白介素22对角蛋白17表达的影响及其分子机制

发布时间：2018-03-12 17:45

本文选题：角蛋白17　切入点：白介素22　出处：《第四军医大学》2011年硕士论文　论文类型：学位论文

【摘要】：银屑病(psoriasis)是临床常见的慢性炎症性皮肤病,其发病机理尚未明确。现阶段认为它是一种主要由Th细胞介导的自身免疫紊乱性疾病,在其发病过程中有Thl7、Th22细胞及细胞因子等共同参与。在银屑病患者皮损中,过度增生的表皮角质形成细胞(KC)的角蛋白表达会发生显著改变,其中包含角蛋白17(K17)的高表达。K17作为“银屑病相关性细胞角蛋白”在正常皮肤中并不表达,而在银屑病患者皮损区则呈现特异性高表达,其表达水平与银屑病发病过程和严重程度有一定相关性。既往本研究组针对K17的研究发现,K17的表达与银屑病的发生和发展密切相关,可能存在“T细胞——细胞因子——K17自身免疫环路”,已证实K17分子可以刺激来自银屑病患者的T淋巴细胞,使之活化、增生,释放γ干扰素(IFN-γ)、白介素17(IL-17)等炎症因子,而IFN-γ、IL-17又可诱导、增加K17的表达,从而形成一个相互促进的环路,导致炎症反应和细胞异常增生等病理改变。白介素22(IL-22)是Thl7的主要效应因子之一,被称为“银屑病前炎症因子”,其水平与银屑病患者疾病严重程度正相关,能够调控KC中多个分化相关蛋白基因表达改变,并强烈抑制KC分化,导致表皮增生,棘层肥厚,以此参与银屑病的发病。目的：探讨不同浓度IL-22刺激KC后是否表达K17；K17产生量与IL-22浓度变化是否存在剂量依赖的关系；IL-22调控K17表达的分子机制。方法：培养HaCaT细胞,并以Ong/mL、12.5ng/mL、25ng/mL、50ng/mL、100ng/mL的IL-22分别作用于培养的HaCaT细胞48h。其中Ong/mL IL-22处理的HaCaT细胞作为阴性对照,250U/mL的IFN-y处理的HaCaT细胞为阳性对照。分别提取HaCaT细胞的RNA和蛋白质,用实时荧光定量聚合酶链反应(Real-time PCR)、ELISA、Western blot、细胞免疫荧光染色等方法检测K17的mRNA和蛋白表达水平,筛选IL-22诱导K17表达的有效浓度,统计分析二者之间是否存在剂量依赖关系。培养HaCaT细胞,并用有效浓度的IL-22分别作用0min,15min,30min,60min后,用Western blot、细胞免疫荧光染色筛选在HaCaT细胞中出现酪氨酸磷酸化的信号分子。抑制实验中,预先选用相应的通路抑制剂处理细胞2h,再用有效浓度的IL-22处理HaCaT细胞12-24h,利用Real-time PCR、细胞免疫荧光染色检测通路抑制剂对IL-22诱导HaCaT细胞表达K17的影响。结果：HaCaT细胞经12.5ng/mL、25ng/mL、50ng/mL、100ng/mL的IL-22作用48小时后,均出现不同程度的K17表达。与未经处理的HaCaT细胞相比,12.5ng/mL组的mRNA及蛋白表达水平无统计学差异,而25ng/mL、50ng/mL、100ng/mL组的mRNA及蛋白表达有统计学差异(P0.05),并且K17表达与IL-22浓度之间存在正向剂量依赖关系。有效浓度25ng/mL的IL-22刺激角质形成细胞15min开始,出现信号转导和转录激活因子3(STAT3)、细胞外信号调节激酶1/2(ERK1/2)通路的磷酸化。使用STAT3、ERK1/2的特异性通路抑制剂Piceatannol、PD-98059处理细胞2h,再用25ng/mL作用12～24小时,K17表达量明显降低。结论：本研究证明IL-22能够以剂量依赖的方式诱导HaCaT细胞表达K17,并且其调控机制是通过STAT3、ERKl/2分子通路实现。这一发现提示IL-22在银屑病病理改变的发生过程中所发挥的作用可能部分地与诱导K17表达相关,丰富并补充了我们之前提出的“T细胞——细胞因子——K17自身免疫环路”假说,并为银屑病的治疗研究提供了新的思路和靶点,为研发更加安全有效的特异性治疗手段奠定了基础。
[Abstract]:Psoriasis (psoriasis) is a common chronic inflammatory skin disease in clinical practice. Its pathogenesis is not clear. At the present stage, it is considered that it is a autoimmune disorder mediated by Th cells. Thl7, Th22 cells and cytokines participate in the pathogenesis.
In psoriatic lesions, hyperplasia of epidermal keratinocytes (KC) expression of keratin will change significantly, which contains keratin 17 (K17) high expression of.K17 as a "no correlation between the expression of cell of psoriasis keratin in normal skin, while in the lesions of patients with psoriasis showed high expression specifically, there is a certain correlation between the expression and the pathogenesis of psoriasis and severity. The previous study group for the K17 study found that closely related to the occurrence and development of psoriasis and the expression of K17, there may be" T cell cytokine K17 autoimmune loop ", it has been confirmed that K17 molecules can stimulate from patients with psoriasis T lymphocytes, the activation, proliferation, release of interferon gamma (IFN- gamma), interleukin 17 (IL-17) and other inflammatory cytokines, IFN- gamma, IL-17 can also induce and increase the expression of K17, thus forming a phase The mutual promoting loop leads to pathological changes such as inflammatory reaction and abnormal cell proliferation.
