白介素21对角质形成细胞角蛋白17表达的影响及其分子机制的研究
发布时间:2019-05-29 19:50
【摘要】:银屑病是一种常见的易反复发作的慢性炎症性皮肤病,其发病机制尚不清楚。目前认为它是一种由辅助性T细胞(Th细胞)为主介导的自身免疫性疾病,多种炎症细胞及细胞因子参与其发病过程。 在银屑病患者皮损中,角质形成细胞(KC)所表达的角蛋白谱会发生改变,其中包括角蛋白17(K17)水平显著升高。K17作为“银屑病增殖相关角蛋白”在正常皮肤中不表达或表达量很低,而在银屑病患者的皮损中则是高表达,同时其表达水平与银屑病发病过程和严重程度有一定相关性。本课题组前期研究发现K17与银屑病的关系非常密切,在银屑病发病过程中可能存在“自身反应性T细胞——细胞因子——K17”作用环路,在这个环路中K17可以激活自身反应性T细胞,使之分泌一些银屑病相关细胞因子如γ-干扰素(IFN-γ)、白介素17(IL-17)等,从而引发局部的表皮增生和免疫炎症反应。同时还可以通过上调KC中K17的表达,,进一步激活自身反应性T细胞,形成一个相互促进的恶性环路,参与银屑病的发生发展。 白介素21(IL-21)主要来源于CD4+T细胞,因其受体分布广泛而被视为一种多效性的前炎症因子,在多种炎症性和自身免疫性疾病中发挥作用。IL-21在银屑病患者皮损中的mRNA和蛋白水平显著高于健康对照,且能够刺激小鼠和银屑病患者正常表皮的KC进行增殖,同时伴有真皮炎症细胞的浸润,与银屑病斑块的病理改变类似,提示IL-21可能在银屑病发生发展过程中发挥一定的作用。那么,IL-21与银屑病严重程度是否相关?其对本课题组前期研究的重点——“银屑病增殖相关角蛋白”K17的表达是否有影响及其调控机制又如何?本实验针对以上问题展开相关研究。 目的:检测斑块型银屑病患者血清IL-21的表达水平及其与疾病严重程度的相关性,研究IL-21对KC表达K17的影响及其表达调控的具体分子机制。 方法:采用酶联免疫吸附实验(ELISA)法检测银屑病患者和健康对照的血清IL-21水平,用统计学方法比较两者有无差异,并分析银屑病患者血清IL-21水平与疾病严重程度评分(PASI评分)是否存在相关性。 用不同浓度的IL-21(0ng/mL、12.5ng/mL、25ng/mL、50ng/mL、100ng/mL)和250U/mL的IFN-γ分别刺激KCs24h和48h提取RNA和蛋白,采用实时荧光定量聚合酶链反应(Real-time PCR)、Western blot和细胞免疫荧光染色分别检测K17mRNA和蛋白的表达水平,统计分析二者之间是否存在剂量依赖关系,并筛选合适的刺激浓度继续后续分子通路实验。 用筛选出的合适浓度IL-21分别刺激KCs0min、10min、20min、30min,用Western blot和细胞免疫荧光染色筛选在KC中出现酪氨酸磷酸化的信号通路分子。 最后为验证前期实验中所激活的信号转导通路是否与KC中K17的上调有关,我们预先采用相应的通路抑制剂处理细胞2h,再用IL-21刺激KCs,利用Western blot、Real-time PCR和细胞免疫荧光染色检测信号通路抑制剂能否抑制IL-21诱导的K17表达。 结果:寻常型银屑病患者血清IL-21水平高于健康对照(P<0.01),且与其PASI评分呈显著正相关(r=0.471,P<0.01)。采用12.5ng/mL、25ng/mL、50ng/mL、100ng/mL IL-21分别刺激KCs24h和48h后,均出现不同程度的K17表达。与空白对照组相比,12.5ng/mL和25ng/mL组的mRNA及蛋白表达水平有一定程度的增加,但无统计学差异,而50ng/mL和100ng/mL组的mRNA及蛋白表达水平显著高于空白对照,且有统计学差异(P<0.05),IL-21浓度和K17的表达量之间存在剂量依赖关系。采用50ng/mL IL-21分别刺激KCs10min、20min和30min,均可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)通路的激活。最后采用STAT3特异性通路抑制剂(Piceatannol)和ERK1/2特异性通路抑制剂(PD-98059)预先处理KCs2h,再用50ng/mL IL-21刺激KCs,K17mRNA和蛋白的表达均受到抑制。 结论:本研究证明寻常型银屑病患者血清IL-21水平高于健康对照,且与PASI评分呈显著正相关,说明IL-21与银屑病关系密切,可能参与银屑病的发生过程。IL-21能够上调K17的表达,且存在剂量依赖关系。其具体调控机制是通过激活STAT3和ERK1/2来实现的,说明IL-21在银屑病中所发挥的作用可能与K17有关。IL-21可在角蛋白水平上参与银屑病的发生发展,同时也丰富和补充了本课题组前期研究所提出的“自身反应性T细胞——细胞因子——K17”的环路假说,为银屑病的发病机理研究和治疗提供了新的策略。
[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.
【学位授予单位】:第四军医大学
【学位级别】:硕士
【学位授予年份】:2012
【分类号】:R758.63
本文编号: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.
【学位授予单位】:第四军医大学
【学位级别】:硕士
【学位授予年份】:2012
【分类号】:R758.63
【共引文献】
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