NF-κB寡核苷酸诱骗策略构建胶原诱导性关节炎大鼠耐受性DC
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摘要
目的:探讨NF-κB寡核苷酸诱骗剂(NF-κB ODN Decoy)策略构建胶原诱导性关节炎(CIA)大鼠脾脏来源的稳定的耐受性树突状细胞(DC),并对其成熟状态进行评估。方法:取CIA大鼠脾脏细胞,运用粒细胞-巨噬细胞集落刺激因子(GM-CSF)和白细胞介素(IL)-4两种细胞因子诱导培养DC,并分为4组:单纯DC组、Decoy-DC组、LPS刺激DC组、LPS刺激Decoy-DC组。对4组细胞进行形态学观察、活力评价、表型鉴定及耐受性评估。结果:各组DC活力均>90%。OX-62表达率>55%。Decoy-DC组表面共刺激分子CD80和CD86阳性率和平均荧光强度、刺激淋巴细胞增殖能力及IFN-γ分泌能力均显著低于Control-DC组(P<0. 05),而IL-10分泌能力显著高于Control-DC组(P<0. 05)。LPS刺激Decoy-DC组CD80和CD86阳性率和平均荧光强度、刺激淋巴细胞增殖能力及IFN-γ分泌能力均显著低于LPS-DC组(P<0. 05),而IL-10分泌能力显著高于LPS-DC组(P<0. 05)。LPS-Decoy-DC组CD80和CD86阳性率和平均荧光强度、刺激淋巴细胞增殖能力与Decoy-DC组比,无显著性差异(P>0. 05)。结论:CIA大鼠脾脏细胞经NF-κB ODN Decoy策略成功构建出形态、表型及功能稳定的耐受性DC。
Objective: To investigate the strategy of NF-κB ODN Decoy was used to construct the stable tolerant dendritic cells( DC) derived from spleen during collagen induced arthritis( CIA) in rats,and assessed its maturation status. Methods: Spleen cells from CIA rats were induced by DC with GM-CSF and IL-4 and divided into 4 groups: DC group; Decoy-DC group; LPS-stimulated DC group; LPS-stimulated Decoy-DC group. The Morphological observation,viability evaluation,phenotypic identification and tolerance assessment of all 4 groups cells were performed. Results: The DC viability of each group was >90%,and the expression rate of OX-62 was>55%.Compared with the Control-DC group,the positive rate and Mean Fluorescence Intensity of co-stimulatory molecules CD80,CD86 and the ability of lymphocyte proliferation and secretion of IFN-γ in the Decoy-DC group were significantly decreased( P<0. 05); secretion of IL-10 were significantly increased( P<0. 05). Compared with the LPS-DC group,the positive rate and Mean Fluorescence Intensity of co-stimulatory molecules CD80,CD86 and the ability of lymphocyte proliferation and secretion of IFN-γ in the LPS-Decoy-DC group were significantly decreased( P < 0. 05); secretion of IL-10 were significantly increased( P < 0. 05). The positive rate and Mean Fluorescence Intensity of co-stimulatory molecules CD80,CD86 and the ability of lymphocyte proliferation were no significant difference between LPS-Decoy-DC group and Decoy-DC group( P>0. 05). Conclusion: The morphological,phenotypic and functionally stable tolerant DC were constructed by the strategy of NF-κB ODN Decoy in rats spleen during CIA diseases.
Objective: To investigate the strategy of NF-κB ODN Decoy was used to construct the stable tolerant dendritic cells( DC) derived from spleen during collagen induced arthritis( CIA) in rats,and assessed its maturation status. Methods: Spleen cells from CIA rats were induced by DC with GM-CSF and IL-4 and divided into 4 groups: DC group; Decoy-DC group; LPS-stimulated DC group; LPS-stimulated Decoy-DC group. The Morphological observation,viability evaluation,phenotypic identification and tolerance assessment of all 4 groups cells were performed. Results: The DC viability of each group was >90%,and the expression rate of OX-62 was>55%.Compared with the Control-DC group,the positive rate and Mean Fluorescence Intensity of co-stimulatory molecules CD80,CD86 and the ability of lymphocyte proliferation and secretion of IFN-γ in the Decoy-DC group were significantly decreased( P<0. 05); secretion of IL-10 were significantly increased( P<0. 05). Compared with the LPS-DC group,the positive rate and Mean Fluorescence Intensity of co-stimulatory molecules CD80,CD86 and the ability of lymphocyte proliferation and secretion of IFN-γ in the LPS-Decoy-DC group were significantly decreased( P < 0. 05); secretion of IL-10 were significantly increased( P < 0. 05). The positive rate and Mean Fluorescence Intensity of co-stimulatory molecules CD80,CD86 and the ability of lymphocyte proliferation were no significant difference between LPS-Decoy-DC group and Decoy-DC group( P>0. 05). Conclusion: The morphological,phenotypic and functionally stable tolerant DC were constructed by the strategy of NF-κB ODN Decoy in rats spleen during CIA diseases.
