子宫内膜再生细胞在溃疡性结肠炎模型中免疫调节的相关机制研究

发布时间：2018-08-19 07:11

【摘要】：目的:本课题探讨子宫内膜再生细胞(ERC)在溃疡性结肠炎(UC)小鼠模型中免疫调节的作用及相关机制。自经血中分离鉴定ERC,观察其表面程序性死亡受体-配体1(PD-L1)的表达情况;建立UC的小鼠动物模型;探讨ERC在UC小鼠模型体内的分布情况;将ERC应用于UC的体内体外实验,研究ERC细胞表面的PD-L1免疫调节分子在UC治疗中的作用。方法:1)以月事杯收集健康育龄期女性志愿者的月经血,利用标准的Ficoll方法分离月经血样品中的单个核细胞,进行细胞培养,应用流式细胞术鉴定ERC的表面抗原。2)选用SPF级的BALB/c小鼠,将其饮用不同浓度的葡聚糖硫酸钠(DSS)溶液建立UC小鼠动物模型,观察造模期间实验动物的一般情况、疾病活动指数(DAI)及组织病理学改变,选择最佳的DSS浓度并对小鼠动物模型进行评估。3)应用PKH26(Paul Karl Horan 26)对ERC进行体外标记,将标记后的ERC应用于UC动物模型,观察ERC在UC动物模型体内的分布情况。4)应用PD-L1抗体对ERC细胞表面的PD-L1进行封闭,在体内实验中,将ERC及PD-L1抗体封闭后的ERC应用于UC动物模型(分为四组,(1)正常对照组:自由饮用不含DSS的饮用水;(2)未治疗组:自由饮用3%DSS溶液+经静脉注射PBS缓冲液;(3)ERC组:自由饮用3%DSS溶液+静脉注射以PBS缓冲液重悬的ERC;(4)PD-L1封闭的ERC组:自由饮用3%DSS溶液+静脉注射以PBS缓冲液重悬的PD-L1封闭的ERC),第2、5、8天,ERC组经尾静脉注射ERC(1×106/0.2ml/只),PD-L1封闭的ERC组经尾静脉注射PD-L1封闭的ERC(1×106/0.2ml/只),第8天,所有组均改为饮用水,实验期间每天观察并记录各组实验动物的一般状况,进行DAI评分,第15天,处死全部小鼠,测量自然状态下结直肠长度,显微镜下观察结直肠的病理改变,采用流式细胞术检测脾脏CD3~+CD4~+T、CD3~+CD8~+T、CD4~+CD25~+Foxp3~+Tregs、CD11c~+MHC-II~+DCs、F4/80~+M、F4/80~+CD206~+M细胞数量;采用ELISA及q PCR的方法检测结直肠正常组织及病变组织TNF-α、IFN-γ、IL-10、TGF-β的蛋白水平及m RNA转录水平。在体外实验中,从正常小鼠中分离脾细胞,将ERC与脾细胞进行共培养或者将PD-L1封闭的ERC与脾细胞进行共培养,应用不同的细胞刺激因子对脾细胞进行刺激(分为四组,(1)未治疗组:单纯脾细胞培养;(2)ERC治疗组:ERC与脾细胞进行共培养;(3)PD-L1封闭的ERC治疗组:PD-L1封闭的ERC与脾细胞进行共培养;(4)transwell培养组:ERC与脾细胞进行非接触的共培养),应用流式细胞术分析不同组中CD3~+CD4~+T、CD3~+CD8~+T、CD4~+CD25~+Foxp3~+Tregs、CD11c~+MHC-II~+DCs、F4/80~+M、F4/80~+CD206~+M细胞的数量;分析上述指标的变化,评价PD-L1在ERC治疗小鼠UC中的作用。结果:1)ERC能够表达PD-L1、CD90、CD105,不表达CD34、CD45;随着刺激因子IFN-γ浓度的增加,ERC表面的PD-L1表达相应增加。2)随着DSS浓度的增加及造模时间的延长,造模小鼠的一般状况逐渐变差、进食进水逐渐减少、体重逐渐下降、DAI评分逐渐增加,小鼠的病死率增加,其病理学改变包括:粘膜固有层中性粒细胞、巨噬细胞、淋巴细胞的浸润,腺窝扭曲变形,固有层增厚,粘膜下层淋巴滤泡形成,小鼠肠道的炎症逐渐加重。3)将PKH26荧光标记的ERC于造模第5天经尾静脉注入UC小鼠动物模型体内,在ERC注入48h时进行组织取材,荧光显微镜观察显示荧光强度为肺脏肝脏脾脏结肠组织,肾脏没有观察到荧光,模型组结直肠病变组织荧光强度高于正常组结直肠组织的荧光强度。4)体内实验显示,尾静脉注射ERC对小鼠结直肠炎症有明显的保护作用,封闭ERC表面的PD-L1明显降低ERC对小鼠的结直肠炎症保护作用;与未治疗组比较,ERC组脾脏CD3~+CD4~+T、CD3~+CD8~+T细胞数量明显减少(P0.05),CD4~+CD25~+Foxp3~+Tregs细胞数量明显增加(P0.05),CD11c~+MHC-II~+DCs细胞数量显著减少(P0.05),F4/80~+M细胞数量明显减少(P0.05),F4/80~+CD206~+M相对增加(P0.05),结直肠病变组织内TNF-α、IFN-γ的m RNA转录水平及蛋白表达水平降低(P0.05),IL-10、TGF-β的m RNA转录水平及蛋白表达水平升高(P0.05);与ERC组相比,PD-L1封闭的ERC治疗组脾脏CD3~+CD4~+T、CD3~+CD8~+细胞数量相对增加(P0.05)、CD4~+CD25~+Foxp3~+Tregs细胞数量相对减少(P0.05)、CD11c~+MHC-II~+DCs细胞数量相对增加(P0.05),结直肠组织内TNF-α、IFN-γ的m RNA转录水平及蛋白表达水平相对增加(P0.05),IL-10、TGF-β的m RNA转录水平及蛋白表达水平相对减少(P0.05)。体外实验显示,与正常对照组相比,经不同的刺激因子刺激后,ERC共培养组CD3~+CD4~+T、CD3~+CD8~+T、CD11c~+MHC-II~+DCs、F4/80~+M细胞数量相对减少,CD4~+CD25~+Foxp3~+Tregs、F4/80~+CD206~+M细胞数量相对增加;PD-L1封闭的ERC共培养组与ERC共培养组相比,CD3~+CD4~+T、CD3~+CD8~+T、CD11c~+MHC-II~+DCs、F4/80~+M细胞数量相对增加,CD4~+CD25~+Foxp3~+Tregs、F4/80~+CD206~+M细胞数量相对减少;transwell培养组与ERC共培养组相比,CD3~+CD4~+T、CD3~+CD8~+T、CD11c~+MHC-II~+DCs、F4/80~+M细胞数量相对增加,CD4~+CD25~+Foxp3~+Tregs、F4/80~+CD206~+M细胞数量相对减少。结论:1)自经血中分离的ERC是一种类似于MSC的再生细胞,ERC不但来源丰富、获取无创,而且ERC表面表达在诱导免疫耐受方面发挥重要作用的免疫调节分子PD-L1。2)3%的DSS为构建BALB/c小鼠UC动物模型的理想浓度,该浓度不但能够很好的模拟人UC肠道病理改变,而且没有小鼠的病死发生。3)将ERC通过小鼠尾静脉注入UC模型小鼠体内后,ERC能够向肠道炎症部位迁移,而且ERC在肠道的分布高于正常对照组。4)PD-L1在ERC有效控制UC动物模型肠道炎症的发生发展中发挥重要作用,体内体外实验均显示ERC通过PD-L1抑制DCs的成熟分化及CD4~+T和CD8~+T细胞的增殖、促进Tregs细胞及M2细胞的产生,并且体外实验中还显示ERC表面的PD-L1通过直接接触的方式起作用,进而发挥免疫调节的作用。
