谷氨酸信号通路在淋巴细胞及白癜风免疫发病机制中的作用研究

发布时间：2018-03-01 19:28

本文关键词： 谷氨酸信号通路淋巴细胞白癜风　出处：《大连医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：研究背景: 已有研究发现,淋巴细胞表达代谢型谷氨酸受体,并且他们在淋巴细胞功能中起着双向调节作用,细胞通过谷氨酸信号通路调节免疫应答。研究还证实NMDA可以改变淋巴细胞内Ca2+通透性和活性氧簇的表达水平,且NMDA的这种作用是与淋巴细胞的活性密切相关的。用NMDA和白介素-2共同作用于淋巴细胞,使NMDAR1的表达升高。现已证实NMDAR拮抗剂抑制淋巴细胞的增殖,并且这种作用是由于抑制了细胞的活性而引起的。以上现象都说明谷氨酸信号通路与免疫系统的功能有关。白癜风是一种由于黑素细胞损害导致皮肤和粘膜色素脱失的疾病,其病因及发病机制迄今仍未阐明。目前主要学说有:黑素细胞自身破坏学说,自身免疫学说,神经化学因子学说和遗传因素学说。随着分子生物学和免疫学的发展,对白癜风自身免疫学说的研究日益增多。有研究表明:白癜风患者外周血单个核细胞释放肿瘤坏死因子和干扰素减少,可能与白癜风患者皮损处炎症反应减少有关。还有研究检测患者血清中白介素- 2受体(IL-2R),发现在白癜风病人中,IL-2R的水平明显高于正常人,白癜风患者的IL-2R水平与疾病的活动性相关,IL-2R水平可用来作为衡量白癜风的严重程度和进展情况的一个指标。以上现象提示淋巴细胞活性在白癜风发病机制中起重要作用。白癜风色素脱失可能与淋巴细胞内谷氨酸信号通路改变有关。有必要进一步探讨谷氨酸信号通路在白癜风免疫学发病机制中的作用。目的:探讨谷氨酸信号通路在淋巴细胞及白癜风免疫发病机制的作用。方法: 1.人外周血淋巴细胞的分离和培养。 2.流式细胞术测定离子型谷氨酸受体NMDAR的非竞争性拮抗剂MK801对淋巴细胞表面活化标志CD25的表达的影响,MK801对CD25+ IFN-γ+细胞的作用及NMDAR的激动剂NMDA和MK801对淋巴细胞内活性氧簇(ROS)水平的影响。 3. Real time PCR和流式细胞术方法测定白癜风患者和正常人淋巴细胞中的NMDAR1和NMDAR2A的表达差异。 4.免疫组化方法观察白癜风患者和正常人皮损中离子型谷氨酸受体NMDAR1和NMDAR2A的表达。结果: 1.流式细胞术发现外周血淋巴细胞经MK-801作用,使淋巴细胞内CD25的表达下降。 2. MK-801作用于淋巴细胞,使淋巴细胞内CD25+ IFN-γ+的细胞比例升高。 3.用NMDA作用于淋巴细胞,使细胞内ROS水平明显升高。 4.流式细胞术分析发现白癜风患者外周血淋巴细胞中NMDAR1的表达比正常人高。结论: 1.非竞争性离子型谷氨酸受体NMDAR的拮抗剂MK-801使淋巴细胞内CD25的表达降低,提示谷氨酸信号通路作用于淋巴细胞活化的早期。 2. MK-801使活化淋巴细胞的IFN-γ分泌量增加。 3.离子型谷氨酸受体NMDAR的激动剂NMDA可使淋巴细胞内的ROS水平升高。 4.白癜风患者PBL中NMDAR1的表达比正常人高。 5.稳定期白癜风患者与正常人相比较,皮损的表皮细胞内谷氨酸受体NMDAR1和NMDAR2A的表达无差异。
[Abstract]:Research background:
It has been found that the expression of metabotropic glutamate receptors in lymphocytes, and their lymphocyte function plays dual roles in regulating cell immune response through glutamate signaling. The study also confirmed that NMDA can change the expression level of lymphocyte Ca2+ permeability and reactive oxygen species, and this function of NMDA is related with lymphocyte activity. Using NMDA and interleukin -2 interaction in lymphocytes, the expression of NMDAR1 was increased. It has been proved that NMDAR antagonists inhibit lymphocyte proliferation, and this effect is due to inhibition of cellular activity. These phenomena have shown that glutamate signaling pathway and the immune system function.
Vitiligo is a melanocyte damage that leads to skin and mucosal depigmentation disease, its etiology and pathogenesis has not yet been elucidated. At present the main theories are: melanocyte destruction doctrine, autoimmune theory, theory of theory of chemical factors and genetic factors. With the development of molecular biology and immunology, more and more research on vitiligo itself immune theory increased. Studies have shown that: to reduce vitiligo patients peripheral blood mononuclear cells release tumor necrosis factor and interferon, and skin lesions of patients with vitiligo may reduce inflammatory response related. And detection of serum interleukin - 2 receptor (IL-2R), found in vitiligo patients, IL-2R levels were significantly higher than the normal IL-2R, activity level and disease in patients with vitiligo is related to the level of IL-2R may be used as a measure of severity and progression of vitiligo One of the indicators. These results suggest that lymphocytes play an important role in the pathogenesis of vitiligo. The depigmentation of vitiligo may and glutamate signaling pathways in lymphocyte change. It is necessary to further explore the role of glutamate signaling pathway in the pathogenesis of vitiligo in immunology.
Objective: To investigate the role of glutamate signaling pathway in the immune pathogenesis of lymphocyte and vitiligo.
Method:
The isolation and culture of peripheral blood lymphocytes from 1. people.
MK801 non competitive antagonist 2. flow cytometry determination of ionotropic glutamate receptor NMDAR on the lymphocyte surface activation marker expression of CD25, NMDAR and MK801 of CD25+ IFN- + cells gamma agonists NMDA and MK801 on reactive oxygen species in lymphocyte (ROS) levels.
3. Real time PCR and flow cytometry were used to determine the difference in the expression of NMDAR1 and NMDAR2A in the lymphocytes of patients with vitiligo and normal human lymphocytes.
4. immunohistochemical method was used to observe the expression of NMDAR1 and NMDAR2A in the skin lesions of patients with vitiligo and normal human skin.
Result:
1. flow cytometry found the effect of MK-801 on peripheral blood lymphocytes, which decreased the expression of CD25 in lymphocytes.
2. MK-801 acts on lymphocytes and increases the proportion of CD25+ IFN- gamma + cells in lymphocytes.
3. the effect of NMDA on the lymphocyte and the level of ROS in the cells increased significantly.
The analysis of 4. flow cytometry showed that the expression of NMDAR1 in peripheral blood lymphocytes of patients with vitiligo was higher than that of normal people.
Conclusion:
1., MK-801, a noncompetitive glutamic acid receptor NMDAR antagonist, reduced the expression of CD25 in lymphocytes, suggesting that glutamate signaling pathway acts on the early stage of lymphocyte activation.
2. MK-801 increased the secretion of IFN- gamma in activated lymphocytes.
The agonist NMDA of the 3. ionotropic glutamate receptor NMDAR can increase the level of ROS in the lymphocyte.
4. the expression of NMDAR1 in PBL was higher in patients with vitiligo than in normal people.
5. there was no difference in the expression of glutamate receptor NMDAR1 and NMDAR2A in the epidermis of the epidermis of the epidermis of the epidermis of the skin lesions.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R758.41

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