Kupffer细胞中GITRL通过抑制PDL-1调控炎症反应的研究
发布时间：2018-05-06 02:37
本文选题：脓毒症 + 糖皮质激素诱导的肿瘤坏死因子配体（GITRL） ； 参考：《重庆医科大学》2012年硕士论文
【摘要】：目的：通过分别转染GITRLsiRNA和pEGFP-N1-GITRL重组质粒进入Kupffer细胞（KCs），干扰KCs中GITRL的基础表达，并与T细胞体外共培养，在LPS刺激下观察KCs中PDL1表达情况，对T细胞的活度影响分析，，初步探讨GITRL在KCs中通过PDL~(-1)调控炎症反应机制。 方法：1、采用不连续密度梯度离心、选择性贴壁法分离小鼠KCs与尼龙毛柱过滤纯化脾淋巴细胞提取T细胞。2、随机将KCs分为四组转染，将GITRLsiRNA和pEGFP-N1-GITRL重组质粒，及其分别对应的controlsiRNA和空载质粒pEGFP-N1-control转入KCs中。转染24小时后对各组（siRNA：异硫氰酸荧光素标记；重组质粒: pEGFP载体为自带绿色荧光载体)通过荧光显微镜鉴定转染效果转染效率初步鉴定;实时荧光定量逆转录-聚合酶链反应（RT-PCR）和蛋白质免疫印迹（Westernbolt）技术进一步对转染各组细胞从基因和蛋白表达水平鉴定。转染后各组细胞和正常KCs细胞分别与T细胞体外共培养（1:10）。3、将共培养细胞分为6组：Control组，未转染处理过的KCs与T细胞共培养体系只加培养基；LPS组，未转染处理过的KCs与T细胞同培养体系加入LPS(1ug／m1)；GITRL siRNA组, ControlsiRNA组, pEGFP-N1-GITRL组， pEGFP-N1control组，对应分别转染后的KCs与T细胞共培养,处理后同LPS组。24小时后，细胞免疫荧光法, Westernbolt检测KCs的GITRL、PDL1表达, MTT法和Annexin-V/FITC流式法分析T细胞活性，ELISA检测上清液肿瘤坏死因子TNFα分泌水平，激光共聚焦观察KCsP65浆核分布情况。 结果：1、吞墨实验和F4／80免疫荧光染色鉴定KCs细胞活率和纯度分别为94％和95％，CD3/CD4双抗染色后流式分析T细胞纯度为73.8%。2、转染siRNA和GITRL重组质粒24h后荧光显微镜下观察并鉴定KCs中GITRLsiRNA和pEGFP-N1-GITRL转染效果分别达90％和85%，RT-PCR和Westernbolt检测显示基因沉默组（RT-PCR:0.23±0.13;WB:0.05±0.01）与过表达组（RT-PCR:2.19±0.16;WB:0.86±0.036）均与对照组有显著性差异（P0.05）。3、在LPS刺激24小时后，LPS组,LPS+Control siRNA组及pEGFP-N1control组共培养体系中KCs上GITRL（IF:0.26×10~(-1)±0.63×10-3;WB:1.44±0.5）,T细胞活性(0.78±0.02)和上清液肿瘤坏死因子TNFα(603.50±5.96)分泌表达明显上调（P0.05），PDL1(IF:0.15×10~(-1)±0.10×10~(~(-2));WB:1.44±0.04)显著下调（P0.05）; pEGFP-N1-GITRL组则出现了GITRL(IF:0.24×10~(-1)±0.29×10~(~(-2));WB1.77±0.05),T细胞活性(0.99±0.00)和上清液肿瘤坏死因子TNFα(908.83±2.64)分泌更加显著的上调，而PDL1(IF：0.817×10~(~(-2))±0.75×10-3;WB:1.04±0.07)的表达也进一步的降低。GITRL siRNA组在被LPS刺激后GITRL(IF:0.11×10~(-1)±0.11×10~(~(-2));WB:1.11±0.04),T细胞增值(0.50±0.04)和分泌的肿瘤坏死因子TNFα(252.83±4.49)的表达明显被下降（P0.05），对PDL1(IF:0.29×10~(-1)±0.81×10-3;WB:1.84±0.04)被抑制的程度也明显减弱（0.05）。 结论: KCs上的GITRL上调后可通过抑制PD-L1，激活T细胞活性，促进炎症反应的发展，干扰KCs上GITRL表达可恢复免疫平衡，抑制炎症反应程度。
[Abstract]:Objective: by transfecting GITRLsiRNA and pEGFP-N1-GITRL recombinant plasmids into Kupffer cells to interfere with the basic expression of GITRL in KCs and co-culture with T cells in vitro, the expression of PDL1 in KCs was observed under the stimulation of LPS, and the effect of LPS on the activity of KCs was analyzed. To explore the mechanism of GITRL regulating inflammatory response in KCs. Methods: t lymphocytes were extracted from splenic lymphocytes by using discontinuous density gradient centrifugation and selective adherent method. KCs was randomly divided into four groups. The recombinant plasmids of GITRLsiRNA and pEGFP-N1-GITRL were transfected. The corresponding controlsiRNA and empty plasmid pEGFP-N1-control were transferred into KCs. After 24 hours of transfection, the transfection efficiency of each group of siRNAs: fluorescein isothiocyanate, recombinant plasmid: pEGFP vector was identified by fluorescence microscope. Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot (Western blot) technique were used to identify the gene and protein expression levels in the transfected cells. After transfection, each group of cells and normal KCs cells were co-cultured with T cells in vitro. The co-cultured cells were divided into 6 groups: control group. The untransfected KCs and T cell co-culture system was only supplemented with LPS-treated medium. The untransfected KCs and T cells were co-cultured in the same culture system with LPSN 1ugP / m1GITRL siRNA group, ControlsiRNA group, pEGFP-N1-GITRL group, pEGFP-N1control group, corresponding to the co-culture of KCs and T cells respectively. After treatment, the cells were co-cultured in the same LPS group for 24 hours. The expression of GITRL- PDL1 in KCs was detected by cell immunofluorescence assay, and the expression of TNF 伪 in supernatant was detected by MTT and Annexin-V/FITC flow cytometry. The distribution of KCsP65 cytoplasm was observed by confocal laser. Results the viability and purity of KCs cells were 94% and 95% respectively. The purity of T cells was 73.8% by flow cytometry. 24 hours after transfection of siRNA and GITRL recombinant plasmids, KCs was observed and identified under fluorescence microscope. 涓璆ITRLsiRNA鍜宲EGFP-N1-GITRL杞煋鏁堟灉鍒嗗埆杈
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