脂氧素A4在口腔扁平苔藓NF-κBp65信号通路中的作用研究

发布时间：2018-05-20 03:26

本文选题：脂氧素A4 + 口腔扁平苔藓　；参考：《福建医科大学》2014年硕士论文

【摘要】：目的本课题通过研究脂氧素A4受体（ALX-R）及核因子-Bp65(NF-κBp65)在口腔扁平苔藓（oral lichen planus，OLP）病灶和正常口腔黏膜（nomal oral mucosa，NOM）中的表达情况，以及不同浓度脂氧素A4类似物15-epi-LXA4对脂多糖（LPS）诱导角质形成细胞(HaCaT细胞)所模拟的OLP炎症模型的影响，探讨LXA4对口腔扁平苔藓的可能作用机制。方法第一部分：（1）获取OLP及NOM组织，切片并HE染色，选取符合标准的标本。（2）SP免疫组化法检测ALX-R、NF-κBp65在OLP组织和NOM组织中的表达情况，并比较所测指标在两种组织中的表达差异及其相关关系。第二部分：（1）采用10g/mL的LPS刺激HaCaT细胞，一定程度上模拟OLP局部免疫应答环境，ELISA法观察LPS作用时间长度分别为6、12、24h，所构建的OLP细胞模型中免疫应答产物IL-6的表达状况；（2）运用50、100、200nmol/L的15-epi-LXA4干预处理所建立的OLP细胞模型，分别于6、12、24小时提取细胞培养上清和总RNA，检测分析15-epi-LXA4对OLP细胞模型IL-6分泌和NF-κBp65基因表达的影响。结果第一部分：（1）ALX-R蛋白在OLP和NOM中表达阳性率分别为43.3%、90%，两者具有显著性差异(P＜0.05)；（2）NF-Bp65蛋白在OLP组及NOM中表达强度分别为83.3%、20%，两者具有显著性差异(P0.01)，其中糜烂萎缩型高于网纹型组(P0.05)；（3）OLP和NOM中NF-κBp65表达水平与ALX-R表达水平呈负相关。第二部分：（1）体外OLP炎症模型中，免疫应答产物IL-6表达量明显增加，且表达量随着时间的增加呈上升趋势；（2）一定浓度的15-epi-LXA4能够抑制NF-κBp65和IL-6的表达，200nmol/L的15-epi-LXA4对NF-κBp65和IL-6抑制效果最显著(P＜0.05)。结论 1、ALX-R在NOM中强表达，而在OLP中弱阳性表达，且其表达与NF-κBp65呈负相关，提示ALX-R表达缺陷可能与OLP的转归有一定关系； 2、不同临床类型OLP的ALX-R表达无显著性差异，而NF-κBp65的表达有差异，糜烂萎缩型高于网纹型组，可能与NF-κBp65介导大量炎症因子的表达有关，这与临床中糜烂型OLP炎症更严重相符合； 3、LXA4类似物干预处理LPS诱导的OLP炎症模型，，能抑制NF-κBp65信号通路及其下游炎症介质IL-6的表达，且呈浓度依赖性，这将为口腔扁平苔藓的临床治疗提供可能的新契机，并为新药的研发提供一定的理论依据，但尚需对LXA4在OLP中作用进行更深入的研究。
[Abstract]:Purpose The purpose of this study was to investigate the expression of lipoxygen-A4 receptor ALX-Rand nuclear factor -Bp65NF- 魏 Bp65 in lichen planus oral (OLP) and normal oral mucosa- (NOM) of oral lichen planus (OLP), and to investigate the expression of ALX-R and Bp65NF- 魏 Bp65 in oral lichen planus (OLP) lesions and normal oral mucosa. The effects of different concentrations of lipoxygenA4 analogue 15-epi-LXA4 on OLP inflammatory model induced by lipopolysaccharide (LPS) in keratinocytes were studied. The possible mechanism of LXA4 on oral lichen planus was discussed. Method Part I: 1) OLP and NOM tissues were obtained, sections were stained with HE, and the standard specimens. The immunohistochemical method was used to detect the expression of ALX-RnNF- 魏 Bp65 in OLP and NOM tissues. The expression differences and their correlation between the two tissues were compared. Part two: 1) HaCaT cells were stimulated by LPS of 10g/mL. To a certain extent, the length of action of LPS observed by Elisa in simulated local immune response environment of OLP was 612 ~ 24 h. The expression of IL-6 in the OLP cell model was established. The OLP cell model was established by 15-epi-LXA4 intervention of 50100200nmol/L. The supernatants and total RNAs were extracted from the cultured cells at 6h 12h, respectively. The effects of 15-epi-LXA4 on the secretion of IL-6 and the expression of NF- 魏 Bp65 gene in OLP cell model were detected and analyzed. Result Part one: the positive rate of ALX-R protein expression in OLP and NOM was 43.3% and 90, respectively. There was significant difference between the two groups (P < 0.05). The expression intensity of NF-Bp65 protein in OLP group and NOM group was 83.33%. There was a significant difference between the two groups (P 0.01), and the erosive atrophy type was higher than that in reticular type group. There was a negative correlation between the expression of NF- 魏 Bp65 and the expression of ALX-R in P0. 05 OLP and NOM. Part two: 1) in OLP inflammatory model in vitro, the expression of IL-6 increased significantly, and the expression of 15-epi-LXA4 increased with time. (2) A certain concentration of 15-epi-LXA4 could inhibit the expression of NF- 魏 Bp65 and IL-6. 15-epi-LXA4 of 200nmol / L had the most significant inhibitory effect on NF- 魏 Bp65 and IL-6 (P < 0.05). Conclusion 1ALX-R was strongly expressed in NOM, but weakly positive in OLP, and its expression was negatively correlated with NF- 魏 Bp65, suggesting that ALX-R expression defect may be related to the outcome of OLP; (2) there was no significant difference in the expression of ALX-R in different clinical types of OLP, but the expression of NF- 魏 Bp65 was higher in erosive atrophic type than in reticular type, which may be related to the expression of a large number of inflammatory factors mediated by NF- 魏 Bp65, which was in line with the severity of OLP inflammation in clinical practice. 3LXA4 analogue can inhibit the expression of NF- 魏 Bp65 signaling pathway and its downstream inflammatory mediator IL-6 in a concentration-dependent manner, which may provide a new opportunity for clinical treatment of oral lichen planus. It also provides some theoretical basis for the research and development of new drugs, but the role of LXA4 in OLP needs to be further studied.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R781.5

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