氧糖剥夺再灌注后Src激酶的变化及其可能机制的探讨

发布时间：2018-02-11 04:08

本文关键词： 氧糖剥夺再灌注 Src激酶磷酸化去磷酸化　出处：《中南大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：探讨缺血再灌注过程神经细胞内Src激酶的表达变化和活性改变,探讨其可能的机制和意义。方法：以小鼠神经瘤母细胞株N2a细胞为研究对象,给予氧糖剥夺再灌注干预,体外模拟缺血再灌注过程,采用MTT法检测细胞活性,应用免疫细胞化学方法观察Src激酶在细胞内定位及表达,应用QRT PCR检测Src mRNA的含量变化,同时采用Western blot方法检测Src总蛋白的表达及其不同位点Tyr527和Tyr416的磷酸化水平。结果： 1.MTT结果提示,氧糖剥夺4小时后N2a细胞活性较同期正常组稍有减低,但差异不具有统计学意义；再灌注早期,再灌注6小时组和再灌注12小时组的细胞活性较同期正常组降低,差异均具有统计学差异(P0.05)；再灌注后期即再灌注24小时组,细胞活性较正常组出现显著下降,差异具有显著性统计差异(P0.01)。 2.免疫细胞化学结果提示,Src蛋白定位于N2a细胞的胞浆内,围绕在细胞核周围。氧糖剥夺过程中,Src蛋白的表达分布无明显变化,阳性反应呈浅棕色或棕色。而再灌注过程中,Src蛋白的阳性表达在再灌注12小时和24小时出现增多,部分阳性反应呈深棕色。 3. QRT PCR结果提示,与正常组比较,缺血4小时组和再灌注6小时组Src mRNA的表达出现减低,差异有统计学意义(P0.05)。随着再灌注时间的延长,Src mRNA的表达逐渐回升,其中再灌注24小时组Src mRNA的表达明显上调,与正常组对比,差异具有统计学意义(P0.05)。 4. Western blot结果显示,与正常组比较,氧糖剥夺4小时组、再灌注6小时组和12小时组的Src总蛋白表达水平无明显改变(P0.05),再灌注24小时组Src总蛋白的表达较正常组出现增多,其差异具有统计学意义(P0.05)。另一方面,不同位点发生磷酸化的Src蛋白则变化不同。与正常组相比,Tyr527位点发生磷酸化的Src在氧糖剥夺4小时组和再灌注6小时组的表达无明显变化,在再灌注12小时组和再灌注24小时组则出现了明显的表达下调,差异具有统计学意义(P0.05)。同样与正常组比较,Tyr416位点发生磷酸化的Src蛋白则在氧糖剥夺4小时组的表达无明显变化,而在再灌注6小时组、12小时组和24小时组的表达均出现了上调,差异均具有统计学意义(P0.05)。结论： 1.缺血再灌注损伤引起Src激酶的表达上调； 2.缺血再灌注损伤引起Src激酶的活化,与Tyr416位点磷酸化和Tyr527位点的去磷酸化均有关。
[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.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741

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