本文關(guān)鍵詞： 谷氨酸信號(hào)通路 淋巴細(xì)胞 白癜風(fēng)　出處：《大連醫(yī)科大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景: 已有研究發(fā)現(xiàn),淋巴細(xì)胞表達(dá)代謝型谷氨酸受體,并且他們?cè)诹馨图?xì)胞功能中起著雙向調(diào)節(jié)作用,細(xì)胞通過谷氨酸信號(hào)通路調(diào)節(jié)免疫應(yīng)答。研究還證實(shí)NMDA可以改變淋巴細(xì)胞內(nèi)Ca2+通透性和活性氧簇的表達(dá)水平,且NMDA的這種作用是與淋巴細(xì)胞的活性密切相關(guān)的。用NMDA和白介素-2共同作用于淋巴細(xì)胞,使NMDAR1的表達(dá)升高�，F(xiàn)已證實(shí)NMDAR拮抗劑抑制淋巴細(xì)胞的增殖,并且這種作用是由于抑制了細(xì)胞的活性而引起的。以上現(xiàn)象都說明谷氨酸信號(hào)通路與免疫系統(tǒng)的功能有關(guān)。 白癜風(fēng)是一種由于黑素細(xì)胞損害導(dǎo)致皮膚和粘膜色素脫失的疾病,其病因及發(fā)病機(jī)制迄今仍未闡明。目前主要學(xué)說有:黑素細(xì)胞自身破壞學(xué)說,自身免疫學(xué)說,神經(jīng)化學(xué)因子學(xué)說和遺傳因素學(xué)說。隨著分子生物學(xué)和免疫學(xué)的發(fā)展,對(duì)白癜風(fēng)自身免疫學(xué)說的研究日益增多。有研究表明:白癜風(fēng)患者外周血單個(gè)核細(xì)胞釋放腫瘤壞死因子和干擾素減少,可能與白癜風(fēng)患者皮損處炎癥反應(yīng)減少有關(guān)。還有研究檢測(cè)患者血清中白介素- 2受體(IL-2R),發(fā)現(xiàn)在白癜風(fēng)病人中,IL-2R的水平明顯高于正常人,白癜風(fēng)患者的IL-2R水平與疾病的活動(dòng)性相關(guān),IL-2R水平可用來作為衡量白癜風(fēng)的嚴(yán)重程度和進(jìn)展情況的一個(gè)指標(biāo)。以上現(xiàn)象提示淋巴細(xì)胞活性在白癜風(fēng)發(fā)病機(jī)制中起重要作用。白癜風(fēng)色素脫失可能與淋巴細(xì)胞內(nèi)谷氨酸信號(hào)通路改變有關(guān)。有必要進(jìn)一步探討谷氨酸信號(hào)通路在白癜風(fēng)免疫學(xué)發(fā)病機(jī)制中的作用。 目的:探討谷氨酸信號(hào)通路在淋巴細(xì)胞及白癜風(fēng)免疫發(fā)病機(jī)制的作用。 方法: 1.人外周血淋巴細(xì)胞的分離和培養(yǎng)。 2.流式細(xì)胞術(shù)測(cè)定離子型谷氨酸受體NMDAR的非競(jìng)爭(zhēng)性拮抗劑MK801對(duì)淋巴細(xì)胞表面活化標(biāo)志CD25的表達(dá)的影響,MK801對(duì)CD25+ IFN-γ+細(xì)胞的作用及NMDAR的激動(dòng)劑NMDA和MK801對(duì)淋巴細(xì)胞內(nèi)活性氧簇(ROS)水平的影響。 3. Real time PCR和流式細(xì)胞術(shù)方法測(cè)定白癜風(fēng)患者和正常人淋巴細(xì)胞中的NMDAR1和NMDAR2A的表達(dá)差異。 4.免疫組化方法觀察白癜風(fēng)患者和正常人皮損中離子型谷氨酸受體NMDAR1和NMDAR2A的表達(dá)。 結(jié)果: 1.流式細(xì)胞術(shù)發(fā)現(xiàn)外周血淋巴細(xì)胞經(jīng)MK-801作用,使淋巴細(xì)胞內(nèi)CD25的表達(dá)下降。 2. MK-801作用于淋巴細(xì)胞,使淋巴細(xì)胞內(nèi)CD25+ IFN-γ+的細(xì)胞比例升高。 3.用NMDA作用于淋巴細(xì)胞,使細(xì)胞內(nèi)ROS水平明顯升高。 4.流式細(xì)胞術(shù)分析發(fā)現(xiàn)白癜風(fēng)患者外周血淋巴細(xì)胞中NMDAR1的表達(dá)比正常人高。 結(jié)論: 1.非競(jìng)爭(zhēng)性離子型谷氨酸受體NMDAR的拮抗劑MK-801使淋巴細(xì)胞內(nèi)CD25的表達(dá)降低,提示谷氨酸信號(hào)通路作用于淋巴細(xì)胞活化的早期。 2. MK-801使活化淋巴細(xì)胞的IFN-γ分泌量增加。 3.離子型谷氨酸受體NMDAR的激動(dòng)劑NMDA可使淋巴細(xì)胞內(nèi)的ROS水平升高。 4.白癜風(fēng)患者PBL中NMDAR1的表達(dá)比正常人高。 5.穩(wěn)定期白癜風(fēng)患者與正常人相比較,皮損的表皮細(xì)胞內(nèi)谷氨酸受體NMDAR1和NMDAR2A的表達(dá)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(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R758.41

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