STAT3信号通路在鼻息肉发病机制中作用的初步研究

发布时间：2018-05-21 08:04

本文选题：鼻息肉病 + Th17细胞　；参考：《重庆医科大学》2013年博士论文

【摘要】：目的及背景：鼻息肉（nasal polyposis,NP）是一种鼻部黏膜的炎症性疾病，在鼻科临床上呈多发态势，根据欧洲的鼻窦炎及鼻息肉治疗指南（EP3OS）,鼻息肉被认为是慢性鼻-鼻窦炎（chronic sinusitis，CRS）的一个亚型。由于该病较高的复发率及目前治疗手段无法达到完全根治该病，鼻息肉已成为严重降低人类生活质量并带来大量社会经济负担的全球性健康问题。对NP发病机制的研究在近几十年是鼻部炎症性疾病研究领域的重点，国内外学者们对NP的起病原因及相关机制进行了大量的科学研究，尽管对于其疾病机理仍不能彻底地的予以明确，但是几乎所有的研究均表明：鼻息肉的形成是一个多种因素参与、多步骤发展的过程，慢性持续性炎症与变态反应与之关系密切。而Th淋巴细胞，尤其是Th17细胞在中国人鼻息肉的发病过程中扮演了重要的角色,而且变应性因素在中国人鼻息肉中很可能通过影响Th17细胞的反应发挥了关键作用。作为一种关键的核转录因子，在胞外信号的作用下，STAT3的酪氨酸基团被JAK磷酸化，以二聚体的形式进入胞核内，与胞核中特异的基因片段相结合，调节下游基因及相关蛋白的表达。近年的研究发现STAT3信号通路在IL-6的刺激下参与Th17细胞的增殖活化，由STAT3介导下引起的Th17细胞反应在葡萄膜炎、克罗恩病、哮喘等免疫及炎症性疾病的发病中发挥关键作用。那在中国，作为同样是Th17细胞反应为主的NP中，STAT3信号通路的异常激活是否也存在呢？变应性因素对NP中Th17细胞反应的影响与其与NP中STAT3通路的活化状态的关系有关联吗？为研究上述疑问，本研究以已经明确诊断的NP患者作为实验的对象，并将他们按照个体有无变应性的标准而分为atopic NP和non-atopicNP两个组，以STAT3信号通路相关关键因子的异常激活为支撑点，研究分析在中国西南地区的汉族人群中，不同类型NP的局部炎症特性与免疫机制，以解释在atopic和non-atopic NP发病机制中STAT3信号通路所起的作用，同时探讨变应性因素在NP中的作用与STAT3信号通路是否相关。本实验将从新的研究途径研究NP的发病机制，完善中国人NP是以Th17细胞反应为主这一理论，为NP的研究与治疗提供新的方向与靶点。第一部分STAT3信号通路相关因子在伴和不伴变应性体质鼻息肉患者中表达的研究目的：通对检测atopic和non-atopic NP两组患者息肉组织中STAT3信号通路中代表激活的关键因子及其下游Th17细胞在RNA、蛋白水平的相关因子的表达。观察两组患者鼻腔鼻窦病变局部STAT3信号通路的异常激活状态、Th17细胞反应状态及两组间的异同。旨在探讨STAT3信号通路的异常激活在NP发病机制中的意义，及NP中变应性因素对Th17反应的影响与STAT3信号通路的异常激活之间的关系。方法：对atopic-NP组20例，non-atopic NP组20例及对照的下鼻甲组织10例，采用免疫组化及Western Blot方法检测两组NP组织中pSTAT3及SOCS3的表达；采用ELISA检测NP组织中IL-6、sIL-6R、IL-17的表达；采用real-time PCR检测NP组织中RORc的表达；采用免疫荧光双标方法检测NP局部组织中CD4+RORc+细胞的表达。同时，以相关性分析的方法统计分析息肉组织中Th17细胞及其相关因子与STAT3信号通路的关键因子的关系。结果：NP患者息肉组织中RORc、IL-17及CD4+RORc+细胞的水平比对照组明显上升，在atopic NP组比另一NP组的表达更高，差距有统计学意义；与对照组相比，NP患者息肉组织中IL-6、sIL-6R、pSTAT3及SOCS3的表达显著升高，且在atopic NP组pSTAT3的表达显著高于non-atopic NP组，SOCS3的表达在atopic NP组显著低于non-atopic NP组，而在这两组NP中的IL-6与sIL-6R的水平的差异无统计学意义；在NP组织中，pSTAT3的表达水平与RORc、IL-17及CD4+RORc+细胞水平呈正相关，SOCS3的表达与pSTAT3的水平呈负相关，而IL-6与sIL-6R的水平与息肉组织中SOCS3的表达与pSTAT3的水平相关性无统计学意义。结论：STAT3信号通路的异常激活及其下游Th17细胞反应的亢进存在于NP中，其在NP的发病机制中起到关键作用；且NP患息肉组织中STAT3信号通路关键因子与Th17细胞相关因子基本上消长一致，说明STAT3信号通路的异常激活在Th17细胞反应过度亢进过程中扮演关键角色；Atopic NP存在更为严重的STAT3信号通路的异常激活和更为严重的Th17细胞反应的亢进，说明变应性因素可能通过影响STAT3信号通路的异常激活推动Th17反应的进一步亢进，从而促进有更严重临床病理特征的NP的发生发展。而SOCS3的表达差异可能是引起atopic NP组与non-atopic NP组出现IL-6及sIL-6R的水平与其下游的细胞因子的表达消长不统一的原因。