白细胞介素7及其受体通路异常在多发性硬化症自然杀伤细胞中的研究

发布时间：2018-07-11 16:56

本文选题：多发性硬化 + 自然杀伤细胞　；参考：《天津医科大学》2014年硕士论文

【摘要】：目的：最新研究表明编码IL-7Ra的单核苷酸多肽性(single nucleotide polymorphisms, SNP) rs6897932位点基因型‘C’与多发性硬化症(Multiple Sclerosis, MS)易感性相关。研究证实IL-7/IL-7Ra通路对CD4+、CD8+T细胞、调节性T细胞(Regulatory T cells, Treg)的异常调控参与MS疾病的病理发展,但此通路在MS患者自然杀伤细胞(Natural Killer, NK)中的研究尚未见报导。本研究目的在于探索IL-7/IL-7Ra通路在MS患者外周血NK细胞中是否异常及其生物学作用,包括对NK细胞分泌细胞因子的能力、杀伤肿瘤细胞的能力、增殖功能和抗凋亡能力的调节,并探索其可能的作用机制,同时分析该通路与MS病人NK细胞的功能缺陷的相关性,据此提出MS治疗的新思路。方法：收集MS病人和健康对照的外周血,提取血清,用酶联免疫吸附测定法(Enzyme-linked immunosorbent assay, ELISA)检测细胞因子IL-2、IL-7、IL-15和IL-21的水平；从MS病人和健康对照的外周血中提取单个核细胞,在体外用人IL-7刺激培养,用流式细胞仪检测NK细胞表面IL-7Ra (CD127)的表达及NK细胞内IFN-y的分泌水平；用磁珠分离纯化NK细胞,通过检测乳酸脱氢酶的浓度来鉴定其杀伤肿瘤细胞的能力；用溴脱氧尿嘧啶核苷(Bromodeoxyuridine, BrdU)标记增殖的细胞,通过流式细胞术检测NK细胞的增殖；以7-氨基放线菌素D染色(7-amino-actinomycin D staining,7-AAD)和膜联蛋白(Annexin V)标记调亡或死亡的细胞,检测存活的细胞比例和细胞内抗凋亡因子2-B细胞淋巴瘤(B-cell lymphoma2, Bcl-2)的表达水平。结果：MS病人血清IL-7水平比健康对照显著降低；NK细胞表面表达CD127, MS病人的NK细胞表面CD127的表达水平比健康对照组高；经IL-7刺激后,MS病人和健康对照的NK细胞表达的CD127水平均下降,其中以MS病人的下降幅度明显,在CD3-CD56bright和CD3-CD56dim两个NK细胞亚群都有所体现；CD3-CD56brightNK细胞亚群经IL-7刺激后,MS患者中分泌细胞因子IFN-y的NK细胞比例增加,同时平均每个NK细胞分泌的IFN-γ量显著增加,CD3-CD56dimNK细胞亚群中此刺激效应不显著；经IL-7刺激后,来自MS病人的NK细胞杀伤K562肿瘤细胞的能力增强,体现在以效应细胞和靶细胞比例为2.5：1、5：1、10：1时,MS病人的NK细胞杀伤肿瘤细胞增多；经IL-7刺激后,CD3-CD56brightBrdU+和CD3-CD56dimBrdU+增殖的NK细胞比例在MS病人和健康对照组中没有显著差别；Annexin V-7-AAD-标记的存活的细胞比例在MS病人的CD3-CD56brightNK细胞亚群中显著增加,并且抗凋亡因子Bcl-2表达强度在MS病人的CD3-CD56brightNK细胞亚群中显著增强,而CD3-CD56dimNK细胞亚群变化不显著。结论： 1、IL-7/IL-7Ra通路在MS病人NK细胞中被异常激活； 2、与健康对照相比,IL-7可更大程度上增强MS病人NK细胞分泌细胞因子IFN-y的能力和杀伤肿瘤细胞的能力,IL-7可以通过上调抗凋亡因子Bcl-2的表达来促进NK细胞的存活；但IL-7对MS病人NK细胞的增殖作用不明显。 3、IL-7/IL-7Ra通路对MS病人的NK细胞尤其是CD56bright细胞亚群具有重要的调节作用,推测IL-7/IL-7Ra通路的异常导致了MS病人中NK细胞的功能缺陷,从而进一步促进MS的病理改变。因此靶向针对MS病人NK细胞的IL-7/IL-7Ra通路对MS的治疗具有潜在的价值。
[Abstract]:Objective: the latest research shows that the single nucleotide polymorphisms (SNP) rs6897932 locus genotype 'C' is associated with the susceptibility to multiple sclerosis (Multiple Sclerosis, MS) in IL-7Ra. The purpose of this study is to explore the abnormal and biological effects of IL-7/IL-7Ra pathway in the peripheral blood NK cells of patients with MS, including the ability to secrete NK cell factors and kill the tumor cells in the peripheral blood NK cells of patients with MS. The purpose of this study is not to be reported in the pathological development of MS disease. The purpose of this study is to explore the abnormal and biological effects of IL-7/IL-7Ra pathway in the peripheral blood NK cells of MS patients. The regulation of capacity, proliferation and anti apoptosis ability and the possible mechanism of action are explored, and the correlation between the pathway and the functional defects of NK cells in MS patients is analyzed, and a new idea of MS therapy is proposed accordingly.
