本文選題：角蛋白17　切入點(diǎn)：白介素22　出處：《第四軍醫(yī)大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：銀屑病(psoriasis)是臨床常見的慢性炎癥性皮膚病,其發(fā)病機(jī)理尚未明確�，F(xiàn)階段認(rèn)為它是一種主要由Th細(xì)胞介導(dǎo)的自身免疫紊亂性疾病,在其發(fā)病過程中有Thl7、Th22細(xì)胞及細(xì)胞因子等共同參與。 在銀屑病患者皮損中,過度增生的表皮角質(zhì)形成細(xì)胞(KC)的角蛋白表達(dá)會(huì)發(fā)生顯著改變,其中包含角蛋白17(K17)的高表達(dá)。K17作為“銀屑病相關(guān)性細(xì)胞角蛋白”在正常皮膚中并不表達(dá),而在銀屑病患者皮損區(qū)則呈現(xiàn)特異性高表達(dá),其表達(dá)水平與銀屑病發(fā)病過程和嚴(yán)重程度有一定相關(guān)性。既往本研究組針對(duì)K17的研究發(fā)現(xiàn),K17的表達(dá)與銀屑病的發(fā)生和發(fā)展密切相關(guān),可能存在“T細(xì)胞——細(xì)胞因子——K17自身免疫環(huán)路”,已證實(shí)K17分子可以刺激來自銀屑病患者的T淋巴細(xì)胞,使之活化、增生,釋放γ干擾素(IFN-γ)、白介素17(IL-17)等炎癥因子,而IFN-γ、IL-17又可誘導(dǎo)、增加K17的表達(dá),從而形成一個(gè)相互促進(jìn)的環(huán)路,導(dǎo)致炎癥反應(yīng)和細(xì)胞異常增生等病理改變。 白介素22(IL-22)是Thl7的主要效應(yīng)因子之一,被稱為“銀屑病前炎癥因子”,其水平與銀屑病患者疾病嚴(yán)重程度正相關(guān),能夠調(diào)控KC中多個(gè)分化相關(guān)蛋白基因表達(dá)改變,并強(qiáng)烈抑制KC分化,導(dǎo)致表皮增生,棘層肥厚,以此參與銀屑病的發(fā)病。 目的：探討不同濃度IL-22刺激KC后是否表達(dá)K17；K17產(chǎn)生量與IL-22濃度變化是否存在劑量依賴的關(guān)系；IL-22調(diào)控K17表達(dá)的分子機(jī)制。 方法：培養(yǎng)HaCaT細(xì)胞,并以O(shè)ng/mL、12.5ng/mL、25ng/mL、50ng/mL、100ng/mL的IL-22分別作用于培養(yǎng)的HaCaT細(xì)胞48h。其中Ong/mL IL-22處理的HaCaT細(xì)胞作為陰性對(duì)照,250U/mL的IFN-y處理的HaCaT細(xì)胞為陽性對(duì)照。分別提取HaCaT細(xì)胞的RNA和蛋白質(zhì),用實(shí)時(shí)熒光定量聚合酶鏈反應(yīng)(Real-time PCR)、ELISA、Western blot、細(xì)胞免疫熒光染色等方法檢測(cè)K17的mRNA和蛋白表達(dá)水平,篩選IL-22誘導(dǎo)K17表達(dá)的有效濃度,統(tǒng)計(jì)分析二者之間是否存在劑量依賴關(guān)系。 培養(yǎng)HaCaT細(xì)胞,并用有效濃度的IL-22分別作用0min,15min,30min,60min后,用Western blot、細(xì)胞免疫熒光染色篩選在HaCaT細(xì)胞中出現(xiàn)酪氨酸磷酸化的信號(hào)分子。 抑制實(shí)驗(yàn)中,預(yù)先選用相應(yīng)的通路抑制劑處理細(xì)胞2h,再用有效濃度的IL-22處理HaCaT細(xì)胞12-24h,利用Real-time PCR、細(xì)胞免疫熒光染色檢測(cè)通路抑制劑對(duì)IL-22誘導(dǎo)HaCaT細(xì)胞表達(dá)K17的影響。 結(jié)果：HaCaT細(xì)胞經(jīng)12.5ng/mL、25ng/mL、50ng/mL、100ng/mL的IL-22作用48小時(shí)后,均出現(xiàn)不同程度的K17表達(dá)。與未經(jīng)處理的HaCaT細(xì)胞相比,12.5ng/mL組的mRNA及蛋白表達(dá)水平無統(tǒng)計(jì)學(xué)差異,而25ng/mL、50ng/mL、100ng/mL組的mRNA及蛋白表達(dá)有統(tǒng)計(jì)學(xué)差異(P0.05),并且K17表達(dá)與IL-22濃度之間存在正向劑量依賴關(guān)系。有效濃度25ng/mL的IL-22刺激角質(zhì)形成細(xì)胞15min開始,出現(xiàn)信號(hào)轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄激活因子3(STAT3)、細(xì)胞外信號(hào)調(diào)節(jié)激酶1/2(ERK1/2)通路的磷酸化。使用STAT3、ERK1/2的特異性通路抑制劑Piceatannol、PD-98059處理細(xì)胞2h,再用25ng/mL作用12～24小時(shí),K17表達(dá)量明顯降低。 結(jié)論：本研究證明IL-22能夠以劑量依賴的方式誘導(dǎo)HaCaT細(xì)胞表達(dá)K17,并且其調(diào)控機(jī)制是通過STAT3、ERKl/2分子通路實(shí)現(xiàn)。這一發(fā)現(xiàn)提示IL-22在銀屑病病理改變的發(fā)生過程中所發(fā)揮的作用可能部分地與誘導(dǎo)K17表達(dá)相關(guān),豐富并補(bǔ)充了我們之前提出的“T細(xì)胞——細(xì)胞因子——K17自身免疫環(huán)路”假說,并為銀屑病的治療研究提供了新的思路和靶點(diǎn),為研發(fā)更加安全有效的特異性治療手段奠定了基礎(chǔ)。
[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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R758.63

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