第二部分STAT3抑制剂阻断鼻息肉中STAT3信号通路后相关下游因子表达的实验研究目的：通过不同浓度的STAT3信号通路抑制剂AG490作用于NP组织单细胞悬液，观察阻断NP中STAT3信号通路后对NP局部组织的Th17细胞及其相关因子的影响，并分析不同浓度下Th17细胞相关因子表达有无差异。以期在前一部分实验基础之上进一步探讨STAT3信号途径在NP发病中的作用，从体外研究的方面证明STAT3信号途径在中国人NP的发病中扮演的关键角色。方法：用不同浓度的STAT3通路抑制剂AG490体外作用培养于20例NP组织单细胞悬液（atopic NP10例，non-atopic NP10例）48h，根据浓度分为低浓度组（50μM）与高浓度组（100μM），以PBS代替AG490干预作为对照组。采取流式细胞术分析各组标本中Th17细胞的分布，运用real-time PCR检测各组RORc的水平，以ELISA方法检测各组中IL-17的水平。结果：用AG490处理48h后，AG490作用组的Th17细胞及相关因子（RORc及IL-17）的表达较对照组显著降低，差异有统计学意义；且的Th17细胞及相关因子（RORc及IL-17）的表达水平在高浓度AG490作用组较低浓度AG490作用组的水平有明显降低，差异有统计学意义。结论：在NP中STAT3信号通路的激活与Th17细胞的亢进反应密切相关，AG490阻断STAT3活化可显著下调RORc及IL-17的表达，，抑制NP中的Th17细胞反应。上述结果从体外实验方面证实STAT3信号通路是通过影响Th17细胞反应来促进NP的发生发展，在NP的发病机制中发挥重要作用。第三部分尘螨变应原体外刺激对鼻息肉患者STAT3信号通路相关因子局部表达的影响目的：采用尘螨特异性变应原(HDM)作为刺激原，在体外短时期内刺激并培养患者NP组织单细胞悬液中的单个核细胞，观察变应原体外刺激后，对两组NP患者STAT3信号通路的影响，分析两组间STAT3信号通路相关因子表达变化的差异，在前两部分实验基础上进一步阐述变应性因素对两组患者STAT3信号通路异常激活的影响，体外证实变应性因素是通过影响STAT3信号通路异常激活来促进TH17细胞的亢进反应从而在NP发病机制中发挥作用的。方法：分别以大剂量HDM+PHA及单纯PHA，对atopic和non-atopic NP患者局部NP组织单细胞悬液中细胞予以体外刺激培养48h，采用Western Blot方法检测两组NP组织中pSTAT3的表达，Real-time PCR方法及Western Blot方法检测SOCS3的表达，并分析两组间差异。结果：HDM+PHA刺激后的NP组患者局部NP组织单细胞悬液细胞中pSTAT3及SOCS3的表达较对照组明显上升，且在NP组内，变应性NP组的pSTAT3水平比非变应性NP组明显上升，而变应性NP组SOCS3的表达水平则较非变应性NP组显著下降；此外，在atopic NP组，HDM+PHA刺激使pSTAT3表达水平较PHA刺激显著升高,而SOCS3的表达水平较PHA刺激显著降低；而non-atopic NP组与对照组经HDM+PHA刺激和经PHA刺激对pSTAT3及SOCS3的表达水平影响差异无显著性。结论：伴变应性的NP患者的局部NP组织中HDM可诱导STAT3相关因子表达上调，揭示了STAT3信号通路在变应性上气道炎症中的重要作用，证实了变应性因素可通过影响STAT3信号通路在NP的发生发展中发挥作用；此外，在atopic NP组HDM诱导的STAT3相关抑制因子的表达下调，可以进一步支持前部分实验中关于变应性因素影响STAT3相关抑制因子从而促进更严重的STAT3信号通路的异常激活的假设，在体外实验方面解释了atopic NP组与non-atopic NP组出现IL-6及sIL-6R的表达与其下游因子表达消长不一致的原因。
[Abstract]:Objective and background: nasal polyps (nasal polyposis (NP)) is an inflammatory disease of the nasal mucosa. It has a multiple trend in the clinic. According to the European nasosinusitis and nasal polyp treatment guidelines (EP3OS), nasal polyps are considered as a subtype of chronic nasosinusitis (chronic sinusitis, CRS). Due to the high recurrence rate and current treatment of the disease, the nasal polyps are considered as a subtype. Treatment can not completely cure the disease. Nasal polyps have become a global health problem that seriously reduces the quality of life of human beings and brings a lot of social and economic burden.
