本文選題：p38絲裂原活化蛋白激酶 + N-甲基-D-天冬氨酸　； 參考：《遼寧醫(yī)學(xué)院》2014年碩士論文

【摘要】：目的 觀察N-甲基-D-天門冬氨酸（NMDA）對(duì)原代培養(yǎng)的皮層神經(jīng)元損傷的影響，應(yīng)用NMDA受體（NMDAR）抑制劑MK801及p38絲裂原活化蛋白激酶（p38MAPK）抑制劑SB203580進(jìn)行干預(yù)，從信號(hào)轉(zhuǎn)導(dǎo)通路和凋亡因子兩方面來探討p38MAPK信號(hào)通路在神經(jīng)元損傷中的作用和機(jī)制。 方法 選擇NMDAR特異性激動(dòng)劑NMDA復(fù)制體外培養(yǎng)的神經(jīng)元損傷模型。培養(yǎng)7d的新生SD大鼠皮層神經(jīng)元，隨機(jī)分為5組：對(duì)照組、NMDA損傷組（NMDA50μmol/L）、MK801干預(yù)組（NMDA50μmol/L+MK80110μmol/L）、SB203580干預(yù)組（NMDA50μmol/L+SB20358010μmol/L）和聯(lián)合干預(yù)組（NMDA50μmol/L+MK801和SB203580各10μmol/L）。采用抗神經(jīng)元特異性稀醇化酶(NSE)免疫熒光化學(xué)染色鑒定神經(jīng)元，噻唑藍(lán)（MTT）比色實(shí)驗(yàn)評(píng)價(jià)細(xì)胞生存力，乳酸脫氫酶(LDH)釋放率實(shí)驗(yàn)測(cè)定細(xì)胞損傷程度，吖啶橙/溴化乙錠(AO/EB)雙重?zé)晒馊旧^察細(xì)胞凋亡形態(tài)和數(shù)目，免疫細(xì)胞化學(xué)染色法（IHC）和Western blot法檢測(cè)大鼠皮層神經(jīng)元各組p-p38MAPK，BCL-2和BAX表達(dá)情況。 結(jié)果 同對(duì)照組比較，NMDA損傷組神經(jīng)細(xì)胞細(xì)胞生存力顯著降低，培養(yǎng)上清液中LDH含量明顯增加，凋亡細(xì)胞顯著增多，p-p38MAPK、BAX蛋白表達(dá)明顯增加，而BCL-2蛋白表達(dá)顯著降低（P＜0.05，P＜0.01）；同NMDA損傷組比較，各個(gè)干預(yù)組細(xì)胞生存力提高，培養(yǎng)上清液中LDH釋放率降低，凋亡細(xì)胞減少，，p-p38MAPK及BAX的表達(dá)減少，而BCL-2表達(dá)增多（P＜0.05，P＜0.01），以聯(lián)合干預(yù)組效果最為明顯。 結(jié)論 NMDA可誘導(dǎo)皮質(zhì)神經(jīng)元損傷，MK801、SB203580對(duì)這種損傷具有保護(hù)作用，p38MAPK通路可能參與介導(dǎo)MK801的保護(hù)作用，其共同機(jī)制部分是通過抑制p38MAPK信號(hào)通路介導(dǎo)的凋亡相關(guān)蛋白實(shí)現(xiàn)的。
[Abstract]:Objective to observe the effect of NMDA on primary cultured cortical neuron injury and to observe the effects of NMDA receptor (NMDAR) inhibitor MK801 and p38 mitogen-activated protein kinase (p38MAPK) inhibitor SB203580. To explore the role and mechanism of p38 MAPK signaling pathway in neuronal injury from signal transduction pathway and apoptosis factor. Methods NMDAR specific agonist NMDA was used to induce neuronal injury in vitro. The cortical neurons of neonatal SD rats were cultured for 7 days and were randomly divided into five groups: control group (NMDA50 渭 mol / L) treated with NMDA50 渭 mol / L MK801 (NMDA50 渭 mol / L MK80110 渭 mol / L) and NMDA50 渭 mol / L SB20358010 渭 mol / L (NMDA50 渭 mol / L MK801 and SB203580 10 渭 mol / L each). The neuronal viability was evaluated by immunofluorescence staining against neuron specific dilute alcoholase (NSE), MTT assay and lactate dehydrogenase (LDH) release rate. Acridine orange / ethidium bromide double fluorescent staining was used to observe the morphology and number of apoptosis, and immunocytochemical staining (IHC) and Western blot were used to detect the expression of p-p38MAPKC-2 and Bax in rat cortical neurons. Results compared with the control group, the viability of NMDA injured neurons was significantly decreased, the content of LDH in culture supernatant was increased significantly, and the expression of p-p38 MAPKnBAX protein was significantly increased in apoptotic cells. The expression of BCL-2 protein was significantly decreased (P < 0.05, P < 0.01), the cell viability was increased, the LDH release rate was decreased, and the expression of p-p38 MAPK and Bax was decreased in the culture supernatant. The expression of BCL-2 increased (P < 0.05, P < 0.01), especially in the combined intervention group. Conclusion NMDA can induce cortical neuron injury. MK801 / SB203580 may play a protective role in mediating MK801. The common mechanism is partly through inhibition of apoptosis-related protein mediated by p38 MAPK signaling pathway. [WT5HZ] [WT5 "BZ] [WT5" BZ] [WT5 "BZ] [WT5" BZ] [WT5 "BZ] [WT5" BZ] [WT5 "BZ]
【學(xué)位授予單位】：遼寧醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R741

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