本文選題：伴放線聚集桿菌 + 人牙齦上皮細(xì)胞��； 參考：《廣西醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的:通過檢測不同濃度的伴放線聚集桿菌（A.a）刺激人牙齦上皮細(xì)胞(HGEC)后防御素及炎癥細(xì)胞因子的動態(tài)表達(dá)，探討A.a對牙齦上皮抗感染免疫的影響。 方法:將臨床取材的正常人牙齦組織采用組織塊培養(yǎng)法獲取原代牙齦上皮細(xì)胞。取A.a標(biāo)準(zhǔn)菌株（ATCC43717）復(fù)蘇培養(yǎng)，調(diào)整細(xì)菌濃度分別為5×107cells/ml、1×108cells/ml、2.5×108cells/ml，加入含5×105cells/ml的第3代HGEC中，使感染復(fù)數(shù)（MOI）分別為100:1、200:1、500:1，共培養(yǎng)0h、2h、4h、8h、12h、24h和48h。每個時間點(diǎn)于倒置顯微鏡下觀察HGEC活力，收集共培養(yǎng)的上清液用于ELISA檢測HGEC表達(dá)HBD-1、HBD-2、HBD-3、TNF-α、IL-1β的情況，同期貼壁細(xì)胞用于提取總RNA進(jìn)行Real-TimePCR檢測HBD-1mRNA、HBD-2mRNA、HBD-3mRNA的表達(dá)。所有數(shù)據(jù)來源于3次獨(dú)立實驗。計量指標(biāo)用均數(shù)標(biāo)準(zhǔn)差或中位數(shù)表示；干預(yù)前后不同時間點(diǎn)及感染復(fù)數(shù)下檢測因子表達(dá)水平的組間差異采用區(qū)組設(shè)計方差分析及LSD-t檢驗或Freidman-M檢驗；用Pearson或Spearman檢驗分析感染復(fù)數(shù)、干預(yù)時間與檢測因子表達(dá)量、以及各檢測因子間的相關(guān)性；P㩳0.05時有統(tǒng)計學(xué)意義。 結(jié)果:1、人牙齦上皮細(xì)胞產(chǎn)生HBD-1、HBD-2、HBD-3以及IL-1β、TNF-α隨感染復(fù)數(shù)變化情況。取A.a與HGEC共培養(yǎng)的上清液進(jìn)行ELISA檢測，加入A.a后, HBD-1的表達(dá)僅在感染2h隨MOI變化而變化（R2=0.999,P=0.024）。HBD-2在感染8h表達(dá)量隨MOI增加而減少（R2=0.994,P=0.048）。Real-Time PCR檢測，加入A.a后，，觀察點(diǎn)2h，HBD-1mRNA增長倍數(shù)隨MOI增加而增加（R2=0.996, P=0.040）。 2、人牙齦上皮細(xì)胞產(chǎn)生的HBD-1、HBD-2、HBD-3以及IL-1β、TNF-α隨感染時間變化情況。MOI為100:1時，7個觀察時間點(diǎn)組間比較HGEC產(chǎn)生的HBD-1、HBD-3均無明顯差異（F=0.753, P＞0.05; F=0.485, P＞0.05）；HBD-2在2h即開始增加(P＜0.001)，且0h到24h表達(dá)量隨時間的延長而增加（R2=0.818, P=0.013）。MOI為200:1時，HBD-1在7個觀察點(diǎn)組間比較無差異（F=2.923, P＞0.05）； HBD-3在0h到12h逐漸增加（R2=0.924, P=0.009）；IL-1β表達(dá)在4h到48h隨時間延長表達(dá)量反而減少（R2=0.865, P=0.022），但24h表達(dá)量仍高于干預(yù)前（P＜0.001）。MOI為500:1時，HBD-1表達(dá)在2h后隨著觀察時間的延長，其表達(dá)量逐漸增多（R2=0.901, P=0.014）。Real-TimePCR檢測，三種感染復(fù)數(shù)下， HBD-2mRNA表達(dá)量24h達(dá)到最高峰；HBD-3mRNA表達(dá)量分別在12h、8h和24h達(dá)到最高峰。結(jié)論: A.a誘導(dǎo)正常人牙齦上皮細(xì)胞中HBD-2、HBD-3表達(dá)較HBD-1明顯。
[Abstract]:Objective: to detect the dynamic expression of defensins and inflammatory cytokines in human gingival epithelial cells (HGECs) stimulated by A. actinomycetes at different concentrations, and to investigate the effect of A. a on the anti-infective immunity of gingival epithelium. Methods: primary gingival epithelial cells were obtained from normal human gingival tissue by tissue mass culture. A. a standard strain ATCC43717) was resuscitated and the bacterial concentration was adjusted to 5 脳 10 7 cells / ml 1 脳 10 8 cells / ml respectively, and added to the third generation of HGEC containing 5 脳 105cells/ml. The infected plural number of moi was 100: 1200: 15001, and the total culture time was 0 h 2 h, 4 h, 8 h, 12 h and 48 h, respectively. The activity of HGEC was observed under inverted microscope at each time point. The supernatants of co-culture were collected to detect the expression of HBD-1mRNA-HBD-3TNF- 偽 