IDO基因修饰的小鼠骨髓间充质干细胞诱导免疫耐受的体外实验研究

发布时间：2018-06-27 15:04

本文选题：骨髓间充质干细胞 + IDO　；参考：《苏州大学》2012年硕士论文

【摘要】：目的：研究吲哚胺2，3-双加氧酶（IDO）基因修饰的小鼠骨髓间充质干细胞（IDO-BMSC）体外诱导免疫耐受作用，并探讨其相关的免疫学机制。方法：C57BL/6小鼠的BMSC与BALB/C小鼠骨髓来源的树突状细胞（DC）和脾脏单个核细胞（Splenic mononuclear cells，SMC）在体外按不同细胞比例共同培养4天。根据BMSC中IDO基因修饰与否，将实验分为对照组和基因修饰组，对照组和基因修饰组均设：A组：BMSC与DC共同培养（1：1），B组：BMSC与SMC共同培养（1：4），C组：BMSC与SMC共同培养（1：8），D组：BMSC与SMC共同培养（1：16），E组：BMSC与DC、SMC共同培养（BMSC：SMC=1：4），F组：BMSC与DC、SMC共同培养（BMSC：SMC=1：8），G组： BMSC与DC、SMC共同培养（BMSC：SMC=1：16），培养体系中均加入（1ug/ml）的脂多糖（LPS）。流式细胞术分析共培养前后树突状细胞表面分子表型变化及脾脏单个核细胞中FoxP3+Treg含量。微量样本多指标流式蛋白定量技术（CBA）检测共培养上清中IL-2，IL-4，IL-6，IFN-γ，IL-17，IL-10的浓度。高效液相（HPLC）法检测上清中色氨酸及犬尿氨酸浓度。RT-PCR检测A、E组DC中IDOmRNA的表达。结果：(1)BMSC与DC细胞以1：1的比例共培养(A组)，对照组DC与未成熟DC表型相似，而基因修饰组较对照组能明显抑制DC成熟（P0.05），有统计学意义。(2) B、C、D组中FoxP3+Treg含量比较，基因修饰组FoxP3+Treg高于对照组（P0.05），有统计学意义；E、F、G组中FoxP3+Treg含量增加，基因修饰组高于对照组，P0.05，，有统计学意义。(3)组间比较B、C、D组，基因修饰组较对照组IL-2，IL-6，IFN-γ，IL-17明显下降，而IL-4及IL-10明显上升，P0.05，有统计学意义。组间比较E、F、G组，基因修饰组IL-2，IL-6明显下降，而IL-4，IFN-γ明显升高，P0.05，有统计学意义，IL-17，IL-10改变不明显，P0.05，无统计学意义。(4)基因修饰组IDO活性明显高于对照组，P0.05。且在基因修饰组，E组IDO活性明显高于A组，P0.05,有统计学意义。(5)基因修饰的E组中的DC中检测出IDOmRNA的表达，对照组无IDOmRNA的表达。结论：IDO基因修饰的骨髓间充质干细胞能通过以下四条途径诱导免疫耐受：1、IDO-BMSC能够显著抑制DC细胞的成熟，2、IDO-BMSC可以诱导FoxP3+Treg含量增加，3、IDO-BMSC能够促进抗炎因子，抑制促炎因子的分泌，4、IDO-BMSC通过SMC的桥梁作用诱导DC细胞产生IDO，进而诱导免疫耐受。
[Abstract]:Aim: to investigate the induction of immune tolerance of mouse bone marrow mesenchymal stem cells (IDO-BMSC) modified with indoleamine 2o 3-dioxygenase (IDO) gene in vitro and to explore its immunological mechanism. Methods BMSCs from BMSCs of C57BL / 6 mice were co-cultured with dendritic cells (DC) derived from bone marrow and splenic mononuclear cells (SMC) of BMSCs from BALB / C mice for 4 days. According to the IDO gene modification in BMSC, the experiment was divided into two groups: control group and gene modified group. The control group and the gene modified group were divided into two groups: group A: BMSC co-cultured with DC (1:1); group B: BMSC and SMC co-cultured (1:4); group C: BMSC and SMC co-cultured (1:8); group D: BMSC and SMC co-cultured (1:8); group E: BMSC and DCSMC co-cultured Group F: BMSC: SMC 1: 8 (BMSC: SMC 1: 8) Group G: BMSC: SMC 1: 16. Lipopolysaccharide (1ug/ml) was added to the culture system. The changes of surface molecular phenotype of dendritic cells and the content of FoxP3 Treg in splenic mononuclear cells before and after co-culture were analyzed by flow cytometry. The concentration of IL-2, IL-4, IL-6, IFN- 纬, IL-17 and IL-10 in the supernatant of co culture was detected by flow protein quantitative assay (CBA). High performance liquid chromatography (HPLC) was used to detect the concentration of tryptophan and canine uric acid in supernatant. RT-PCR was used to detect the expression of Ido mRNA in DC of Agne group. Results: (1) BMSC and DC cells were co-cultured at 1:1 (group A). The phenotypes of DC in control group were similar to those in immature DC, while the maturation of DC was significantly inhibited in gene modified group (P0.05). (2) the content of FoxP3 Treg in BMSC / DC group was higher than that in control group. The level of FoxP3 Treg in the gene modified group was higher than that in the control group (P0.05), and the content of FoxP3 Treg in the gene modified group was higher than that in the control group (P 0.05). (3) compared with the control group, the gene modified group was significantly lower than the control group in IL-2, IL-6IFN- 纬 IL-17. The levels of IL-4 and IL-10 increased significantly (P 0.05). Compared with the control group, IL 2 and IL 6 in the gene modified group decreased significantly, while the level of IL 4 hIFN- 纬 increased significantly (P 0 05, P 0 05). (4) the IDO activity in the gene modified group was significantly higher than that in the control group (P 0 05), and there was no significant difference between the two groups in the change of IL-17 and IL 10. (4) the activity of IDO in the gene modified group was significantly higher than that in the control group (P 0. 05). The activity of Ido in group E was significantly higher than that in group A (P 0.05). (5) the expression of Ido mRNA was detected in DC of gene modified group E, but no expression of Ido mRNA was detected in control group. Conclusion the bone marrow mesenchymal stem cells modified with 1: IDO gene can induce immune tolerance through the following four pathways: 1. IDO-BMSC can significantly inhibit the maturation of DC cells. IDO-BMSC can induce the increase of FoxP3 Treg content and the promotion of anti-inflammatory factor by inducing the increase of FoxP3 Treg content. Inhibition of the secretion of proinflammatory factor 4 IDO-BMSC induces DC cells to produce IDO through the bridge of SMC, and then induces immune tolerance.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.1

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