The research on the pathogenesis of NP has been the focus of the research field of the nasal inflammatory disease in recent decades. Scholars at home and abroad have carried out a lot of scientific research on the causes and related mechanisms of the onset of NP. Although the mechanism of its disease is still not completely clear, almost all studies have shown that the formation of nasal polyps is a one. Many factors participate in the process of multi step development, and chronic persistent inflammation and allergy are closely related. Th lymphocytes, especially Th17 cells, play an important role in the pathogenesis of Chinese nasal polyps, and the allergic factors are likely to play a key role in the reaction of Chinese nasal polyps by affecting the reaction of Th17 cells. Bond action.
As a key nuclear transcription factor, the tyrosine group of STAT3 is phosphorylated by JAK, entering the nucleus in the form of two polymer, combining with the specific gene fragments in the nucleus and regulating the expression of the downstream genes and related proteins. In recent years, the present STAT3 signaling pathway is involved in Th17 fines under the stimulation of IL-6. Cell proliferation activation, the Th17 cell response induced by STAT3 plays a key role in the pathogenesis of uveitis, Crohn's disease, asthma and other immune and inflammatory diseases. In China, is the abnormal activation of STAT3 signaling as the same Th17 cell response NP? Allergic factors to Th17 cells in NP Is there any relationship between the effect of reaction and the activation state of STAT3 pathway in NP?
In order to study the above questions, the NP patients who have been clearly diagnosed are selected as the subjects of the experiment, and they are divided into two groups of atopic NP and non-atopicNP according to the standard of the individual unaltered, and the abnormal activation of the key factors of the STAT3 signaling pathway is the support point. The local inflammatory properties and immune mechanisms of the same type of NP are used to explain the role of STAT3 signaling pathway in the pathogenesis of atopic and non-atopic NP, and to explore whether the role of the allergic factors in NP is related to the STAT3 signaling pathway. This experiment will study the pathogenesis of NP from a new approach and improve the NP in China by Th17 cells. This theory provides a new direction and target for the research and treatment of NP.
Part one: the expression of STAT3 signaling pathway related factors in patients with and without allergic rhinitis.
Objective: To investigate the key factors that represent the activation of the STAT3 signaling pathway in the polyps of atopic and non-atopic NP two groups and the expression of related factors in the level of RNA and protein in the downstream Th17 cells. The abnormal activation state of the local STAT3 signaling pathway in the two groups of nasal sinuses, the reaction state of Th17 cells and the two groups were observed. The purpose of this study is to explore the significance of abnormal activation of STAT3 signaling pathway in the pathogenesis of NP, and the relationship between the effect of allergic factors on the response of Th17 and the abnormal activation of the STAT3 signaling pathway in NP.
Methods: 20 cases in group atopic-NP, 20 cases in group non-atopic NP and 10 cases of inferior turbinate tissue in control, the expression of pSTAT3 and SOCS3 in two groups of NP tissues were detected by immunohistochemistry and Western Blot, and the expression of IL-6, sIL-6R, and SOCS3 in the tissues of NP was detected by ELISA. A double standard method was used to detect the expression of CD4+RORc+ cells in the local tissue of NP, and the correlation analysis was used to analyze the relationship between the Th17 cells and their related factors in polyp tissues with the key factors of the STAT3 signaling pathway.
Results: the levels of RORc, IL-17 and CD4+RORc+ cells in the polyp tissues of NP patients were significantly higher than those in the control group, and the expression in atopic NP group was higher than that in the other NP group, and the difference was statistically significant. In the group of non-atopic NP, the expression of SOCS3 in the atopic NP group was significantly lower than that in the non-atopic NP group, but there was no significant difference in the level of IL-6 and sIL-6R in the two groups of NP. There was no significant correlation between the level of R and the expression of SOCS3 in polyp tissue and pSTAT3 level.
Conclusion: the abnormal activation of the STAT3 signaling pathway and the hyperactivity of the downstream Th17 cell reaction exist in NP, which plays a key role in the pathogenesis of NP, and the key factor of STAT3 signaling pathway in NP polyps with Th17 cell related factors is basically the same, indicating that the abnormal activation of the STAT3 signaling pathway is overreactive in Th17 cells. Hyperactivity plays a key role in the process of hyperactivity; Atopic NP has a more serious STAT3 signaling pathway and a more severe Th17 cell response, suggesting that the allergic factors may promote the further hyperactivity of the Th17 reaction by affecting the abnormal activation of the STAT3 signaling pathway, thus promoting a more severe clinical pathological NP. The difference in expression of SOCS3 may be the reason why the level of IL-6 and sIL-6R in the group atopic NP group and the non-atopic NP group and the expression of the downstream cell factors are not uniform.