Methods: the peripheral blood of MS patients and healthy controls was collected and serum was extracted. The levels of cytokine IL-2, IL-7, IL-15 and IL-21 were detected by Enzyme-linked immunosorbent assay (ELISA). The single nucleus cells were extracted from the peripheral blood of the MS patients and the healthy controls, and were cultured in vitro by human IL-7, and used flow cells in vitro. The expression of IL-7Ra (CD127) on the surface of NK cells and the secretion level of IFN-y in NK cells were detected by the instrument. NK cells were isolated and purified by magnetic beads. The ability to kill tumor cells was identified by detecting the concentration of lactate dehydrogenase, and the proliferating cells were labeled with bromodeoxyuridine (Bromodeoxyuridine, BrdU), and NK fine was detected by flow cytometry. Cell proliferation, 7- amino actinomycin D staining (7-amino-actinomycin D staining, 7-AAD) and membrane associated protein (Annexin V) were used to mark the cells of the dead or dead cells to detect the proportion of surviving cells and the expression level of the intracellular anti apoptotic factor 2-B cell lymphoma (B-cell lymphoma2, Bcl-2).
Results: the serum IL-7 level of MS patients was significantly lower than that of the healthy controls; the expression of CD127 on the surface of NK cells and the expression level of CD127 on the surface of NK cells in MS patients were higher than those in the healthy control group. After IL-7 stimulation, the CD127 level of the MS patients and the NK cells in the healthy controls were all decreased, and the decrease of the MS patients was obvious. CD3-CD56dim two NK cell subsets were all reflected; the proportion of NK cells secreted cytokine IFN-y in MS patients increased after IL-7 stimulation, and the average of IFN- gamma secreted by each NK cell increased significantly, and this stimulation effect in CD3-CD56dimNK cell subgroups should not be significant; IL-7 stimulation, from MS patients The ability of K cells to kill K562 tumor cells is enhanced, which is reflected in the increase of NK cells in the MS patients when the proportion of the effect cells and target cells is 2.5:1,5:1,10:1; after IL-7 stimulation, the proportion of NK cell proliferation of CD3-CD56brightBrdU+ and CD3-CD56dimBrdU+ is not significantly different between the MS patients and the healthy control group; Annexin The percentage of surviving cells marked by V-7-AAD- increased significantly in the CD3-CD56brightNK cell subgroup of MS patients, and the expression intensity of anti apoptotic factor Bcl-2 was significantly enhanced in the CD3-CD56brightNK cell subgroup of MS patients, while the changes in the CD3-CD56dimNK cell subgroup were not significant.
Conclusion:
1, the IL-7/IL-7Ra pathway was activated in NK cells of MS patients.
2, compared with the healthy control, IL-7 could enhance the ability of NK cells to secrete cytokine IFN-y and the ability to kill the tumor cells in MS patients. IL-7 can promote the survival of NK cells by up regulation of the expression of anti apoptotic factor Bcl-2, but IL-7 does not use an obvious effect on the proliferation of NK cells in MS patients.
3, the IL-7/IL-7Ra pathway plays an important role in regulating the NK cells in MS patients, especially the CD56bright cell subsets. It is speculated that the abnormalities of the IL-7/IL-7Ra pathway lead to the functional defects of NK cells in the MS patients, thus further promoting the pathological changes of MS. Therefore, the IL-7/IL-7Ra pathway aimed at the NK cells of the MS patients has potential for the treatment of MS. Value.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R744.51

【共引文献】