[Abstract]:Objective: To investigate the role of endometrial regeneration cells (ERC) in the immune regulation of mice with ulcerative colitis (UC) and its related mechanism. Methods: 1) The menstrual blood of healthy female volunteers of childbearing age was collected with a menstrual cup, and the mononuclear cells were isolated by standard Ficoll method. The cells were cultured and identified by flow cytometry. To determine the surface antigen of ERC. 2) SPF BALB / c mice were used to establish UC mice model by drinking different concentrations of sodium dextran sulfate (DSS) solution. The general conditions, disease activity index (DAI) and histopathological changes of the experimental animals were observed during the modeling period. The best concentration of DSS was selected and the animal model was evaluated. 3) The animal model was established by P KH26 (Paul Karl Horan 26) was used to label ERC in vitro. The labeled ERC was applied to UC animal model to observe the distribution of ERC in vivo. 4) PD-L1 antibody was used to block the surface of ERC cells. In vivo, ERC blocked by ERC and PD-L1 antibody was applied to UC animal model (divided into four groups: (1) Normal. Control group: free drinking water without DSS; (2) untreated group: free drinking 3% DSS solution + intravenous PBS buffer; (3) ERC group: free drinking 3% DSS solution + intravenous PBS buffer suspension of ERC; (4) PD-L1 blocked ERC group: free drinking 3% DSS solution + intravenous PBS buffer suspension of PD-L1 blocked ERC, 2, 5, 8 On the 8th day, all groups were changed to drinking water. During the experiment, the general condition of experimental animals in each group was observed and recorded daily, and the DAI score was made. On the 15th day, all the mice were sacrificed. Colon length, colon pathological changes were observed under microscope, CD3~+CD4~+T, CD3~+CD8~+T, CD4~+CD25~+Foxp3~+Tregs, CD11c~+MHC-II~+DCs, F4/80~+CD206~+M cells in spleen were detected by flow cytometry, and TNF-a, IFN-gamma, IL-10, TGF-beta in normal and pathological tissues were detected by ELISA and q-PCR. In vitro, spleen cells were isolated from normal mice, and ERC was co-cultured with spleen cells or PD-L1-blocked ERC was co-cultured with spleen cells. Splenocytes were stimulated with different cytokines (divided into four groups: (1) untreated group: spleen cells were cultured alone; (2) ERC treatment group: ERC and spleen Cells were co-cultured; (3) PD-L1-blocked ERC treatment group: PD-L1-blocked ERC and spleen cells were co-cultured; (4) Transwell culture group: ERC and spleen cells were co-cultured without contact, using flow cytometry analysis of CD3~+CD4~+T, CD3~+CD8~+T, CD4~+CD25~+Foxp3~+Tregs, CD11c~+MHC-II~+DCs, F4/80~+M, F4/80~+M, CD206~+ cells in different groups. Results: 1) ERC could express PD-L1, CD90, CD105, but not CD34 and CD45; with the increase of IFN-gamma concentration, the expression of PD-L1 on the surface of ERC increased correspondingly. 2) With the increase of DSS concentration and the prolongation of modeling time, the general condition of model mice increased gradually. Pathological changes include: the infiltration of neutrophils, macrophages, lymphocytes, pits distorted, lamina propria thickened, lymphatic follicles formed in submucosa, intestinal inflammation aggravated gradually. 