The second part is the experimental study of STAT3 inhibitor blocking the expression of downstream related factors after STAT3 signaling pathway in nasal polyps.
Aim: To observe the effect of blocking the STAT3 signaling pathway in NP on the Th17 cells and its related factors in the local tissues of NP, and to analyze the difference in the expression of Th17 cells related factors at different concentrations after the inhibition of the STAT3 signaling pathway inhibitor AG490 on the single cell suspension of NP tissue, and to analyze the difference in the expression of the related factors of Th17 cells under different concentrations. Objective to explore the role of STAT3 signaling pathway in the pathogenesis of NP, and to demonstrate the pivotal role of STAT3 signaling pathway in the pathogenesis of NP in vitro.
Methods: 20 cases of NP tissue single cell suspension (atopic NP10, non-atopic NP10 case) 48h were cultured with different concentration of STAT3 pathway inhibitor AG490 in vitro. The concentration was divided into low concentration group (50 micron) and high concentration group (100 mu M), and PBS instead of AG490 intervention was used as the control group. The levels of RORc in each group were detected by real-time PCR, and the level of IL-17 in each group was detected by ELISA.
Results: after the treatment of 48h with AG490, the expression of Th17 cells and related factors (RORc and IL-17) in the AG490 group was significantly lower than that of the control group, and the difference was statistically significant. The levels of Th17 cells and related factors (RORc and IL-17) were significantly lower in the lower concentration AG490 action group of the high concentration AG490 action group, and the difference was statistically significant. Learning meaning.
Conclusion: the activation of STAT3 signaling pathway in NP is closely related to the hyperactivity of Th17 cells. AG490 blocking the activation of STAT3 can significantly reduce the expression of RORc and IL-17 and inhibit the Th17 cell response in NP. These results confirm that the STAT3 signaling pathway promotes the occurrence and development of NP by influencing Th17 cell response in vitro. It plays an important role in the mechanism of disease.
The third part is the influence of dust mites in vitro stimulation on local expression of STAT3 signaling pathway related factors in patients with nasal polyps.
Objective: to use dust mite specific allergens (HDM) as a stimulator, to stimulate and cultivate mononuclear cells in a single cell suspension of NP tissue in short period in vitro, to observe the effect of allergen to the STAT3 signaling pathway in two groups of NP patients in vitro, and to analyze the difference in the expression of STAT3 signaling pathway related factors between the two groups, in the first two On the basis of some experiments, the effect of allergic factors on the abnormal activation of STAT3 signaling pathway in two groups of patients was further elaborated. In vitro, the allergic factors were proved to play a role in the pathogenesis of NP by affecting the abnormal activation of the STAT3 signaling pathway to promote the hyperactivity of the TH17 cells.
Methods: the cells in the local NP tissue suspension of atopic and non-atopic NP were stimulated and cultured in vitro by large dose of HDM+PHA and simple PHA. The expression of pSTAT3 in the two groups of NP tissues was detected by Western Blot method. The expression of the two groups was detected and the differences between the groups were analyzed.
Results: the expression of pSTAT3 and SOCS3 in the single cell suspension cells of group NP after HDM+PHA stimulation was significantly higher than that in the control group, and in the NP group, the pSTAT3 level in the allergic NP group was significantly higher than that in the non allergic NP group, while the SOCS3 expression level in the allergic NP group was significantly lower than that in the non allergic NP group. The expression level of pSTAT3 was significantly higher than that of PHA stimulated by HDM+PHA stimulation, while the expression level of SOCS3 was significantly lower than that of PHA, but there was no significant difference between the non-atopic NP group and the control group by HDM+PHA stimulation and PHA stimulation on the expression level of pSTAT3 and SOCS3.
Conclusion: HDM can induce the up-regulated expression of STAT3 related factors in the local NP tissues of patients with allergic NP, which reveals the important role of STAT3 signaling pathway in allergic airway inflammation, and confirms that the allergic factors can play a role in the occurrence and development of NP by affecting the STAT3 signaling pathway; furthermore, STAT3 in atopic NP group HDM induces STAT3. Down regulation of related inhibitory factors can further support the hypothesis that the STAT3 related factors affect the abnormal activation of the more serious STAT3 signaling pathway in the previous experiments. In vitro experiments, the expression of IL-6 and sIL-6R in the atopic NP group and the non-atopic NP group and the downstream factor table are explained. The cause of the disagreement.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R765.25

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