本文選題：多發(fā)性硬化 + 自然殺傷細(xì)胞　； 參考：《天津醫(yī)科大學(xué)》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(diǎn)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(shí)IL-7/IL-7Ra通路對(duì)CD4+、CD8+T細(xì)胞、調(diào)節(jié)性T細(xì)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(xì)胞(Natural Killer, NK)中的研究尚未見(jiàn)報(bào)導(dǎo)。本研究目的在于探索IL-7/IL-7Ra通路在MS患者外周血NK細(xì)胞中是否異常及其生物學(xué)作用,包括對(duì)NK細(xì)胞分泌細(xì)胞因子的能力、殺傷腫瘤細(xì)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(jī)制,同時(shí)分析該通路與MS病人NK細(xì)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(duì)照的外周血,提取血清,用酶聯(lián)免疫吸附測(cè)定法(Enzyme-linked immunosorbent assay, ELISA)檢測(cè)細(xì)胞因子IL-2、IL-7、IL-15和IL-21的水平；從MS病人和健康對(duì)照的外周血中提取單個(gè)核細(xì)胞,在體外用人IL-7刺激培養(yǎng),用流式細(xì)胞儀檢測(cè)NK細(xì)胞表面IL-7Ra (CD127)的表達(dá)及NK細(xì)胞內(nèi)IFN-y的分泌水平；用磁珠分離純化NK細(xì)胞,通過(guò)檢測(cè)乳酸脫氫酶的濃度來(lái)鑒定其殺傷腫瘤細(xì)胞的能力；用溴脫氧尿嘧啶核苷(Bromodeoxyuridine, BrdU)標(biāo)記增殖的細(xì)胞,通過(guò)流式細(xì)胞術(shù)檢測(cè)NK細(xì)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(biāo)記調(diào)亡或死亡的細(xì)胞,檢測(cè)存活的細(xì)胞比例和細(xì)胞內(nèi)抗凋亡因子2-B細(xì)胞淋巴瘤(B-cell lymphoma2, Bcl-2)的表達(dá)水平。 結(jié)果：MS病人血清IL-7水平比健康對(duì)照顯著降低；NK細(xì)胞表面表達(dá)CD127, MS病人的NK細(xì)胞表面CD127的表達(dá)水平比健康對(duì)照組高；經(jīng)IL-7刺激后,MS病人和健康對(duì)照的NK細(xì)胞表達(dá)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(gè)NK細(xì)胞亞群都有所體現(xiàn)；CD3-CD56brightNK細(xì)胞亞群經(jīng)IL-7刺激后,MS患者中分泌細(xì)胞因子IFN-y的NK細(xì)胞比例增加,同時(shí)平均每個(gè)NK細(xì)胞分泌的IFN-γ量顯著增加,CD3-CD56dimNK細(xì)胞亞群中此刺激效應(yīng)不顯著；經(jīng)IL-7刺激后,來(lái)自MS病人的NK細(xì)胞殺傷K562腫瘤細(xì)胞的能力增強(qiáng),體現(xiàn)在以效應(yīng)細(xì)胞和靶細(xì)胞比例為2.5：1、5：1、10：1時(shí),MS病人的NK細(xì)胞殺傷腫瘤細(xì)胞增多；經(jīng)IL-7刺激后,CD3-CD56brightBrdU+和CD3-CD56dimBrdU+增殖的NK細(xì)胞比例在MS病人和健康對(duì)照組中沒(méi)有顯著差別；Annexin V-7-AAD-標(biāo)記的存活的細(xì)胞比例在MS病人的CD3-CD56brightNK細(xì)胞亞群中顯著增加,并且抗凋亡因子Bcl-2表達(dá)強(qiáng)度在MS病人的CD3-CD56brightNK細(xì)胞亞群中顯著增強(qiáng),而CD3-CD56dimNK細(xì)胞亞群變化不顯著。 結(jié)論： 1、IL-7/IL-7Ra通路在MS病人NK細(xì)胞中被異常激活； 2、與健康對(duì)照相比,IL-7可更大程度上增強(qiáng)MS病人NK細(xì)胞分泌細(xì)胞因子IFN-y的能力和殺傷腫瘤細(xì)胞的能力,IL-7可以通過(guò)上調(diào)抗凋亡因子Bcl-2的表達(dá)來(lái)促進(jìn)NK細(xì)胞的存活；但I(xiàn)L-7對(duì)MS病人NK細(xì)胞的增殖作用不明顯。 3、IL-7/IL-7Ra通路對(duì)MS病人的NK細(xì)胞尤其是CD56bright細(xì)胞亞群具有重要的調(diào)節(jié)作用,推測(cè)IL-7/IL-7Ra通路的異常導(dǎo)致了MS病人中NK細(xì)胞的功能缺陷,從而進(jìn)一步促進(jìn)MS的病理改變。因此靶向針對(duì)MS病人NK細(xì)胞的IL-7/IL-7Ra通路對(duì)MS的治療具有潛在的價(jià)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(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R744.51

【共引文獻(xiàn)】

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