白介素21對(duì)角質(zhì)形成細(xì)胞角蛋白17表達(dá)的影響及其分子機(jī)制的研究
發(fā)布時(shí)間：2019-05-29 19:50
【摘要】：銀屑病是一種常見的易反復(fù)發(fā)作的慢性炎癥性皮膚病，其發(fā)病機(jī)制尚不清楚。目前認(rèn)為它是一種由輔助性T細(xì)胞（Th細(xì)胞）為主介導(dǎo)的自身免疫性疾病，多種炎癥細(xì)胞及細(xì)胞因子參與其發(fā)病過(guò)程。 在銀屑病患者皮損中，角質(zhì)形成細(xì)胞（KC）所表達(dá)的角蛋白譜會(huì)發(fā)生改變，其中包括角蛋白17（K17）水平顯著升高。K17作為“銀屑病增殖相關(guān)角蛋白”在正常皮膚中不表達(dá)或表達(dá)量很低，而在銀屑病患者的皮損中則是高表達(dá)，同時(shí)其表達(dá)水平與銀屑病發(fā)病過(guò)程和嚴(yán)重程度有一定相關(guān)性。本課題組前期研究發(fā)現(xiàn)K17與銀屑病的關(guān)系非常密切，在銀屑病發(fā)病過(guò)程中可能存在“自身反應(yīng)性T細(xì)胞——細(xì)胞因子——K17”作用環(huán)路，在這個(gè)環(huán)路中K17可以激活自身反應(yīng)性T細(xì)胞，使之分泌一些銀屑病相關(guān)細(xì)胞因子如γ-干擾素（IFN-γ）、白介素17（IL-17）等，從而引發(fā)局部的表皮增生和免疫炎癥反應(yīng)。同時(shí)還可以通過(guò)上調(diào)KC中K17的表達(dá)，，進(jìn)一步激活自身反應(yīng)性T細(xì)胞，形成一個(gè)相互促進(jìn)的惡性環(huán)路，參與銀屑病的發(fā)生發(fā)展。 白介素21（IL-21）主要來(lái)源于CD4+T細(xì)胞，因其受體分布廣泛而被視為一種多效性的前炎癥因子，在多種炎癥性和自身免疫性疾病中發(fā)揮作用。IL-21在銀屑病患者皮損中的mRNA和蛋白水平顯著高于健康對(duì)照，且能夠刺激小鼠和銀屑病患者正常表皮的KC進(jìn)行增殖，同時(shí)伴有真皮炎癥細(xì)胞的浸潤(rùn)，與銀屑病斑塊的病理改變類似，提示IL-21可能在銀屑病發(fā)生發(fā)展過(guò)程中發(fā)揮一定的作用。那么，IL-21與銀屑病嚴(yán)重程度是否相關(guān)？其對(duì)本課題組前期研究的重點(diǎn)——“銀屑病增殖相關(guān)角蛋白”K17的表達(dá)是否有影響及其調(diào)控機(jī)制又如何？本實(shí)驗(yàn)針對(duì)以上問題展開相關(guān)研究。 目的：檢測(cè)斑塊型銀屑病患者血清IL-21的表達(dá)水平及其與疾病嚴(yán)重程度的相關(guān)性，研究IL-21對(duì)KC表達(dá)K17的影響及其表達(dá)調(diào)控的具體分子機(jī)制。 方法：采用酶聯(lián)免疫吸附實(shí)驗(yàn)（ELISA）法檢測(cè)銀屑病患者和健康對(duì)照的血清IL-21水平，用統(tǒng)計(jì)學(xué)方法比較兩者有無(wú)差異，并分析銀屑病患者血清IL-21水平與疾病嚴(yán)重程度評(píng)分（PASI評(píng)分）是否存在相關(guān)性。 用不同濃度的IL-21（0ng/mL、12.5ng/mL、25ng/mL、50ng/mL、100ng/mL）和250U/mL的IFN-γ分別刺激KCs24h和48h提取RNA和蛋白，采用實(shí)時(shí)熒光定量聚合酶鏈反應(yīng)（Real-time PCR）、Western blot和細(xì)胞免疫熒光染色分別檢測(cè)K17mRNA和蛋白的表達(dá)水平，統(tǒng)計(jì)分析二者之間是否存在劑量依賴關(guān)系，并篩選合適的刺激濃度繼續(xù)后續(xù)分子通路實(shí)驗(yàn)。 用篩選出的合適濃度IL-21分別刺激KCs0min、10min、20min、30min，用Western blot和細(xì)胞免疫熒光染色篩選在KC中出現(xiàn)酪氨酸磷酸化的信號(hào)通路分子。 最后為驗(yàn)證前期實(shí)驗(yàn)中所激活的信號(hào)轉(zhuǎn)導(dǎo)通路是否與KC中K17的上調(diào)有關(guān)，我們預(yù)先采用相應(yīng)的通路抑制劑處理細(xì)胞2h,再用IL-21刺激KCs，利用Western blot、Real-time PCR和細(xì)胞免疫熒光染色檢測(cè)信號(hào)通路抑制劑能否抑制IL-21誘導(dǎo)的K17表達(dá)。 結(jié)果：尋常型銀屑病患者血清IL-21水平高于健康對(duì)照（P＜0.01），且與其PASI評(píng)分呈顯著正相關(guān)（r=0.471，P＜0.01）。采用12.5ng/mL、25ng/mL、50ng/mL、100ng/mL IL-21分別刺激KCs24h和48h后，均出現(xiàn)不同程度的K17表達(dá)。與空白對(duì)照組相比，12.5ng/mL和25ng/mL組的mRNA及蛋白表達(dá)水平有一定程度的增加，但無(wú)統(tǒng)計(jì)學(xué)差異，而50ng/mL和100ng/mL組的mRNA及蛋白表達(dá)水平顯著高于空白對(duì)照，且有統(tǒng)計(jì)學(xué)差異（P＜0.05），IL-21濃度和K17的表達(dá)量之間存在劑量依賴關(guān)系。采用50ng/mL IL-21分別刺激KCs10min、20min和30min，均可出現(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）通路的激活。最后采用STAT3特異性通路抑制劑（Piceatannol）和ERK1/2特異性通路抑制劑（PD-98059）預(yù)先處理KCs2h，再用50ng/mL IL-21刺激KCs，K17mRNA和蛋白的表達(dá)均受到抑制。 結(jié)論：本研究證明尋常型銀屑病患者血清IL-21水平高于健康對(duì)照，且與PASI評(píng)分呈顯著正相關(guān)，說(shuō)明IL-21與銀屑病關(guān)系密切，可能參與銀屑病的發(fā)生過(guò)程。IL-21能夠上調(diào)K17的表達(dá)，且存在劑量依賴關(guān)系。其具體調(diào)控機(jī)制是通過(guò)激活STAT3和ERK1/2來(lái)實(shí)現(xiàn)的，說(shuō)明IL-21在銀屑病中所發(fā)揮的作用可能與K17有關(guān)。IL-21可在角蛋白水平上參與銀屑病的發(fā)生發(fā)展，同時(shí)也豐富和補(bǔ)充了本課題組前期研究所提出的“自身反應(yīng)性T細(xì)胞——細(xì)胞因子——K17”的環(huán)路假說(shuō)，為銀屑病的發(fā)病機(jī)理研究和治療提供了新的策略。
