【摘要】：目的:觀察銀屑病患者中濾泡性輔助性T細胞(T follicular helper cells,Tfh cells)頻率、Tfh 細胞功能性分子(ICOS、PD-1、HLA-DR、Ki-67、IL-21 IL-17、IFN-γ、IL-10)、Tfh 細胞亞群(Tfhl、Tfh2、Tfh17)、CD19+B 細胞頻率及 CD19+B 細胞表面IL-21R的表達特點,分析各檢測指標與銀屑病疾病嚴重程度的相關性,并觀察在銀屑病患者中Tfh細胞主要功能性細胞因子IL-21是否可以誘導CD4+T淋巴細胞向輔助性T細胞17(T helper 17 cells,Th17 cells)分化及Tfh17細胞亞群與B細胞表面IL-21R表達水平問的關系,探討Tfh細胞在銀屑病免疫發(fā)病機制中的作用及臨床意義。方法:入組32位尋常型銀屑病患者和13位健康對照者,分別定義為銀屑病組和健康對照組。運用流式細胞技術檢測兩組人員外周血中Tfh細胞頻率、Tfh細胞上功能性分子(ICOS、PD-1、HLA-DR、Ki-67、IL-21、IL-17、IFN-γ、IL-10)、Tfh 細胞亞群(Tfh1、Tfh2、Tfh17)、CD19+B細胞頻率及CD19+B細胞表面IL-21R表達的特征;運用免疫組織化學和免疫熒光的方法觀察銀屑病患者皮損區(qū)、非皮損區(qū)和健康對照者皮膚組織中Tfh細胞的浸潤特點。分析患者外周血中Tfh細胞頻率、皮損內(nèi)Tfh細胞浸潤數(shù)量、Tfh細胞亞群(Tfh1、Tfh2、Tfh17)、(Tfh17+Tfh2)/Tfh1比值與銀屑病面積嚴重度指數(shù)(Psoriasisareaseverityindex,PASI)的相關性,并觀察Tfh17細胞亞群與B細胞表面IL-21R表達水平間的關系。此外,對24位中重度銀屑病患者(PASI評分10)采用阿維A膠囊、中藥湯劑口服及卡泊三醇軟膏、糠酸莫米松乳膏外用治療,觀察患者治療1月后Tfh細胞頻率及其功能分子IL-21、IL-17、IFN-γ的變化特點。IL-21為Tfh細胞主要的功能性分子,為進一步探討Tfh細胞在銀屑病免疫發(fā)病機制中的作用,將IL-21與銀屑病患者及健康對照者外周血中CD4+T淋巴細胞體外共培養(yǎng),觀察IL-21是否可以促進CD4+T淋巴細胞向Th17細胞分化。結(jié)果:銀屑病患者外周血中Tfh細胞頻率顯著高于健康對照組(P0.05),且與疾病嚴重程度呈正相關。與健康對照組相比,銀屑病患者外周血中Tfh細胞上功能性分子(ICOS、PD-1、HLA-DR、Ki-67、IL-21、IL-17、IFN-y)的表達水平明顯升高(均P0.05),但Tfh細胞上IL-10的表達水平與健康對照組無明顯差別(P0.05)。我們的結(jié)果還顯示銀屑病患者外周血中存在Tfh細胞亞群失衡現(xiàn)象。銀屑病患者外周血中Tfh17細胞亞群頻率較健康對照組明顯升高(P0.05),而Tfh1細胞亞群頻率及Tfh2細胞亞群頻率在銀屑病組和健康對照組間無統(tǒng)計學差異(P0.05)。(Tfh17+Tfh2)/Tfh1比值在銀屑病組中也明顯升高,差異具有統(tǒng)計學意義(P0.05)。與銀屑病嚴重程度相關性分析中發(fā)現(xiàn),銀屑病患者外周血中Tfh細胞頻率、Tfh17細胞頻率與PASI評分呈明顯正性相關,而Tfh1細胞頻率、Tfh2細胞頻率、(Tfh17+Tfh2)/Tfh1比值與PASI評分間相關性無統(tǒng)計學意義。對B細胞的檢測結(jié)果顯示,CD19+B細胞頻率在銀屑病患者和健康對照者間無統(tǒng)計學差異(P0.05),但是CD19+B細胞上IL-21R表達的水平在銀屑病患者外周血中顯著升高(P0.05),且與PASI評分呈正性相關。在銀屑病患者外周血中CD19+B細胞上IL-21R表達的水平與Tfh17細胞頻率間也有明顯正性相關性。與健康對照者皮膚組織及銀屑病患者非皮損區(qū)相比,銀屑病患者皮損區(qū)Tfh細胞浸潤數(shù)量明顯升高,差異具有統(tǒng)計學意義(P0.05),但與PASI評分無明顯相關性,僅有隨PASI評分升高而升高的趨勢。在隨訪研究中我們發(fā)現(xiàn),中重度銀屑病患者經(jīng)1月治療后,Tfh細胞頻率、Tfh細胞分泌的細胞因子IL-21、IL-17、IFN-γ的水平較治療前明顯降低(P0.05)。將部分健康對照者及銀屑病患者外周血中CD4+T細胞純化出來并分別與IL-21體外共培養(yǎng),結(jié)果顯示經(jīng)IL-21細胞因子刺激后CD4+T細胞中Th17細胞比例明顯升高(P0.05)。結(jié)論:在銀屑病患者外周血中Tfh細胞不僅頻率增多,而且功能處于活化狀態(tài),其與銀屑病疾病嚴重程度也具有顯著相關性。在銀屑病患者外周血中還存在Tfh細胞亞群失衡現(xiàn)象。在Tfh細胞的3個亞群中,主要是Tfh17細胞參與銀屑病的發(fā)生發(fā)展,且與B細胞表面IL-21R的表達水平呈明顯相關性。經(jīng)1月治療,隨著銀屑病患者皮損臨床表現(xiàn)的好轉(zhuǎn),患者外周血中Tfh細胞頻率降低,其功能性分子表達下降。此外,IL-21是Tfh細胞主要的功能性細胞因子,在銀屑病中可以促進CD4+T細胞向Th17細胞分化。因此,Tfh細胞在銀屑病免疫發(fā)病機制中具有重要作用,可能通過誘導CD4+T細胞向Th17細胞的分化及促進B細胞的活化參與疾病的發(fā)生發(fā)展。我們的研究結(jié)果為銀屑病發(fā)病機制的闡明提供了新思路,也為銀屑病的治療提供了新的潛在性靶點。
[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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R758.63

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