DC与蜕膜NK细胞的相互作用在弓形虫感染致不良妊娠结局中的作用机制研究

发布时间：2018-05-03 12:39

  本文选题：IL-12 + IL-12受体　； 参考：《滨州医学院》2014年硕士论文

【摘要】：目的：探索树突状细胞(DC)与蜕膜自然杀伤细胞(uNK)之间的相互作用在刚地弓形虫(Toxoplasma gondii)感染致不良妊娠结局中分子免疫机制。方法：体外纯化的来自正常妊娠的C57BL/6小鼠的uNK细胞和正常小鼠骨髓诱导而来的DCs以5：1的比例在弓形虫感染或不感染的情况下进行共培养24小时。设transwell小室隔开组和直接接触组。同时建立弓形虫感染C57BL/6孕鼠模型。体内将24只C57BL/6孕鼠按完全随机分组法分为感染组(12只)和对照组(12只),感染组小鼠于孕8天每只腹腔注射400个RH株刚地弓形虫速殖子,对照组注射等量无菌PBS,所有孕鼠均于孕14天时处死,观察胎盘及胎鼠的发育情况。用流式细胞仪分析uNK细胞上白细胞介素12受体(IL-12R)、抑制性受体NKG2A和活化性受体NKG2D的表达情况。用ELISA方法测定细胞培养上清和胎盘中IL-12和IFN-y的水平。共培养组并加设抗IL-12抗体组。用流式细胞仪检测uNK细胞的杀伤活性。结果：体内实验,在孕14d感染组小鼠的胎盘出血坏死较多,未感染的对照组小鼠胎盘血供丰富。感染组小鼠的死胎率比对照组小鼠显着增加(p0.01)。弓形虫感染后IL-12R、NKG2A和NKG2D的表达水平均上调,但活化性受体NKG2D比抑制性受体NKG2A升高更为明显。共培养组在弓形虫感染后NKG2D的表达水平及uNK细胞的细胞毒性比单独uNK细胞感染组升高明显,且共培养组上清液中的IL-12和IFN-y水平比单独的uNK细胞组、单纯DC组升高明显。在共培养组加入抗IL-12抗体时,IFN-γ的增强不明显。在感染孕鼠的胎盘中IL-12和IFN-y的水平较正常孕鼠组也显著增加。结论：弓形虫感染后,DC细胞通过分泌IL-12与uNK细胞表面的IL-12R相互作用,从而促进uNK细胞的活化、IFN-γ分泌增多和细胞毒性增强从而导致不良妊娠结局的发生,这可能是弓形虫感染致不良妊娠结局的重要的免疫机制之一。
[Abstract]:Aim: to explore the molecular immune mechanism of the interaction between dendritic cell (DC) and decidua natural killer (NK) in the adverse pregnancy outcome caused by Toxoplasma gondii infection (Toxoplasma gondii). Methods: purified uNK cells from normal pregnant C57BL/6 mice and DCs from bone marrow of normal mice were cultured for 24 hours with or without Toxoplasma gondii infection at 5:1. Transwell compartment group and direct contact group were established. At the same time, the pregnant rat model of C57BL/6 infection with Toxoplasma gondii was established. Twenty-four pregnant C57BL/6 mice were randomly divided into infected group (n = 12) and control group (n = 12). Four hundred Toxoplasma gondii Tachyzoites of RH strain were injected intraperitoneally in infected mice on the 8th day of gestation. All the pregnant rats were killed on the 14th day of gestation. The development of placenta and fetal mice were observed. The expression of interleukin-12 receptor (IL-12), inhibitory receptor (NKG2A) and activated receptor (NKG2D) in uNK cells were analyzed by flow cytometry. The levels of IL-12 and IFN-y in cell culture supernatant and placenta were measured by ELISA method. Co-culture group and anti-IL-12 antibody group were added. The cytotoxicity of uNK cells was detected by flow cytometry. Results: in vivo experiment, the placenta hemorrhage and necrosis were more in the infected mice on the 14th day of gestation, and the placental blood supply was abundant in the uninfected control group. The stillbirth rate of infected mice was significantly higher than that of control group (P 0.01). After Toxoplasma gondii infection, the expression of IL-12 RN NKG2A and NKG2D were up-regulated, but the activated receptor NKG2D was significantly higher than the inhibitory receptor NKG2A. The expression of NKG2D and the cytotoxicity of uNK cells in co-culture group were significantly higher than those in uNK cell group, and the levels of IL-12 and IFN-y in supernatant of co-culture group were significantly higher than those in uNK cell group and DC group. The increase of IFN- 纬 was not obvious when anti-IL-12 antibody was added in co-culture group. The levels of IL-12 and IFN-y in the placenta of infected pregnant mice were significantly higher than those of normal pregnant mice. Conclusion: after Toxoplasma gondii infection, DC cells secrete IL-12 and interact with IL-12R on the surface of uNK cells, thus promoting the activation of uNK cells to increase the secretion of IFN- 纬 and the enhancement of cytotoxicity, resulting in the occurrence of adverse pregnancy outcomes. This may be an important immune mechanism for adverse pregnancy outcomes caused by Toxoplasma gondii infection.
【学位授予单位】：滨州医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.251
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本文编号：1838516