[Abstract]:Psoriasis is a common chronic inflammatory skin disease, and its pathogenesis is not clear. It is believed that it is an autoimmune disease, which is mediated by helper T cells (Th cells), and a variety of inflammatory cells and cytokines are involved in their pathogenesis. In the psoriatic lesions, the keratin spectrum expressed by the keratinocytes (KC) may change, including a significant increase in the level of the keratin 17 (K17). High. K17 is not expressed or expressed as a "psoriasis-related keratin" in normal skin, and it is highly expressed in the skin of patients with psoriasis, and its expression level is related to the course and severity of psoriasis. The results of this study show that the relationship between K17 and psoriasis is very close, and there may be a "Autoreactive T-cell _ Cytokine _ K17" loop in the course of the onset of psoriasis. In this loop, K17 can activate the self-reactive T. The cells are made to secrete a number of psoriasis-related cytokines such as interferon-interferon (IFN-1), interleukin-17 (IL-17), and the like, leading to local epidermal hyperplasia and immune inflammation. At the same time, by up-regulating the expression of K17 in the KC, the self-reactive T cells can be further activated to form a mutually reinforcing malignant loop and participate in the generation of psoriasis. The interleukin-21 (IL-21) is mainly derived from CD4 + T cells, which is considered to be a pleiotropic preinflammatory factor due to its wide receptor distribution, and is developed in a variety of inflammatory and autoimmune diseases The level of mRNA and protein of IL-21 in the skin of patients with psoriasis was significantly higher than that of healthy control, and it was able to stimulate the proliferation of the KC in the normal epidermis of the mice and the patients with psoriasis, with the infiltration of the dermal inflammatory cells and the pathological changes of the psoriasis. Similar, it is suggested that IL-21 may play a role in the course of the development of psoriasis The effect of IL-21 and psoriasis is, however, No correlation? It has an effect on the expression of the key _ "psoriasis-related keratin" K17 in the earlier stage of the research group and its regulation and control mechanism What? This experiment is for the above problems. Objective: To study the expression level of serum IL-21 and its correlation with the severity of the disease in patients with plaque psoriasis, and to study the effect of IL-21 on the expression of K17 and the expression and control of the expression of IL-21. Methods: The levels of serum IL-21 in patients with psoriatic and healthy controls were detected by enzyme-linked immunosorbent assay (ELISA), and the difference was compared with the statistical method, and the level of IL-21 and severity of the disease (PASI score) in patients with psoriasis were analyzed. Whether there was a correlation or not. The RNA and protein were extracted with different