本文關(guān)鍵詞： 氧糖剝奪再灌注 Src激酶 磷酸化 去磷酸化　出處：《中南大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：探討缺血再灌注過(guò)程神經(jīng)細(xì)胞內(nèi)Src激酶的表達(dá)變化和活性改變,探討其可能的機(jī)制和意義。 方法：以小鼠神經(jīng)瘤母細(xì)胞株N2a細(xì)胞為研究對(duì)象,給予氧糖剝奪再灌注干預(yù),體外模擬缺血再灌注過(guò)程,采用MTT法檢測(cè)細(xì)胞活性,應(yīng)用免疫細(xì)胞化學(xué)方法觀察Src激酶在細(xì)胞內(nèi)定位及表達(dá),應(yīng)用QRT PCR檢測(cè)Src mRNA的含量變化,同時(shí)采用Western blot方法檢測(cè)Src總蛋白的表達(dá)及其不同位點(diǎn)Tyr527和Tyr416的磷酸化水平。 結(jié)果： 1.MTT結(jié)果提示,氧糖剝奪4小時(shí)后N2a細(xì)胞活性較同期正常組稍有減低,但差異不具有統(tǒng)計(jì)學(xué)意義；再灌注早期,再灌注6小時(shí)組和再灌注12小時(shí)組的細(xì)胞活性較同期正常組降低,差異均具有統(tǒng)計(jì)學(xué)差異(P0.05)；再灌注后期即再灌注24小時(shí)組,細(xì)胞活性較正常組出現(xiàn)顯著下降,差異具有顯著性統(tǒng)計(jì)差異(P0.01)。 2.免疫細(xì)胞化學(xué)結(jié)果提示,Src蛋白定位于N2a細(xì)胞的胞漿內(nèi),圍繞在細(xì)胞核周?chē)�。氧糖剝奪過(guò)程中,Src蛋白的表達(dá)分布無(wú)明顯變化,陽(yáng)性反應(yīng)呈淺棕色或棕色。而再灌注過(guò)程中,Src蛋白的陽(yáng)性表達(dá)在再灌注12小時(shí)和24小時(shí)出現(xiàn)增多,部分陽(yáng)性反應(yīng)呈深棕色。 3. QRT PCR結(jié)果提示,與正常組比較,缺血4小時(shí)組和再灌注6小時(shí)組Src mRNA的表達(dá)出現(xiàn)減低,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。隨著再灌注時(shí)間的延長(zhǎng),Src mRNA的表達(dá)逐漸回升,其中再灌注24小時(shí)組Src mRNA的表達(dá)明顯上調(diào),與正常組對(duì)比,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。 4. Western blot結(jié)果顯示,與正常組比較,氧糖剝奪4小時(shí)組、再灌注6小時(shí)組和12小時(shí)組的Src總蛋白表達(dá)水平無(wú)明顯改變(P0.05),再灌注24小時(shí)組Src總蛋白的表達(dá)較正常組出現(xiàn)增多,其差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。另一方面,不同位點(diǎn)發(fā)生磷酸化的Src蛋白則變化不同。與正常組相比,Tyr527位點(diǎn)發(fā)生磷酸化的Src在氧糖剝奪4小時(shí)組和再灌注6小時(shí)組的表達(dá)無(wú)明顯變化,在再灌注12小時(shí)組和再灌注24小時(shí)組則出現(xiàn)了明顯的表達(dá)下調(diào),差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。同樣與正常組比較,Tyr416位點(diǎn)發(fā)生磷酸化的Src蛋白則在氧糖剝奪4小時(shí)組的表達(dá)無(wú)明顯變化,而在再灌注6小時(shí)組、12小時(shí)組和24小時(shí)組的表達(dá)均出現(xiàn)了上調(diào),差異均具有統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論： 1.缺血再灌注損傷引起Src激酶的表達(dá)上調(diào)； 2.缺血再灌注損傷引起Src激酶的活化,與Tyr416位點(diǎn)磷酸化和Tyr527位點(diǎn)的去磷酸化均有關(guān)。
[Abstract]:Aim: to investigate the expression and activity of Src kinase in neuronal cells during ischemia reperfusion, and to explore its possible mechanism and significance. Methods: the mouse neuroblastoma cell line N2a was treated with oxygen glucose deprivation and reperfusion. The activity of N2a cells was determined by MTT assay. The localization and expression of Src kinase in cells were observed by immunocytochemistry, the content of Src mRNA was detected by QRT PCR, and the expression of Src total protein and the phosphorylation levels of Tyr527 and Tyr416 at different sites were detected by Western blot method. Results:. 1. The MTT results showed that the N2a cell activity was slightly lower than that in the control group after 4 hours of oxygen glucose deprivation, but the difference was not statistically significant, and in the early reperfusion period, the activity of N2a cells in the 6-hour reperfusion group and 12-hour reperfusion group was lower than that in the normal group. The difference was statistically significant (P 0.05), and the cell activity in the late reperfusion group was significantly lower than that in the normal group (P 0.01). 2. The immunocytochemical results showed that the SRC protein was located in the cytoplasm of N2a cells and surrounded the nucleus. The positive reaction was light brown or brown, while the positive expression of Src protein increased at 12 and 24 hours after reperfusion, and some of the positive reactions were dark brown. 3. The results of QRT PCR showed that the expression of Src mRNA decreased in the 4-hour ischemia group and the 6-hour reperfusion group compared with the normal group, and the difference was statistically significant (P 0.05). The expression of SRC mRNA increased gradually with the prolongation of the reperfusion time. The expression of Src mRNA was up-regulated in 24 h reperfusion group, and the difference was statistically significant compared with the normal group (P 0.05). 4. The results of Western blot showed that compared with the normal group, the expression of total Src protein in the 4-hour oxygen glucose deprivation group, the 6-hour reperfusion group and the 12-hour reperfusion group had no significant changes, but the expression of total Src protein in the 24-hour reperfusion group was higher than that in the normal group. The difference was statistically significant (P 0.05). On the other hand, the phosphorylation of Src protein at different sites was different. There was no significant change in the expression of Src phosphorylated at site 527 of oxygen deprivation for 4 hours and reperfusion for 6 hours, compared with the normal group. After 12 hours of reperfusion and 24 hours of reperfusion, there was a significant down-regulation of the expression of Src protein, and the difference was statistically significant (P 0.05). The expression of phosphorylated Src protein at the site of Tyr416 was similar to that in the control group, but there was no significant change in the expression of Src protein in the 4-hour group of oxygen glucose deprivation. However, the expression of P0. 05 was up-regulated in both 12 h and 24 h groups in 6 h reperfusion group, and the difference was statistically significant (P 0. 05%, P 0. 05%, P 0. 05%, P 0. 05%). Conclusion:. 1. The expression of Src kinase was up-regulated by ischemia-reperfusion injury. 2. The activation of Src kinase induced by ischemia reperfusion injury is related to the phosphorylation of Tyr416 site and dephosphorylation of Tyr527 site.
【學(xué)位授予單位】：中南大學(xué)
【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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