γ-干扰素刺激培养imDC过继治疗抗-GBM肾炎的实验研究

发布时间：2018-01-03 18:03

本文关键词：γ-干扰素刺激培养imDC过继治疗抗-GBM肾炎的实验研究　出处：《第三军医大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的:经IFN-γ刺激培养的imDC,过继转入抗-GBM肾炎模型,观察经IFN-γ刺激后的imDC对抗-GBM肾炎肾脏病变的抑制效果。方法:以合成多肽序列pCol (28-40)作为抗原,包入脂质体内制成抗原制剂,建立抗-GBM肾炎大鼠模型,具体为:第0天(d0)脂质体抗原制剂0. 25 ml,于脚掌和尾根部皮下注射初次致敏;第7天(d7)脂质体抗原制剂0. 1 ml,同部位加强致敏;采集wistar大鼠骨髓,采用经典的GM-CSF+IL-4方案制备imDC,经imDCIFN-γ刺激培养,以合成多肽pCol (28-40)多肽作为外源性抗原处理imDCIFN-γ,流式细胞仪检测表面MHC II类分子,及其共刺激分子CD80, CD86和DC表面相对特异性标记OX62的表达情况,在体外与脾脏分离的CD4+T共培养,采用MTT比色法检测CD4+T增殖情况,Annexin-V法检测CD4+T凋亡情况,酶联免疫吸附法检测脾细胞培养液中IL-4、IL-13、IFN-γ的含量,观察体外抗原特异性T细胞功能活性状态;并于首次致敏前7天(-d7)过继转入抗-GBM肾炎模型,间隔7天随机选取相关样本进行生化指标(24小时尿白蛋白,血清肌酐,血尿素氮)肾脏病理学、免疫学指标,病理学、免疫学检查,观察对抗-GBM肾炎肾小球病变抑制效果及抗原特异性免疫耐受的相关机制. 结果:(1)光镜下观察imDC IFN-γ细胞呈圆形或卵圆形,细胞膜略不规则,有少量的短小突起或伪足。流式细胞仪检测imDC和imDC IFN-γ表面低表达的MHC II类分子(14.01%)及共刺激分子CD80(4.78%)、CD86(19.79%),同样低表达DC表面相对特异性标记OX62( 15.31%)(p0.05, n=4)的水平都显著低于mDC; (2)抗-GBM肾炎大鼠模型组大鼠尿蛋白,血肌酐,血尿素氮短期内明显升高(P 0.05,n=10)、HE染色肾小球系膜细胞数增多,炎细胞浸润,PAS染色均有细胞性新月体形成(占肾小囊50%)、免疫荧光可见大量IgG线样沉积;电镜可见足突融合,基底膜增厚,内皮细胞增生,免疫复合物沉积。 (3)体外共培养的脾淋巴细胞,imDC组与对照组增殖无显著性差异;而imDCIFN-γ组与对照组增殖明显受抑制,对照组OD值(0.1950±0.0160.);imDC组OD值(0.1974±0.017);imDCIFN-γ组为OD值(0.836±0.012)(p0.05, n=5);imDCIFN-γ组凋亡率为(12.98+0.02),与对照组和imDC组相比凋亡均明显增高(p0.05, n=5);上调了Th2型细胞因子IL一4及IL-13的表达,而下调Th1型细胞因子IFN-γ;Th1型细胞因子明显向Th2型细胞因子偏移。进一步体内实验发现,过继转入imDC IFN-γ后均能明显降低尿蛋白,血肌酐,血尿素氮水平,同时期亦减少炎细胞浸润,细胞新月体形成比率和速度减少,免疫荧光检测发现IgG沉积减少,荧光强度减弱;过继转入imDCIFN-γ能够抑制细胞性新月体形成,能明显缓解抗-GBM肾炎病情的进展,对抗-GBM肾炎具有保护作用结论: 1. imDC IFN-γ对外源性抗原多肽pCol(28-40)摄取及提呈能力明显下降;可抑制T细胞增殖,促进T细胞凋亡;实验还表明IFN-γ刺激培养的未成熟树突状细胞促使Th1型细胞因子向Th2型细胞因子偏移,诱导形成特异性抗原免疫耐受。 2.成功建立了抗-GBM肾炎大鼠模型 3. IFN-γ刺激培养的imDC过继转入模型大鼠,可诱导形成抗原特异性免疫耐受,能明显抑制抗-GBM肾炎病情的进展,对抗-GBM肾炎具有保护作用。
[Abstract]:Objective: To observe the inhibition effect of imDC on the renal disease of -GBM nephritis after IFN- gamma stimulated by imDC stimulated by IFN- gamma stimulated imDC.
