滤泡性辅助性T细胞及其亚群在寻常型银屑病患者中的特征及意义

发布时间：2018-07-18 08:31

【摘要】：目的:观察银屑病患者中滤泡性辅助性T细胞(T follicular helper cells,Tfh cells)频率、Tfh 细胞功能性分子(ICOS、PD-1、HLA-DR、Ki-67、IL-21 IL-17、IFN-γ、IL-10)、Tfh 细胞亚群(Tfhl、Tfh2、Tfh17)、CD19+B 细胞频率及 CD19+B 细胞表面IL-21R的表达特点,分析各检测指标与银屑病疾病严重程度的相关性,并观察在银屑病患者中Tfh细胞主要功能性细胞因子IL-21是否可以诱导CD4+T淋巴细胞向辅助性T细胞17(T helper 17 cells,Th17 cells)分化及Tfh17细胞亚群与B细胞表面IL-21R表达水平问的关系,探讨Tfh细胞在银屑病免疫发病机制中的作用及临床意义。方法:入组32位寻常型银屑病患者和13位健康对照者,分别定义为银屑病组和健康对照组。运用流式细胞技术检测两组人员外周血中Tfh细胞频率、Tfh细胞上功能性分子(ICOS、PD-1、HLA-DR、Ki-67、IL-21、IL-17、IFN-γ、IL-10)、Tfh 细胞亚群(Tfh1、Tfh2、Tfh17)、CD19+B细胞频率及CD19+B细胞表面IL-21R表达的特征;运用免疫组织化学和免疫荧光的方法观察银屑病患者皮损区、非皮损区和健康对照者皮肤组织中Tfh细胞的浸润特点。分析患者外周血中Tfh细胞频率、皮损内Tfh细胞浸润数量、Tfh细胞亚群(Tfh1、Tfh2、Tfh17)、(Tfh17+Tfh2)/Tfh1比值与银屑病面积严重度指数(Psoriasisareaseverityindex,PASI)的相关性,并观察Tfh17细胞亚群与B细胞表面IL-21R表达水平间的关系。此外,对24位中重度银屑病患者(PASI评分10)采用阿维A胶囊、中药汤剂口服及卡泊三醇软膏、糠酸莫米松乳膏外用治疗,观察患者治疗1月后Tfh细胞频率及其功能分子IL-21、IL-17、IFN-γ的变化特点。IL-21为Tfh细胞主要的功能性分子,为进一步探讨Tfh细胞在银屑病免疫发病机制中的作用,将IL-21与银屑病患者及健康对照者外周血中CD4+T淋巴细胞体外共培养,观察IL-21是否可以促进CD4+T淋巴细胞向Th17细胞分化。结果:银屑病患者外周血中Tfh细胞频率显著高于健康对照组(P0.05),且与疾病严重程度呈正相关。与健康对照组相比,银屑病患者外周血中Tfh细胞上功能性分子(ICOS、PD-1、HLA-DR、Ki-67、IL-21、IL-17、IFN-y)的表达水平明显升高(均P0.05),但Tfh细胞上IL-10的表达水平与健康对照组无明显差别(P0.05)。我们的结果还显示银屑病患者外周血中存在Tfh细胞亚群失衡现象。银屑病患者外周血中Tfh17细胞亚群频率较健康对照组明显升高(P0.05),而Tfh1细胞亚群频率及Tfh2细胞亚群频率在银屑病组和健康对照组间无统计学差异(P0.05)。(Tfh17+Tfh2)/Tfh1比值在银屑病组中也明显升高,差异具有统计学意义(P0.05)。与银屑病严重程度相关性分析中发现,银屑病患者外周血中Tfh细胞频率、Tfh17细胞频率与PASI评分呈明显正性相关,而Tfh1细胞频率、Tfh2细胞频率、(Tfh17+Tfh2)/Tfh1比值与PASI评分间相关性无统计学意义。对B细胞的检测结果显示,CD19+B细胞频率在银屑病患者和健康对照者间无统计学差异(P0.05),但是CD19+B细胞上IL-21R表达的水平在银屑病患者外周血中显著升高(P0.05),且与PASI评分呈正性相关。在银屑病患者外周血中CD19+B细胞上IL-21R表达的水平与Tfh17细胞频率间也有明显正性相关性。与健康对照者皮肤组织及银屑病患者非皮损区相比,银屑病患者皮损区Tfh细胞浸润数量明显升高,差异具有统计学意义(P0.05),但与PASI评分无明显相关性,仅有随PASI评分升高而升高的趋势。在随访研究中我们发现,中重度银屑病患者经1月治疗后,Tfh细胞频率、Tfh细胞分泌的细胞因子IL-21、IL-17、IFN-γ的水平较治疗前明显降低(P0.05)。将部分健康对照者及银屑病患者外周血中CD4+T细胞纯化出来并分别与IL-21体外共培养,结果显示经IL-21细胞因子刺激后CD4+T细胞中Th17细胞比例明显升高(P0.05)。结论:在银屑病患者外周血中Tfh细胞不仅频率增多,而且功能处于活化状态,其与银屑病疾病严重程度也具有显著相关性。在银屑病患者外周血中还存在Tfh细胞亚群失衡现象。在Tfh细胞的3个亚群中,主要是Tfh17细胞参与银屑病的发生发展,且与B细胞表面IL-21R的表达水平呈明显相关性。经1月治疗,随着银屑病患者皮损临床表现的好转,患者外周血中Tfh细胞频率降低,其功能性分子表达下降。此外,IL-21是Tfh细胞主要的功能性细胞因子,在银屑病中可以促进CD4+T细胞向Th17细胞分化。因此,Tfh细胞在银屑病免疫发病机制中具有重要作用,可能通过诱导CD4+T细胞向Th17细胞的分化及促进B细胞的活化参与疾病的发生发展。我们的研究结果为银屑病发病机制的阐明提供了新思路,也为银屑病的治疗提供了新的潜在性靶点。
[Abstract]:Objective: To observe the frequency of T follicular helper cells (Tfh cells) in the patients with psoriasis, Tfh cell functional molecules (ICOS, PD-1, HLA-DR, Ki-67), follicular cell subsets and the expression characteristics of the cell surface The correlation of the severity of psoriasis, and whether the main functional cytokine IL-21 of Tfh cells in patients with psoriasis can induce CD4+T lymphocyte to differentiate into auxiliary T cell 17 (T helper 17 cells, Th17 cells) and the relationship between Tfh17 cell subsets and B cell surface IL-21R expression level. The role and clinical significance of the immune pathogenesis. Methods: 32 patients with psoriasis vulgaris and 13 healthy controls were