concentrations of IL-21 (0 ng/ mL, 12.5 ng/ mL,25 ng/ mL,50 ng/ mL,100 ng/ mL) and 250 U/ mL of IFN-1, respectively, and the K17mRN was detected by real-time fluorescence quantitative polymerase chain reaction (Real-time PCR), Western blot and cell immunofluorescence staining. The level of expression of A and of the protein, the presence or absence of a dose-dependent relationship between the two, and the selection of appropriate stimulation concentrations The follow-up molecular pathway experiment was carried out. The appropriate concentration of IL-21 was used to stimulate KCs0min,10 min,20 min and 30 min respectively. In order to verify that the signal transduction pathway activated in the early stage of the experiment is related to the regulation of K17 in the KC, the corresponding channel inhibitor is used to treat the cells for 2h, and the KCs are stimulated with IL-21. Whether the signal pathway inhibitor can be inhibited by Western blot, Real-time PCR and cell immunofluorescence staining The results showed that the level of serum IL-21 in patients with psoriasis was higher than that of healthy control (P <0.01), and it was positively correlated with its PASI score (P <0.01). R = 0.471, P <0.01). Use 12.5 ng/ mL,25 ng/ mL,50 ng/ mL,100 ng/ mL IL-21 to stimulate KCs24h and 48 h, respectively. The expression of mRNA and protein in the group of 12.5 ng/ mL and 25 ng/ mL was higher than that of the blank control group, but there was no statistical difference, while the expression of mRNA and protein in the group of 50 ng/ mL and 100 ng/ mL was significantly higher than that of the blank control group. with statistical difference (P <0.05), IL-21 concentration and K1 There was a dose-dependent relationship between the expression levels of 7. The signal transduction and the transcription activation factor 3 (STAT3), the extracellular signal-regulated kinase 1, and the signal transduction and transcription activation factor 3 (STAT3), respectively, were stimulated with 50 ng/ mL of IL-21. Activation of the/2 (ERK1/2) pathway. Finally, the KCs2h was pre-treated with the STAT3-specific pathway inhibitor (Piceatanol) and the ERK1/2-specific pathway inhibitor (PD-98059), and the KCs, K17 were stimulated with 50 ng/ mL of IL-21. The results showed that the level of serum IL-21 in patients with psoriasis vulgaris was higher than that of healthy control, and it was positively correlated with PASI score, which indicated that IL-21 and psoriasis It is closely related and may be involved in the process of psoriasis. IL-21 can be The expression of K17 is adjusted and a dose-dependent relationship is present. The specific regulatory mechanism is achieved by activating STAT3 and ERK1/2, indicating that IL-21 is in silver The role of IL-21 can be related to K17. IL-21 can participate in the development of psoriasis at the level of keratin, and also enrich and supplement the loop hypothesis of the "Autoreactive T-cell _ Cytokine _ K17" put forward by the previous research group.
