干扰小分子蛋白Sulfiredoxin-1对大鼠星形胶质细胞氧化应激损伤影响的初步研究

发布时间：2018-05-03 08:59

本文选题：Sulfiredoxin-1 + 星形胶质细胞　；参考：《重庆医科大学》2015年硕士论文

【摘要】：背景：脑卒中等脑缺血性脑血管疾病发病率、致残率与死亡率一直居高不下,给社会及家庭造成严重的负担。脑缺血再灌注损伤使机体产生氧化应激,造成机体活性氧的释放,使机体的内源性抗氧化防御系统清除能力的平衡失调。星形胶质细胞的损伤在脑缺血疾病中起着至关重要的作用。小分子蛋白Sulfiredoxin-1 (Srxnl),属于内源性保守的Sulfiredoxin抗氧化家族的成员,具有重要的神经保护作用,可能与抗氧化、抗凋亡以及抗炎症等作用有关。本课题拟采用慢病毒载体干扰Srxnl基因表达,在体外以OGD诱导大鼠星形胶质细胞氧化应激损伤来模拟脑缺血再灌注,初步探讨干扰Sulfiredoxin-1对OGD诱导的星形胶质细胞氧化应激损伤的影响。目的：初步探讨干扰Sulfiredoxin-1对OGD诱导的星形胶质细胞氧化应激损伤的影响。方法：(1)筛选氧糖剥夺(OGD)最佳时间,用不同时间的OGD(0 h,2h,4h,6h,8h),复氧24 h,作用于星形胶质细胞,运用MTS和LDH检测筛选出最佳OGD作用时间。(2)构建三条小发夹Srxn1干扰慢病毒载体(shRNA-Srxn1-LV3 298, shRNA-Srxn1-LV3 385, shRNA-Srxn1-LV3 456)及一条阴性载体(LV3 NC),运用慢病毒感染技术将各个载体瞬时转染至星形胶质细胞中,倒置荧光显微镜下观察细胞转染效率,运用Western blot和QPCR检测干扰效率,筛选出最佳干扰载体。(3)运用筛选出的最佳OGD时间作用于转染后的细胞以模拟氧化应激损伤模型。运用MTS和LDH测定细胞的活性和损伤程度。(4)建立完整的Srxn1干扰的星形胶质细胞损伤模型,并运用Western blot检测Srxn1的蛋白水平,QPCR检测Srxn1的]mRNA水平。结果：(1)MTS和LDH显示,OGD 6 h/复氧24 h为最佳的氧化应激模型。(2)倒置荧光显微镜观察结果显示,慢病毒瞬时感染大鼠星形胶质细胞效果显著,其转染效率可达到85%以上。(3) Western blot和QPCR检测结果显示,与阴性对照组相比较,shRNA-Srxn1-LV3 298及shRNA-Srxn1-LV3 385的干扰效率不明显,shRNA-Srxn1-LV3 456的干扰效率显著(p0.01)。(4)MTS和LDH检测结果显示,干扰Srxn1后能导致细胞活性明显下降,细胞损伤显著加重(p0.05,p0.01)。结论：Srxn1能保护大鼠星形胶质细胞抵御OGD诱导的氧化应激损伤。
[Abstract]:Background: the morbidity, disability rate and mortality rate of cerebral ischemic cerebrovascular diseases such as stroke have been high all the time. Cerebral ischemia-reperfusion injury resulted in oxidative stress, the release of reactive oxygen species, and the imbalance of the clearance ability of endogenous antioxidant defense system. The injury of astrocytes plays an important role in cerebral ischemic diseases. Sulfiredoxin-1, a member of the endogenous conservative Sulfiredoxin antioxidant family, has important neuroprotective effects, which may be related to anti-oxidation, anti-apoptosis and anti-inflammatory effects. In this study, lentivirus vector was used to interfere the expression of Srxnl gene and OGD was used to induce oxidative stress injury of astrocytes in vitro to simulate cerebral ischemia-reperfusion injury. To investigate the effect of interfering Sulfiredoxin-1 on oxidative stress injury of astrocytes induced by OGD. Aim: to investigate the effect of interfering Sulfiredoxin-1 on oxidative stress of astrocytes induced by OGD. Methods the optimal time for screening oxygen glucose deprivation (Ogd) was determined by 1: 1). The astrocytes were treated with different time of OGD(0 hu 2h, 4hh, 6h, 6h, 8h, reoxygenation, 24 h, respectively. Three small hairpin Srxn1 interfering lentivirus vectors, shRNA-Srxn1-LV3298,shRNA-Srxn1-LV3 385, shRNA-Srxn1-LV3 456) and a negative vector, LV3NCX, were constructed by MTS and LDH detection. The vectors were transiently transfected into astrocytes by lentivirus infection technique. The transfection efficiency was observed under inverted fluorescence microscope. The interference efficiency was detected by Western blot and QPCR. The best interference vector was screened. The best OGD time was used to simulate the oxidative stress injury model. MTS and LDH were used to determine the activity and damage degree of astrocytes. A complete Srxn1 interference model of astrocyte injury was established. The protein level of Srxn1 was detected by Western blot and the mRNA level of Srxn1 was detected by QPCR. Results OGD 6 h / 24 h reoxygenation was the best oxidative stress model. The results of fluorescence microscopy showed that the transient infection of rat astrocytes by lentivirus had a remarkable effect. The results of Western blot and QPCR detection showed that the interference efficiency of shRNA-Srxn1-LV3298 and shRNA-Srxn1-LV3 385 was less than that of the negative control group. The interference efficiency of shRNA-Srxn1-LV3456 was significantly higher than that of the negative control group. After interfering with Srxn1, the cell activity decreased significantly, and the cell damage increased significantly (p 0.05, P 0.01). Conclusion:% Srxn1 can protect astrocytes from oxidative stress induced by OGD.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R743.3

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