本文選題：多發(fā)性硬化 + 自然殺傷細胞��； 參考：《天津醫(yī)科大學》2014年碩士論文

【摘要】：目的：最新研究表明編碼IL-7Ra的單核苷酸多肽性(single nucleotide polymorphisms, SNP) rs6897932位點基因型‘C’與多發(fā)性硬化癥(Multiple Sclerosis, MS)易感性相關(guān)。研究證實IL-7/IL-7Ra通路對CD4+、CD8+T細胞、調(diào)節(jié)性T細胞(Regulatory T cells, Treg)的異常調(diào)控參與MS疾病的病理發(fā)展,但此通路在MS患者自然殺傷細胞(Natural Killer, NK)中的研究尚未見報導。本研究目的在于探索IL-7/IL-7Ra通路在MS患者外周血NK細胞中是否異常及其生物學作用,包括對NK細胞分泌細胞因子的能力、殺傷腫瘤細胞的能力、增殖功能和抗凋亡能力的調(diào)節(jié),并探索其可能的作用機制,同時分析該通路與MS病人NK細胞的功能缺陷的相關(guān)性,據(jù)此提出MS治療的新思路。 方法：收集MS病人和健康對照的外周血,提取血清,用酶聯(lián)免疫吸附測定法(Enzyme-linked immunosorbent assay, ELISA)檢測細胞因子IL-2、IL-7、IL-15和IL-21的水平；從MS病人和健康對照的外周血中提取單個核細胞,在體外用人IL-7刺激培養(yǎng),用流式細胞儀檢測NK細胞表面IL-7Ra (CD127)的表達及NK細胞內(nèi)IFN-y的分泌水平；用磁珠分離純化NK細胞,通過檢測乳酸脫氫酶的濃度來鑒定其殺傷腫瘤細胞的能力；用溴脫氧尿嘧啶核苷(Bromodeoxyuridine, BrdU)標記增殖的細胞,通過流式細胞術(shù)檢測NK細胞的增殖；以7-氨基放線菌素D染色(7-amino-actinomycin D staining,7-AAD)和膜聯(lián)蛋白(Annexin V)標記調(diào)亡或死亡的細胞,檢測存活的細胞比例和細胞內(nèi)抗凋亡因子2-B細胞淋巴瘤(B-cell lymphoma2, Bcl-2)的表達水平。 結(jié)果：MS病人血清IL-7水平比健康對照顯著降低；NK細胞表面表達CD127, MS病人的NK細胞表面CD127的表達水平比健康對照組高；經(jīng)IL-7刺激后,MS病人和健康對照的NK細胞表達的CD127水平均下降,其中以MS病人的下降幅度明顯,在CD3-CD56bright和CD3-CD56dim兩個NK細胞亞群都有所體現(xiàn)；CD3-CD56brightNK細胞亞群經(jīng)IL-7刺激后,MS患者中分泌細胞因子IFN-y的NK細胞比例增加,同時平均每個NK細胞分泌的IFN-γ量顯著增加,CD3-CD56dimNK細胞亞群中此刺激效應不顯著；經(jīng)IL-7刺激后,來自MS病人的NK細胞殺傷K562腫瘤細胞的能力增強,體現(xiàn)在以效應細胞和靶細胞比例為2.5：1、5：1、10：1時,MS病人的NK細胞殺傷腫瘤細胞增多；經(jīng)IL-7刺激后,CD3-CD56brightBrdU+和CD3-CD56dimBrdU+增殖的NK細胞比例在MS病人和健康對照組中沒有顯著差別；Annexin V-7-AAD-標記的存活的細胞比例在MS病人的CD3-CD56brightNK細胞亞群中顯著增加,并且抗凋亡因子Bcl-2表達強度在MS病人的CD3-CD56brightNK細胞亞群中顯著增強,而CD3-CD56dimNK細胞亞群變化不顯著。 結(jié)論： 1、IL-7/IL-7Ra通路在MS病人NK細胞中被異常激活； 2、與健康對照相比,IL-7可更大程度上增強MS病人NK細胞分泌細胞因子IFN-y的能力和殺傷腫瘤細胞的能力,IL-7可以通過上調(diào)抗凋亡因子Bcl-2的表達來促進NK細胞的存活；但IL-7對MS病人NK細胞的增殖作用不明顯。 3、IL-7/IL-7Ra通路對MS病人的NK細胞尤其是CD56bright細胞亞群具有重要的調(diào)節(jié)作用,推測IL-7/IL-7Ra通路的異常導致了MS病人中NK細胞的功能缺陷,從而進一步促進MS的病理改變。因此靶向針對MS病人NK細胞的IL-7/IL-7Ra通路對MS的治療具有潛在的價值。
[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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R744.51

【共引文獻】

相關(guān)期刊論文 前2條

1 蘇寧;施福東;;白細胞介素7對多發(fā)性硬化癥患者不同亞群自然殺傷細胞的影響[J];山東醫(yī)藥;2014年32期

2 朱玉靜;張繆佳;柯瑤;吳云娟;;RA患者IL-7檢測及其臨床意義的初步探討[J];中國現(xiàn)代藥物應用;2013年22期

相關(guān)博士學位論文 前1條

1 張曉穎;鋅指蛋白A20在急性髓性白血病樹突狀細胞中的作用[D];中國人民解放軍軍事醫(yī)學科學院;2013年

相關(guān)碩士學位論文 前4條

1 趙楠;白細胞介素21增強CIK細胞抗白血病作用的研究[D];天津醫(yī)科大學;2013年

2 黨靜;不明原因復發(fā)性自然流產(chǎn)中Tim-3-Gal-9通路介導的免疫失衡機制研究[D];華中科技大學;2013年

3 王曉松;IL-7R基因單核苷酸多態(tài)性，，sIL-7R血清水平與系統(tǒng)性紅斑狼瘡的相關(guān)性研究[D];安徽醫(yī)科大學;2014年

4 馮鳳蘭;白細胞介素7剪接變異體的克隆及其表達料[D];廣東藥學院;2012年

本文編號：2115949