本文選題：膿毒癥 + 糖皮質(zhì)激素誘導的腫瘤壞死因子配體（GITRL）��； 參考：《重慶醫(yī)科大學》2012年碩士論文

【摘要】：目的：通過分別轉(zhuǎn)染GITRLsiRNA和pEGFP-N1-GITRL重組質(zhì)粒進入Kupffer細胞（KCs），干擾KCs中GITRL的基礎(chǔ)表達，并與T細胞體外共培養(yǎng)，在LPS刺激下觀察KCs中PDL1表達情況，對T細胞的活度影響分析，，初步探討GITRL在KCs中通過PDL~(-1)調(diào)控炎癥反應(yīng)機制。 方法：1、采用不連續(xù)密度梯度離心、選擇性貼壁法分離小鼠KCs與尼龍毛柱過濾純化脾淋巴細胞提取T細胞。2、隨機將KCs分為四組轉(zhuǎn)染，將GITRLsiRNA和pEGFP-N1-GITRL重組質(zhì)粒，及其分別對應(yīng)的controlsiRNA和空載質(zhì)粒pEGFP-N1-control轉(zhuǎn)入KCs中。轉(zhuǎn)染24小時后對各組（siRNA：異硫氰酸熒光素標記；重組質(zhì)粒: pEGFP載體為自帶綠色熒光載體)通過熒光顯微鏡鑒定轉(zhuǎn)染效果轉(zhuǎn)染效率初步鑒定;實時熒光定量逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)（RT-PCR）和蛋白質(zhì)免疫印跡（Westernbolt）技術(shù)進一步對轉(zhuǎn)染各組細胞從基因和蛋白表達水平鑒定。轉(zhuǎn)染后各組細胞和正常KCs細胞分別與T細胞體外共培養(yǎng)（1:10）。3、將共培養(yǎng)細胞分為6組：Control組，未轉(zhuǎn)染處理過的KCs與T細胞共培養(yǎng)體系只加培養(yǎng)基；LPS組，未轉(zhuǎn)染處理過的KCs與T細胞同培養(yǎng)體系加入LPS(1ug／m1)；GITRL siRNA組, ControlsiRNA組, pEGFP-N1-GITRL組， pEGFP-N1control組，對應(yīng)分別轉(zhuǎn)染后的KCs與T細胞共培養(yǎng),處理后同LPS組。24小時后，細胞免疫熒光法, Westernbolt檢測KCs的GITRL、PDL1表達, MTT法和Annexin-V/FITC流式法分析T細胞活性，ELISA檢測上清液腫瘤壞死因子TNFα分泌水平，激光共聚焦觀察KCsP65漿核分布情況。 結(jié)果：1、吞墨實驗和F4／80免疫熒光染色鑒定KCs細胞活率和純度分別為94％和95％，CD3/CD4雙抗染色后流式分析T細胞純度為73.8%。2、轉(zhuǎn)染siRNA和GITRL重組質(zhì)粒24h后熒光顯微鏡下觀察并鑒定KCs中GITRLsiRNA和pEGFP-N1-GITRL轉(zhuǎn)染效果分別達90％和85%，RT-PCR和Westernbolt檢測顯示基因沉默組（RT-PCR:0.23±0.13;WB:0.05±0.01）與過表達組（RT-PCR:2.19±0.16;WB:0.86±0.036）均與對照組有顯著性差異（P0.05）。3、在LPS刺激24小時后，LPS組,LPS+Control siRNA組及pEGFP-N1control組共培養(yǎng)體系中KCs上GITRL（IF:0.26×10~(-1)±0.63×10-3;WB:1.44±0.5）,T細胞活性(0.78±0.02)和上清液腫瘤壞死因子TNFα(603.50±5.96)分泌表達明顯上調(diào)（P0.05），PDL1(IF:0.15×10~(-1)±0.10×10~(~(-2));WB:1.44±0.04)顯著下調(diào)（P0.05）; pEGFP-N1-GITRL組則出現(xiàn)了GITRL(IF:0.24×10~(-1)±0.29×10~(~(-2));WB1.77±0.05),T細胞活性(0.99±0.00)和上清液腫瘤壞死因子TNFα(908.83±2.64)分泌更加顯著的上調(diào)，而PDL1(IF：0.817×10~(~(-2))±0.75×10-3;WB:1.04±0.07)的表達也進一步的降低。GITRL siRNA組在被LPS刺激后GITRL(IF:0.11×10~(-1)±0.11×10~(~(-2));WB:1.11±0.04),T細胞增值(0.50±0.04)和分泌的腫瘤壞死因子TNFα(252.83±4.49)的表達明顯被下降（P0.05），對PDL1(IF:0.29×10~(-1)±0.81×10-3;WB:1.84±0.04)被抑制的程度也明顯減弱（0.05）。 結(jié)論: KCs上的GITRL上調(diào)后可通過抑制PD-L1，激活T細胞活性，促進炎癥反應(yīng)的發(fā)展，干擾KCs上GITRL表達可恢復免疫平衡，抑制炎癥反應(yīng)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 偽 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

本文編號：1850430