3) PK H26 fluorescence labeled ERC was injected into UC mice by tail vein on the 5th day of modeling. The tissues were taken at 48h of ERC injection. Fluorescence microscopy showed that the fluorescence intensity was lung, liver, spleen, colon, kidney and no fluorescence was observed. The fluorescence intensity of colorectal lesions in model group was higher than that in normal group. LIGHT INTENSITY.4) In vivo, caudal vein injection of ERC had significant protective effect on colorectal inflammation in mice, and blocked surface of ERC by PD-L1 significantly decreased the protective effect of ERC on colorectal inflammation in mice. Compared with untreated group, the number of splenic CD3~+CD4~+T, CD3~+CD8~+T cells and CD4~+CD25~+Foxp3~+Tregs cells in ERC group decreased significantly (P 0.05), and the number of CD4~+CD25~+Foxp3 The number of CD11c~+MHC-II~+DCs decreased significantly (P 0.05), F4/80~+M cells decreased significantly (P 0.05), F4/80~+CD206~+M increased relatively (P 0.05), TNF-a, IFN-gamma m RNA transcription and protein expression decreased (P 0.05), IL-10, TGF-beta m RNA transcription and protein expression increased in colorectal lesions. Compared with the ERC group, the number of CD3~+CD4~+T, CD3~+CD8~+ cells in spleen and CD4~+CD25~+Foxp3~+Tregs cells in PD-L1-blocked ERC group increased (P 0.05), the number of CD4~+CD25~+Foxp3~+Tregs cells decreased (P 0.05), the number of CD11c~+MHC-II~+DCs cells increased (P 0.05), the level of TNF-a, IFN-gamma m RNA transcription and protein expression in colorectal tissues increased relatively. Compared with the normal control group, the number of CD3~+CD4~+T, CD3~+CD8~+T, CD11c~+MHC-II~+DCs, F4/80~+M cells and the number of CD4~+CD25~+Foxp3~+Tregs, F4/80~+M cells in ERC co-culture group were decreased after stimulation with different stimuli. The number of CD3 + CD4 + T, CD3 + CD4 + T, CD3 + CD8 + T, CD11c + MHC - II + DCs, F4 / 80 + M cells were relatively increased, CD4 + CD25 + Foxp3 + Tregs, F4 / 80 + Tregs, F4 / 80 + CD206 + M cells were relatively decreased; the number of CD3 + CD3 + CD4 + CD4 + CD4 + CD4 + T, CD3 + CD3 + CD3 + CD3, CD3 + CD3 + CD3 + CD8 + CD8 + CD8 + CD8 + CD8 + DCs, CD11c + MHC - II + DCs, F4 / 80 + M cells were relatively increased, CD4 + CD4 + CD4 + CD4 + CD25 + CD25 + Foxp3 + Tregs, F4 / 80 + CD80 s, F4/ The number of CD4~+CD25~+Foxp3~+Tregs and F4/80~+CD206~+M cells were relatively decreased. CONCLUSION: 1) ERC isolated from menstrual blood is a kind of MSC-like regenerative cells. ERC is not only abundant in source, but also noninvasive, and the expression of ERC on the surface plays an important role in inducing immune tolerance PD-L1.2) 3%. DSS is an ideal concentration for constructing UC animal model of BALB/c mice. It can not only simulate the pathological changes of human UC intestine, but also has no mortality in mice. In vivo and in vitro experiments showed that ERC inhibited the maturation and differentiation of DCs and the proliferation of CD4~+T and CD8~+T cells, promoted the production of Tregs cells and M2 cells, and in vitro experiments also showed that PD-L1 on the surface of ERC acted through direct contact. And then play the role of immunomodulation.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R574.62;R-332

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