defined as psoriasis group and healthy control group. The frequency of Tfh cells in peripheral blood of two groups of people was detected by flow cytometry, and the functional molecules on Tfh cells (ICOS, PD-1, HLA-DR, Ki-67, IL-21, IL-17, IFN- gamma, I) were used. L-10), the Tfh cell subgroup (Tfh1, Tfh2, Tfh17), the frequency of CD19+B cells and the characteristics of IL-21R expression on the surface of CD19+B cells; using immunohistochemical and immunofluorescence methods to observe the characteristics of Tfh cell infiltration in the skin lesions of patients with psoriasis, non skin lesions and healthy controls. The frequency of Tfh cells in peripheral blood and skin lesions in the peripheral blood of patients were analyzed. The relationship between the number of Tfh cell infiltration, the Tfh cell subgroup (Tfh1, Tfh2, Tfh17), the (Tfh17+Tfh2) /Tfh1 ratio and the area severity index of psoriasis (Psoriasisareaseverityindex, PASI), and the relationship between the Tfh17 cell subgroup and the B cell surface IL-21R expression level were observed. In addition, the 24 patients with moderate and severe psoriasis were treated with AVI glue. Capsule, Chinese medicine decoction oral and Calcipotriol Ointment, Mometasone Furoate Cream external use, observe the changes of Tfh cell frequency and its functional molecules IL-21, IL-17, IFN- gamma after January,.IL-21 is the main functional molecule of Tfh cells, to further explore the role of Tfh cells in the pathogenesis of psoriasis, IL-21 and silver CD4+T lymphocyte culture in peripheral blood of patients and healthy controls was co cultured in vitro to observe whether IL-21 could promote the differentiation of CD4+T lymphocytes into Th17 cells. Results: the frequency of Tfh cells in peripheral blood of patients with psoriasis was significantly higher than that of the healthy control group (P0.05), and was positively correlated with the severity of the disease. The expression level of functional molecules (ICOS, PD-1, HLA-DR, Ki-67, IL-21, IL-17, IFN-y) on Tfh cells in peripheral blood was significantly increased (all P0.05), but the IL-10 expression on Tfh cells was not significantly different from that in the healthy control group (P0.05). Our results also showed that there was a subgroup imbalance in the peripheral blood of patients with psoriasis. The frequency of Tfh17 cell subsets in the peripheral blood was significantly higher than that in the healthy control group (P0.05), while the frequency of Tfh1 cell subsets and the frequency of Tfh2 cell subsets were not statistically different between the psoriasis group and the healthy control group (P0.05). The (Tfh17+Tfh2) /Tfh1 ratio was also significantly increased in the psoriasis group, and the difference was statistically significant (P0.05). The severity of psoriasis was significant. The correlation analysis showed that the frequency of Tfh cells in peripheral blood and the frequency of Tfh17 cells in the peripheral