IL-1 尾 by ELISA, and the total RNA was extracted from the adherent cells to detect the expression of HBD-1mRNA-HBD-2mRNA-HBD-3 mRNA by Real-TimePCR. All data were obtained from three independent experiments. The mean standard deviation or median was used to measure the index, the difference of the expression level of factors in different time points before and after intervention and in the complex number of infection was analyzed by block design variance analysis and LSD-t test or Freidman-M test, and the complex number of infection was analyzed by Pearson or Spearman test. The time of intervention was significantly higher than that of P0. 05 when the time of intervention was higher than that of the expression of the detected factors (P < 0. 05). Results Human gingival epithelial cells produced HBD-1, HBD-2, HBD-3 and IL-1 尾 -TNF- 偽 with the complex number of infections. The supernatants of A. a and HGEC were taken for ELISA detection. After adding A. a, the expression of HBD-1 changed with the change of MOI only at 2 hours after infection. The expression of R2O0.999Pu 0.024N. HBD-2 decreased with the increase of MOI at 8h, and the expression of R2O0.994P0.048.Real-Time PCR was detected after adding A. The increase of HBD-1 mRNA in the observation site increased with the increase of MOI. (2) when the changes of HBD-1 and IL-1 尾 TNF- 偽 with infection time were 100: 1, the HBD-1 HBD-3 produced by human gingival epithelial cells and IL-1 尾 TNF- 偽 were 100: 1. There was no significant difference in HBD-1 HBD-3 produced by HGEC between the seven observation time points. F0. 485, P > 0. 05, HBD-2 began to increase at 2 h, and the expression of HBD-1 was increased with 0 h to 24 h. When P=0.013).MOI was 200: 1, there was no difference between the seven observation point groups (F = 2.923, P > 0.05), HBD-3 increased gradually from 0h to 12h, the expression of IL-1 尾 decreased after 4h to 48h, but the expression of HBD-3 was still higher than that of intervention at 24h to 48h. P < 0.001).MOI = 500: 1, the expression of HBD-1 was increased with the prolongation of observation time 2 hours later. The expression of HBD-2mRNA reached the highest peak at 24 h and reached the peak at 12 h, 8 h and 24 h, respectively, and the expression of HBD-3 mRNA reached the highest peak at 12 h, 8 h and 24 h, respectively. Conclusion: the expression of HBD-2 and HBD-3 in normal gingival epithelial cells induced by A. a was significantly higher than that in HBD-1.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R781.4

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1 張旭;束蓉;羅禮君;葛琳華;李昊妍;;β-防御素-2多肽在不同類型牙周炎及健康牙齦組織中的表達(dá)[J];上�？谇会t(yī)學(xué);2006年01期

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