伴放线聚集杆菌对正常人牙龈上皮细胞中β防御素表达的影响
发布时间：2018-04-29 16:41
本文选题：伴放线聚集杆菌 + 人牙龈上皮细胞 ； 参考：《广西医科大学》2014年硕士论文
【摘要】：目的:通过检测不同浓度的伴放线聚集杆菌（A.a）刺激人牙龈上皮细胞(HGEC)后防御素及炎症细胞因子的动态表达，探讨A.a对牙龈上皮抗感染免疫的影响。 方法:将临床取材的正常人牙龈组织采用组织块培养法获取原代牙龈上皮细胞。取A.a标准菌株（ATCC43717）复苏培养，调整细菌浓度分别为5×107cells/ml、1×108cells/ml、2.5×108cells/ml，加入含5×105cells/ml的第3代HGEC中，使感染复数（MOI）分别为100:1、200:1、500:1，共培养0h、2h、4h、8h、12h、24h和48h。每个时间点于倒置显微镜下观察HGEC活力，收集共培养的上清液用于ELISA检测HGEC表达HBD-1、HBD-2、HBD-3、TNF-α、IL-1β的情况，同期贴壁细胞用于提取总RNA进行Real-TimePCR检测HBD-1mRNA、HBD-2mRNA、HBD-3mRNA的表达。所有数据来源于3次独立实验。计量指标用均数标准差或中位数表示；干预前后不同时间点及感染复数下检测因子表达水平的组间差异采用区组设计方差分析及LSD-t检验或Freidman-M检验；用Pearson或Spearman检验分析感染复数、干预时间与检测因子表达量、以及各检测因子间的相关性；P㩳0.05时有统计学意义。 结果:1、人牙龈上皮细胞产生HBD-1、HBD-2、HBD-3以及IL-1β、TNF-α随感染复数变化情况。取A.a与HGEC共培养的上清液进行ELISA检测，加入A.a后, HBD-1的表达仅在感染2h随MOI变化而变化（R2=0.999,P=0.024）。HBD-2在感染8h表达量随MOI增加而减少（R2=0.994,P=0.048）。Real-Time PCR检测，加入A.a后，，观察点2h，HBD-1mRNA增长倍数随MOI增加而增加（R2=0.996, P=0.040）。 2、人牙龈上皮细胞产生的HBD-1、HBD-2、HBD-3以及IL-1β、TNF-α随感染时间变化情况。MOI为100:1时，7个观察时间点组间比较HGEC产生的HBD-1、HBD-3均无明显差异（F=0.753, P＞0.05; F=0.485, P＞0.05）；HBD-2在2h即开始增加(P＜0.001)，且0h到24h表达量随时间的延长而增加（R2=0.818, P=0.013）。MOI为200:1时，HBD-1在7个观察点组间比较无差异（F=2.923, P＞0.05）； HBD-3在0h到12h逐渐增加（R2=0.924, P=0.009）；IL-1β表达在4h到48h随时间延长表达量反而减少（R2=0.865, P=0.022），但24h表达量仍高于干预前（P＜0.001）。MOI为500:1时，HBD-1表达在2h后随着观察时间的延长，其表达量逐渐增多（R2=0.901, P=0.014）。Real-TimePCR检测，三种感染复数下， HBD-2mRNA表达量24h达到最高峰；HBD-3mRNA表达量分别在12h、8h和24h达到最高峰。结论: A.a诱导正常人牙龈上皮细胞中HBD-2、HBD-3表达较HBD-1明显。
[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- 伪 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- 伪 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- 伪 with infection time were 100: 1, the HBD-1 HBD-3 produced by human gingival epithelial cells and IL-1 尾 TNF- 伪 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.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R781.4
【参考文献】
相关期刊论文 前1条
1 张旭;束蓉;罗礼君;葛琳华;李昊妍;;β-防御素-2多肽在不同类型牙周炎及健康牙龈组织中的表达[J];上海口腔医学;2006年01期
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