本文關鍵詞： 谷氨酸信號通路 淋巴細胞 白癜風　出處：《大連醫(yī)科大學》2011年碩士論文　論文類型：學位論文

【摘要】：研究背景: 已有研究發(fā)現(xiàn),淋巴細胞表達代謝型谷氨酸受體,并且他們在淋巴細胞功能中起著雙向調節(jié)作用,細胞通過谷氨酸信號通路調節(jié)免疫應答。研究還證實NMDA可以改變淋巴細胞內Ca2+通透性和活性氧簇的表達水平,且NMDA的這種作用是與淋巴細胞的活性密切相關的。用NMDA和白介素-2共同作用于淋巴細胞,使NMDAR1的表達升高�，F(xiàn)已證實NMDAR拮抗劑抑制淋巴細胞的增殖,并且這種作用是由于抑制了細胞的活性而引起的。以上現(xiàn)象都說明谷氨酸信號通路與免疫系統(tǒng)的功能有關。 白癜風是一種由于黑素細胞損害導致皮膚和粘膜色素脫失的疾病,其病因及發(fā)病機制迄今仍未闡明。目前主要學說有:黑素細胞自身破壞學說,自身免疫學說,神經化學因子學說和遺傳因素學說。隨著分子生物學和免疫學的發(fā)展,對白癜風自身免疫學說的研究日益增多。有研究表明:白癜風患者外周血單個核細胞釋放腫瘤壞死因子和干擾素減少,可能與白癜風患者皮損處炎癥反應減少有關。還有研究檢測患者血清中白介素- 2受體(IL-2R),發(fā)現(xiàn)在白癜風病人中,IL-2R的水平明顯高于正常人,白癜風患者的IL-2R水平與疾病的活動性相關,IL-2R水平可用來作為衡量白癜風的嚴重程度和進展情況的一個指標。以上現(xiàn)象提示淋巴細胞活性在白癜風發(fā)病機制中起重要作用。白癜風色素脫失可能與淋巴細胞內谷氨酸信號通路改變有關。有必要進一步探討谷氨酸信號通路在白癜風免疫學發(fā)病機制中的作用。 目的:探討谷氨酸信號通路在淋巴細胞及白癜風免疫發(fā)病機制的作用。 方法: 1.人外周血淋巴細胞的分離和培養(yǎng)。 2.流式細胞術測定離子型谷氨酸受體NMDAR的非競爭性拮抗劑MK801對淋巴細胞表面活化標志CD25的表達的影響,MK801對CD25+ IFN-γ+細胞的作用及NMDAR的激動劑NMDA和MK801對淋巴細胞內活性氧簇(ROS)水平的影響。 3. Real time PCR和流式細胞術方法測定白癜風患者和正常人淋巴細胞中的NMDAR1和NMDAR2A的表達差異。 4.免疫組化方法觀察白癜風患者和正常人皮損中離子型谷氨酸受體NMDAR1和NMDAR2A的表達。 結果: 1.流式細胞術發(fā)現(xiàn)外周血淋巴細胞經MK-801作用,使淋巴細胞內CD25的表達下降。 2. MK-801作用于淋巴細胞,使淋巴細胞內CD25+ IFN-γ+的細胞比例升高。 3.用NMDA作用于淋巴細胞,使細胞內ROS水平明顯升高。 4.流式細胞術分析發(fā)現(xiàn)白癜風患者外周血淋巴細胞中NMDAR1的表達比正常人高。 結論: 1.非競爭性離子型谷氨酸受體NMDAR的拮抗劑MK-801使淋巴細胞內CD25的表達降低,提示谷氨酸信號通路作用于淋巴細胞活化的早期。 2. MK-801使活化淋巴細胞的IFN-γ分泌量增加。 3.離子型谷氨酸受體NMDAR的激動劑NMDA可使淋巴細胞內的ROS水平升高。 4.白癜風患者PBL中NMDAR1的表達比正常人高。 5.穩(wěn)定期白癜風患者與正常人相比較,皮損的表皮細胞內谷氨酸受體NMDAR1和NMDAR2A的表達無差異。
[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.

【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R758.41

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