Interleukin 22 (IL-22) is one of the main effect factors of Thl7, known as the "psoriasis proinflammatory factor", its level is positively correlated with the severity of psoriasis, can change a plurality of differentiation related protein gene expression and regulation of KC, and strongly inhibit the differentiation of KC, leading to Pi Zengsheng, acanthosis, in order to participate in psoriasis the onset of the disease.
Objective: To investigate whether different concentrations of IL-22 stimulate the expression of K17 after KC stimulation, whether there is a dose-dependent relationship between K17 production and IL-22 concentration, and IL-22 to regulate the molecular mechanism of K17 expression.
Methods: HaCaT cells were cultured with Ong/mL, 12.5ng/mL, 25ng/mL, 50ng/mL, 100ng/mL, IL-22 were incubated with HaCaT cells 48h. Ong/mL IL-22 treated HaCaT cells as negative control, IFN-y treated HaCaT cells 250U/mL as positive control. HaCaT was extracted from RNA cells and proteins by fluorescence quantitative polymerase chain the reaction (Real-time PCR), ELISA, Western, blot, mRNA and protein expression cell immunofluorescence staining method to detect K17, the effective concentration of screening IL-22 induced the expression of K17, whether there is a dose dependent relationship between the statistical analysis between the two.
HaCaT cells were cultured and 0min, 15min, 30min and 60min were treated with effective concentration of IL-22 respectively. Western tyrosine phosphorylated signal molecules in HaCaT cells were screened by Western blot and cellular immunofluorescence staining.
In the inhibition experiment, pretreatment of corresponding pathway inhibitors was used to treat cell 2h, then IL-22 cells were treated with effective concentration of 12-24h. Real-time PCR and cell immunofluorescence staining were used to detect the effect of pathway inhibitors on IL-22 induced HaCaT cell expression of K17 12-24h.
Results: HaCaT cells were treated with 12.5ng/mL, 25ng/mL, 50ng/mL, IL-22, 100ng/mL after 48 hours, there were different degrees of expression. K17 and untreated HaCaT cells compared with no significant difference between the expression of mRNA and protein in 12.5ng/mL group and 25ng/mL, 50ng/mL, mRNA and 100ng/mL protein expression was statistically different (P0.05), there is a positive dose-response relationship and the expression of K17 and IL-22 concentration. The effective concentration of 25ng/mL IL-22 stimulated keratinocyte 15min, signal transducer and activator of transcription 3 (STAT3), extracellular signal regulated kinase 1/2 (ERK1/2) phosphorylation. STAT3 specific inhibitor Piceatannol ERK1/2 the cells treated with PD-98059 2h, and 25ng/mL for 12~24 hours, the expression of K17 was significantly reduced.
Conclusion: This study demonstrated that the expression of K17 HaCaT cells induced by IL-22 in a dose dependent manner, and its regulation mechanism is through STAT3, ERKl/2 molecular pathway. This finding suggests that IL-22 plays in the pathogenesis of psoriasis in effect may be partially induced K17 expression, rich and added before us the T cell cytokine K17 autoimmune loop "hypothesis, and provides new ideas and targets for the treatment of psoriasis, specific treatment for the development of more safe and effective laid the foundation.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R758.63

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