本文關(guān)鍵詞：γ-干擾素刺激培養(yǎng)imDC過繼治療抗-GBM腎炎的實驗研究　出處：《第三軍醫(yī)大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： γ-干擾素 未成熟樹突狀細(xì)胞 抗-GBM腎炎 免疫耐受 多肽pCol (28-40)

【摘要】： 目的:經(jīng)IFN-γ刺激培養(yǎng)的imDC,過繼轉(zhuǎn)入抗-GBM腎炎模型,觀察經(jīng)IFN-γ刺激后的imDC對抗-GBM腎炎腎臟病變的抑制效果。 方法:以合成多肽序列pCol (28-40)作為抗原,包入脂質(zhì)體內(nèi)制成抗原制劑,建立抗-GBM腎炎大鼠模型,具體為:第0天(d0)脂質(zhì)體抗原制劑0. 25 ml,于腳掌和尾根部皮下注射初次致敏;第7天(d7)脂質(zhì)體抗原制劑0. 1 ml,同部位加強致敏;采集wistar大鼠骨髓,采用經(jīng)典的GM-CSF+IL-4方案制備imDC,經(jīng)imDCIFN-γ刺激培養(yǎng),以合成多肽pCol (28-40)多肽作為外源性抗原處理imDCIFN-γ,流式細(xì)胞儀檢測表面MHC II類分子,及其共刺激分子CD80, CD86和DC表面相對特異性標(biāo)記OX62的表達(dá)情況,在體外與脾臟分離的CD4+T共培養(yǎng),采用MTT比色法檢測CD4+T增殖情況,Annexin-V法檢測CD4+T凋亡情況,酶聯(lián)免疫吸附法檢測脾細(xì)胞培養(yǎng)液中IL-4、IL-13、IFN-γ的含量,觀察體外抗原特異性T細(xì)胞功能活性狀態(tài);并于首次致敏前7天(-d7)過繼轉(zhuǎn)入抗-GBM腎炎模型,間隔7天隨機選取相關(guān)樣本進行生化指標(biāo)(24小時尿白蛋白,血清肌酐,血尿素氮)腎臟病理學(xué)、免疫學(xué)指標(biāo),病理學(xué)、免疫學(xué)檢查,觀察對抗-GBM腎炎腎小球病變抑制效果及抗原特異性免疫耐受的相關(guān)機制. 結(jié)果:(1)光鏡下觀察imDC IFN-γ細(xì)胞呈圓形或卵圓形,細(xì)胞膜略不規(guī)則,有少量的短小突起或偽足。流式細(xì)胞儀檢測imDC和imDC IFN-γ表面低表達(dá)的MHC II類分子(14.01%)及共刺激分子CD80(4.78%)、CD86(19.79%),同樣低表達(dá)DC表面相對特異性標(biāo)記OX62( 15.31%)(p0.05, n=4)的水平都顯著低于mDC; (2)抗-GBM腎炎大鼠模型組大鼠尿蛋白,血肌酐,血尿素氮短期內(nèi)明顯升高(P 0.05,n=10)、HE染色腎小球系膜細(xì)胞數(shù)增多,炎細(xì)胞浸潤,PAS染色均有細(xì)胞性新月體形成(占腎小囊50%)、免疫熒光可見大量IgG線樣沉積;電鏡可見足突融合,基底膜增厚,內(nèi)皮細(xì)胞增生,免疫復(fù)合物沉積。 (3)體外共培養(yǎng)的脾淋巴細(xì)胞,imDC組與對照組增殖無顯著性差異;而imDCIFN-γ組與對照組增殖明顯受抑制,對照組OD值(0.1950±0.0160.);imDC組OD值(0.1974±0.017);imDCIFN-γ組為OD值(0.836±0.012)(p0.05, n=5);imDCIFN-γ組凋亡率為(12.98+0.02),與對照組和imDC組相比凋亡均明顯增高(p0.05, n=5);上調(diào)了Th2型細(xì)胞因子IL一4及IL-13的表達(dá),而下調(diào)Th1型細(xì)胞因子IFN-γ;Th1型細(xì)胞因子明顯向Th2型細(xì)胞因子偏移。進一步體內(nèi)實驗發(fā)現(xiàn),過繼轉(zhuǎn)入imDC IFN-γ后均能明顯降低尿蛋白,血肌酐,血尿素氮水平,同時期亦減少炎細(xì)胞浸潤,細(xì)胞新月體形成比率和速度減少,免疫熒光檢測發(fā)現(xiàn)IgG沉積減少,熒光強度減弱;過繼轉(zhuǎn)入imDCIFN-γ能夠抑制細(xì)胞性新月體形成,能明顯緩解抗-GBM腎炎病情的進展,對抗-GBM腎炎具有保護作用 結(jié)論: 1. imDC IFN-γ對外源性抗原多肽pCol(28-40)攝取及提呈能力明顯下降;可抑制T細(xì)胞增殖,促進T細(xì)胞凋亡;實驗還表明IFN-γ刺激培養(yǎng)的未成熟樹突狀細(xì)胞促使Th1型細(xì)胞因子向Th2型細(xì)胞因子偏移,誘導(dǎo)形成特異性抗原免疫耐受。 2.成功建立了抗-GBM腎炎大鼠模型 3. IFN-γ刺激培養(yǎng)的imDC過繼轉(zhuǎn)入模型大鼠,可誘導(dǎo)形成抗原特異性免疫耐受,能明顯抑制抗-GBM腎炎病情的進展,對抗-GBM腎炎具有保護作用。
[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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 劉加洪,王英年,趙云峰,姚艷紅,張克佳;Th1-Th2平衡失調(diào)與人類疾病關(guān)系及其相關(guān)治療研究現(xiàn)狀[J];青島大學(xué)醫(yī)學(xué)院學(xué)報;2002年04期

2 王海權(quán);徐皓;肇毅;沈歷宗;陳濤;華一兵;吳文溪;;聯(lián)合應(yīng)用未成熟樹突狀細(xì)胞和CD40L單克隆抗體延長大鼠移植腸存活時間[J];南京醫(yī)科大學(xué)學(xué)報(自然科學(xué)版);2006年07期

，

本文編號：1374970