blood of patients with psoriasis were positively correlated with the PASI score, while the frequency of Tfh1 cells, the frequency of Tfh2 cells, and the correlation between the ratio of (Tfh17+Tfh2) /Tfh1 and PASI score were not statistically significant. The test results of B cells showed that the frequency of CD19+B cells was in the patients with psoriasis and health. There was no statistical difference between the healthy controls (P0.05), but the level of IL-21R expression on CD19+B cells increased significantly in peripheral blood (P0.05) in patients with psoriasis and was positively correlated with the PASI score. The level of IL-21R expression on CD19+B cells in the peripheral blood of psoriasis patients was also positively correlated with the Tfh17 cell frequency. The number of Tfh cell infiltration in psoriatic lesions was significantly higher in patients with psoriasis than in the non skin lesions of psoriasis. The difference was statistically significant (P0.05), but there was no significant correlation between the PASI score and the increase in the PASI score. In the follow-up study, we found that patients with moderate and severe psoriasis were treated in January, Tfh Cell frequency, the level of cytokine IL-21, IL-17, IFN- gamma secreted by Tfh cells was significantly lower than that before treatment (P0.05). CD4+T cells from peripheral blood of some healthy controls and patients with psoriasis were purified and co cultured with IL-21 in vitro. The results showed that the proportion of Th17 cells in CD4+T cells increased significantly (P0.) after IL-21 cell stimulation (P0.) 05) 05) conclusion: in the peripheral blood of psoriatic patients, the frequency of the peripheral blood is not only increased, but also the function is in the state of activation. It also has a significant correlation with the severity of psoriasis. In the peripheral blood of psoriasis, there is a Tfh cell subgroup imbalance in the peripheral blood of patients with psoriasis. Among the 3 subgroups of Tfh cells, the major Tfh17 cells are involved in the occurrence of psoriasis. In January, the frequency of Tfh cells in peripheral blood decreased and its functional molecular expression decreased. In addition, IL-21 was the main functional cell factor of Tfh cells, which could promote CD4+T cells to Th1 in psoriasis, in addition to the improvement of the skin lesions in patients with psoriasis in January. 7 cells are differentiated. Therefore, Tfh cells play an important role in the pathogenesis of psoriasis, and may be involved in the development of the disease by inducing the differentiation of CD4+T cells into Th17 cells and promoting the activation of B cells. Our results provide new ideas for the elucidation of the pathogenesis of psoriasis, and also provide new treatment for the treatment of psoriasis. Potential targets.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R758.63

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