expression of IL-8 and RANTES in the serum and saliva of the oral lichen planus with positive and negative HP infection was detected by ELISA. The relationship between HP infection and NF-B pathway in the pathogenesis of oral lichen planus was analyzed and the expression of the key factors was analyzed. The relationship between OLP and HP infection was discussed by the rapid urease test in the detection of Helicobacter pylori in the dental plaque of the oral lichen planus. Results: The levels of IL-8 and RANTES in the serum and saliva of 1 oral lichen planus were significantly higher than those in the control group. The level of IL-8 in the saliva of the oral lichen planus was higher than that of the normal lichen planus, P 0.05;3. The oral lichen planus was divided into HP infection positive and HP infection negative. There was no significant difference in the expression of IL-8 and RANTES in the patients with positive HP infection in saliva, P0.05. Conclusion: The level of IL-8 and RANTES in the serum and saliva of the oral lichen planus is higher than that of the control group, suggesting that the incidence of the oral lichen planus may be related to the inflammatory response mediated by the NF-B pathway. Blocking the activation of the NF-B inflammatory pathway may be a new target for the treatment of oral lichen planus, providing a new thought and method for the treatment of oral lichen planus. It is suggested that the incidence of oral lichen planus may be associated with HP infection in the oral cavity and further mediate the immune inflammatory response. The effect of the second part of the drug on the proliferation of gingival fibroblasts is that the mechanism of drug-induced gingival hyperplasia is not yet completely clear. Some studies have shown that the stimulation of plaque in gingivitis and periodontitis may contribute to the development of gingival hyperplasia. In this paper, the gingival fibroblasts were cultured in the gingival tissues of healthy people, and nifedipine, IL-1 and the two drugs were applied to the gingival fibroblasts, and the effects of different drugs on the proliferation of gingival fibroblasts were observed. To further study the relationship between drug-induced gingival hyperplasia and drug-induced gingival hyperplasia and gingivitis, and provide a new way for the treatment of drug-induced gingival hyperplasia. Methods:1. Primary culture of gingival fibroblasts: the gingival tissue mass was inoculated into a 25 m L cell culture flask according to the primary cell culture method, and the primary cell was observed. The cells were passaged at about 80% of the bottom of the flask. The subculture was digested with 0.25% trypsin, and the first passage was carried out at a ratio of 1:1, and the passage was carried out at a ratio of 1:3. The 5th generation of gingival fibroblasts was used for the experiment. The rest of the cells were cryopreserved.2 Gingival fibroblast identification: the morphological changes of primary cells were observed under an inverted microscope; the HE staining and the immunohistochemical results of primary cells (anti-keratin and anti-keratin) were observed under light microscope. The changes of the number of cells under different conditions were determined by MTT method. The group A was the blank control group without any drug. The group B was the nifedipine group, and it was divided into 3 concentration sub-groups, and the B1 group was added nifedipine 1200. m u.g/ L and the B2 group was added with nifedipine 360. m In group B3, Nifedipine 108. mu.g/ L was added; group C was IL-1, IL-1 and 10 ng/ ml were added; the D group was nifedipine + IL-1, divided into 3 subgroups, D1 group = B1 group + C group, D2 group = B2 group + C group, D3 group = B3 group + C group), The absorbance value (OD value) of each hole at 490 nm was measured on an enzyme-linked immunoassay. This experiment was repeated three times. The software was used to analyze the data. Results: The free time of the primary cultured cells from the tissue mass was about 10 days, and the adherent stretching cells of the primary cultured cells were observed under the inverted microscope. The cell morphology of the primary cultured cells was long and the number of cells around the tissue mass in the 11th day was significantly increased, and the cell growth halo was formed around the tissue mass. On the 12th day of primary culture, the cells were found to be long, round or oval, and the number of cells increased significantly. from about 12 to 14 days, the cells are gradually fused; the gingival fibroblasts are characterized in that the morphology of the gingival fibroblasts is gradually transformed to a typical fibroblast-like cell after subculture, Irregular triangular shape (see Fig.1). HE staining showed that the cells were in the form of a shuttle or an irregular triangle, a nucleus or an egg circle, a split image in the nucleus of the nucleus, pink of the cytoplasm, and blue staining of the nucleus (see Fig.2). The immunohistochemical staining of the cells was observed under the light microscope. The expression of the fluorescent protein was strongly positive. It was found to be brown and yellow, which was localized in the cytoplasm of the cell. The positive particles were distributed in the cytoplasm. The positive expression was not found in the cell. The nuclei were stained with hematoxylin to blue. The cells were derived from the human mesenchymal stem cells (see Fig.3), and the control group cells were stained with negative staining. The expression of keratin was negative (see Fig.4);3-cell proliferation experiment: the proliferation of cells after different concentration of culture medium: the changes of OD values in each group after incubation for 0 h,24 h,48 h,72 h, and 96 h are shown in Table 1. Conclusion:1. The expression of anti-keratin in primary cultured cells is positive, the anti-keratin expression is negative and the morphological observation is in line with the gingival fibroblasts, and the cell line is preserved. so as to be used for future experiments. The three different concentrations of nifedipine promote the proliferation of the gingival fibroblast cells to a certain extent, and the cell proliferation effect is most obvious when the concentration of nifedipine is 1200 & mu; g/ L, and the concentration of the local drug of nifedipine can play an important role in the gingival hyperplasia; The proliferation of gingival fibroblasts was inhibited by the concentration of 3IL-1 at 10 ng/ ml. The effect of nifedipine and IL-1 on the proliferation of gingival fibroblasts is not obvious, and the effect of gingival inflammation on the gingival hyperplasia caused by nifedipine is not obvious.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R781.5

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