【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】：2012
【分類號(hào)】：R758.63
本文編號(hào)：2488178
[Abstract]:Psoriasis is a common chronic inflammatory skin disease, and its pathogenesis is not clear. It is believed that it is an autoimmune disease, which is mediated by helper T cells (Th cells), and a variety of inflammatory cells and cytokines are involved in their pathogenesis. In the psoriatic lesions, the keratin spectrum expressed by the keratinocytes (KC) may change, including a significant increase in the level of the keratin 17 (K17). High. K17 is not expressed or expressed as a "psoriasis-related keratin" in normal skin, and it is highly expressed in the skin of patients with psoriasis, and its expression level is related to the course and severity of psoriasis. The results of this study show that the relationship between K17 and psoriasis is very close, and there may be a "Autoreactive T-cell _ Cytokine _ K17" loop in the course of the onset of psoriasis. In this loop, K17 can activate the self-reactive T. The cells are made to secrete a number of psoriasis-related cytokines such as interferon-interferon (IFN-1), interleukin-17 (IL-17), and the like, leading to local epidermal hyperplasia and immune inflammation. At the same time, by up-regulating the expression of K17 in the KC, the self-reactive T cells can be further activated to form a mutually reinforcing malignant loop and participate in the generation of psoriasis. The interleukin-21 (IL-21) is mainly derived from CD4 + T cells, which is considered to be a pleiotropic preinflammatory factor due to its wide receptor distribution, and is developed in a variety of inflammatory and autoimmune diseases The level of mRNA and protein of IL-21 in the skin of patients with psoriasis was significantly higher than that of healthy control, and it was able to stimulate the proliferation of the KC in the normal epidermis of the mice and the patients with psoriasis, with the infiltration of the dermal inflammatory cells and the pathological changes of the psoriasis. Similar, it is suggested that IL-21 may play a role in the course of the development of psoriasis The effect of IL-21 and psoriasis is, however, No correlation? It has an effect on the expression of the key _ "psoriasis-related keratin" K17 in the earlier stage of the research group and its regulation and control mechanism What? This experiment is for the above problems. Objective: To study the expression level of serum IL-21 and its correlation with the severity of the disease in patients with plaque psoriasis, and to study the effect of IL-21 on the expression of K17 and the expression and control of the expression of IL-21. Methods: The levels of serum IL-21 in patients with psoriatic and healthy controls were detected by enzyme-linked immunosorbent assay (ELISA), and the difference was compared with the statistical method, and the level of IL-21 and severity of the disease (PASI score) in patients with psoriasis were analyzed. Whether there was a correlation or not. The RNA and protein were extracted with different concentrations of IL-21 (0 ng/ mL, 12.5 ng/ mL,25 ng/ mL,50 ng/ mL,100 ng/ mL) and 250 U/ mL of IFN-1, respectively, and the K17mRN was detected by real-time fluorescence quantitative polymerase chain reaction (Real-time PCR), Western blot and cell immunofluorescence staining. The level of expression of A and of the protein, the presence or absence of a dose-dependent relationship between the two, and the selection of