引文
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[13]Brenan M,Puklavec M.The MRC OX-62 antigen:a useful marker in the purification of rat veiled cells with the biochemical properties of an integrin[J].J Exp Med,1992,175(6):1457-1465.
[14]徐东亮,唐孝达,李博,等.核因子-κB对小鼠树突状细胞分化成熟及其体外刺激T细胞免疫反应的影响[J].中华器官移植杂志,2004,25(2):89-92.Xu DL,Tang XD,Li P,et al.Effect of NF-κB Decoys on development and maturation of dendritic cells and initiation of T cells immune response in vitro[J].Chin J Organ Transplant,2004,25(2):89-92.
[15]Jiang H,Zhang Y,Yin X,et al.Construction and evaluation of rats'tolerogenic dendritic cells(DC)induced by NF-κB Decoy method[J].Afr Health Sci,2014,14(3):626-633.
[2]姜申易,鲁静.NF-κB在类风湿关节炎中的研究进展[J].中国免疫学杂志,2016,32(1):119-122.Jiang SY,Lu J.Research progress of NF-κB in rheumatoid arthritis[J].Chin J Immunol,2016,32(1):119-122.
[3]Karimi MH,Ebadi P,Pourfathollah AA,et al.Immune modulation through RNA interference-mediated silencing of CD40 in dendritic cells[J].Cell Immunol,2009,259(1):74-81.
[4]尹路,陈春球,陈桂明,等.大鼠小肠系膜淋巴组织抗原刺激同种异体混合淋巴细胞增殖反应研究[J].中华外科杂志,2007,45(9):626-629.Yin L,Chen CQ,Chen GM,et al.Research of rat small intestinal mesentery lymphoid tissue stimulating allograft mixed lymphocyte reaction[J].Chin J Surg,2007,45(9):626-629.
[5]Matsue H,Yang C,Matsue K,et al.Contrasting impacts of immunosuppressive agents(rapamycin,FK506,cyclosporin A,and dexamethasone)on bidirectional dendritic cell-T cell interaction during antigen presentation[J].J Immunol,2002,169(7):3555-3564.
[6]Onaitis M,Kalady MF,Pruitt S,et al.Dendritic cell gene therapy[J].Surg Oncol Clin N Am,2002,11(3):645-660.
[7]Xu MQ,Suo YP,Gong JP,et al.Prolongation of liver allograft survival by dendritic cells modified with NF-kappaB decoy oligodeoxynucleotides[J].World J Gastroenterol,2004,10(16):2361-2368.
[8]Bonham CA,Peng L,Liang X,et al.Marked prolongation of cardiac allograft survival by dendritic cells genetically engineered with NF-kappaB oligodeoxyribonucleotide decoys and adenoviral vectors encoding CTLA4-Ig[J].J Immunol,2002,169(6):3382-3391.
[9]尹向飞.NF-κB诱骗剂诱导大鼠特异性耐受性树突状细胞的构建及性能评价[D].贵阳:贵阳医学院,2010.Yin XF.Construction and evaluatation of antigen-specific tolerogenic dendritic cell treated with nuclear factor-κB ODN decoy[D].Guiyang:Guiyang Medical College,2010.
[10]Voisine C,Hubert FX,TrinitéB,et al.Two phenotypically distinct subsets of spleen dendritic cells in rats exhibit different cytokine production and T cell stimulatory activity[J].J Immunol,2002,169(5):2284-2291.
[11]Hart DN.Dendritic cells:unique leukocyte populations which control the primary immune response[J].Blood,1997,90(9):3245-3287.
[12]Fairchild PJ,Waldmann H.Dendritic cells and prospects for transplantation tolerance[J].Curr Opin Immunol,2000,12(5):528-535.
[13]Brenan M,Puklavec M.The MRC OX-62 antigen:a useful marker in the purification of rat veiled cells with the biochemical properties of an integrin[J].J Exp Med,1992,175(6):1457-1465.
[14]徐东亮,唐孝达,李博,等.核因子-κB对小鼠树突状细胞分化成熟及其体外刺激T细胞免疫反应的影响[J].中华器官移植杂志,2004,25(2):89-92.Xu DL,Tang XD,Li P,et al.Effect of NF-κB Decoys on development and maturation of dendritic cells and initiation of T cells immune response in vitro[J].Chin J Organ Transplant,2004,25(2):89-92.
[15]Jiang H,Zhang Y,Yin X,et al.Construction and evaluation of rats'tolerogenic dendritic cells(DC)induced by NF-κB Decoy method[J].Afr Health Sci,2014,14(3):626-633.