Methods: using synthetic peptide sequence pCol (28-40) as antigen, wrap the body made of lipid antigen preparation, model of anti -GBM nephritis rats for zeroth days (D0) liposome 0.25 ml antigen preparation, on the soles of the feet and tail root of subcutaneous injection of primary sensitization; seventh days (D7) liposome antigen preparation 0.1 ml, the same part of strengthening sensitization; acquisition of Wistar rat bone marrow, using the classical GM-CSF+IL-4 scheme imDC was prepared by imDCIFN- gamma stimulated by synthetic peptide pCol (28-40) peptide as exogenous antigen processing by imDCIFN-, flow cytometry detection of surface MHC class II molecules and costimulatory molecules CD80 CD86 and DC relative expression of surface marker of OX62, co culture in vitro in spleen and CD4+T, proliferation of CD4+T was examined by MTT assay. The detection of CD4+T apoptosis by Annexin-V, ELISA detection method of spleen cells cultured in IL-4, IL-1 3, the content of IFN- gamma, observed in vitro antigen specific T cell function activity; and for the first time in 7 days before sensitization (-d7) adoptive transferred anti -GBM nephritis model, interval of 7 days were randomly selected samples of related biochemical indexes (24 hours urinary albumin, serum creatinine, blood urea nitrogen) renal pathology, immunology index, pathology, immunology examination, observation of mechanism against glomerular lesions -GBM nephritis inhibitory effect and antigen specific immune tolerance.
Results: (1) under the light microscope imDC gamma IFN- cells were round or ovoid, slightly irregular cell membrane, a few short processes or pseudopodia. MHC flow cytometry II molecules imDC and imDC gamma IFN- surface (14.01%) and the low expression of costimulatory molecule CD80 (4.78%), CD86 (19.79%), the same low expression of DC surface marker of OX62 (15.31%) (P0.05, n=4) levels were significantly lower than mDC;
(2) anti -GBM urine protein, rats of model group rats nephritis serum creatinine, blood urea nitrogen increased significantly in the short term (P 0.05, n=10), HE staining of glomerular mesangial cells increased, inflammatory cell infiltration, PAS staining showed cells of crescent formation (accounted for 50%, renal capsule) immunofluorescence showed a large amount of IgG linear deposition; electron microscopy showed that the fusion of foot process, basement membrane thickening, endothelial cell proliferation, immune complex deposition.
(3) co cultured in vitro spleen lymphocytes proliferation in imDC group and control group had no significant difference; while the imDCIFN- gamma group and control group significantly inhibited the proliferation, the control group was (0.1950 + 0.0160.); imDC group was (0.1974 + 0.017); imDCIFN- group (0.836 + gamma value (P0.05, 0.012) n=5; imDCIFN-) gamma group apoptosis rate (12.98+0.02), compared with the control group and imDC group were significantly increased apoptosis (P0.05, n=5); upregulated the expression of Th2 type cytokines IL 4 and IL-13, and down-regulation of Th1 type cytokines IFN-; Th1 type cytokines significantly shift to Th2 type cytokine further in vivo, adoptive transferred to imDC IFN- gamma could reduce the urinary protein, serum creatinine, blood urea nitrogen, and also reduce the infiltration of inflammatory cells, cellular crescent formation ratio and speed reduction, immunofluorescence assay showed that IgG deposition decreased fluorescence intensity; adoptively transferred into imDCIFN- gamma Enough to inhibit the formation of crescent body, can obviously alleviate the progress of anti -GBM nephritis, and protect against -GBM nephritis
Conclusion:
1. imDC IFN- gamma of exogenous antigen peptide pCol (28-40) uptake and presentation ability decreased significantly; inhibited the proliferation of T cells and promote the apoptosis of T cells; experiments also show that immature dendritic cells stimulated the IFN- gamma Th1 type cytokines to Th2 type cytokine induced migration, formation of specific antigen immune tolerance.
2. a rat model of anti -GBM nephritis was successfully established.
3. IFN- gamma stimulated imDC was transferred into the model rats, which could induce the formation of antigen specific immune tolerance. It could significantly inhibit the progression of anti -GBM nephritis and protect against -GBM nephritis.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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