appropriate stimulation concentrations The follow-up molecular pathway experiment was carried out. The appropriate concentration of IL-21 was used to stimulate KCs0min,10 min,20 min and 30 min respectively. In order to verify that the signal transduction pathway activated in the early stage of the experiment is related to the regulation of K17 in the KC, the corresponding channel inhibitor is used to treat the cells for 2h, and the KCs are stimulated with IL-21. Whether the signal pathway inhibitor can be inhibited by Western blot, Real-time PCR and cell immunofluorescence staining The results showed that the level of serum IL-21 in patients with psoriasis was higher than that of healthy control (P <0.01), and it was positively correlated with its PASI score (P <0.01). R = 0.471, P <0.01). Use 12.5 ng/ mL,25 ng/ mL,50 ng/ mL,100 ng/ mL IL-21 to stimulate KCs24h and 48 h, respectively. The expression of mRNA and protein in the group of 12.5 ng/ mL and 25 ng/ mL was higher than that of the blank control group, but there was no statistical difference, while the expression of mRNA and protein in the group of 50 ng/ mL and 100 ng/ mL was significantly higher than that of the blank control group. with statistical difference (P <0.05), IL-21 concentration and K1 There was a dose-dependent relationship between the expression levels of 7. The signal transduction and the transcription activation factor 3 (STAT3), the extracellular signal-regulated kinase 1, and the signal transduction and transcription activation factor 3 (STAT3), respectively, were stimulated with 50 ng/ mL of IL-21. Activation of the/2 (ERK1/2) pathway. Finally, the KCs2h was pre-treated with the STAT3-specific pathway inhibitor (Piceatanol) and the ERK1/2-specific pathway inhibitor (PD-98059), and the KCs, K17 were stimulated with 50 ng/ mL of IL-21. The results showed that the level of serum IL-21 in patients with psoriasis vulgaris was higher than that of healthy control, and it was positively correlated with PASI score, which indicated that IL-21 and psoriasis It is closely related and may be involved in the process of psoriasis. IL-21 can be The expression of K17 is adjusted and a dose-dependent relationship is present. The specific regulatory mechanism is achieved by activating STAT3 and ERK1/2, indicating that IL-21 is in silver The role of IL-21 can be related to K17. IL-21 can participate in the development of psoriasis at the level of keratin, and also enrich and supplement the loop hypothesis of the "Autoreactive T-cell _ Cytokine _ K17" put forward by the previous research group.
【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】：2012
【分類號(hào)】：R758.63
【共引文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前10條
1 張巍;白介素22對(duì)角蛋白17表達(dá)的影響及其分子機(jī)制[D];第四軍醫(yī)大學(xué);2011年
2 邱海霞;雷公藤甲素對(duì)銀屑病患者外周血T細(xì)胞活性的影響[D];暨南大學(xué);2003年
3 陳玉欣;K14反義寡核苷酸在角質(zhì)形成細(xì)胞中的作用研究[D];第四軍醫(yī)大學(xué);2004年
4 郝國(guó)孌;延胡索酸酯對(duì)T細(xì)胞的作用——細(xì)胞表型、凋亡分子和轉(zhuǎn)錄因子的改變[D];浙江大學(xué);2005年
5 王巖;靶向K17的小干涉RNA在角質(zhì)形成細(xì)胞中的作用研究[D];第四軍醫(yī)大學(xué);2005年
6 李敬龍;CK免疫組化染色在Ⅰ期NSCLC患者淋巴結(jié)中的表達(dá)及其臨床意義[D];大連醫(yī)科大學(xué);2005年
7 史曉蔚;特異性置換肽對(duì)銀屑病動(dòng)物模型作用的研究[D];第四軍醫(yī)大學(xué);2006年
8 常婷;靶向角蛋白17反義寡核苷酸和siRNA對(duì)銀屑病SCID小鼠模型的治療作用[D];第四軍醫(yī)大學(xué);2007年
9 王曼姬;利用含T淋巴細(xì)胞和鏈球菌抗原的無(wú)血清培養(yǎng)基體外建立銀屑病皮損模型[D];大連醫(yī)科大學(xué);2009年
10 魯慧;CD147高表達(dá)于銀屑病患者外周血中性粒細(xì)胞并誘導(dǎo)中性粒細(xì)胞趨化[D];中南大學(xué);2009年
本文編號(hào)：2488178
本文鏈接：http://sikaile.net/yixuelunwen/pifb/2488178.html
最近更新
教材專著
