HT22细胞氧糖剥夺再灌注及Grasp65过表达干预后高尔基体的形态变化及其可能机制研究

Morphological alterations of Golgi apparatus after oxygen-glucose deprivation followed by reperfusion and intervening by over-expressed Grasp65 in HT22 cells and their underlying mechanism

目的探讨HT22细胞氧糖剥夺再灌注及Grasp65过表达干预后高尔基体的形态变化及其可能机制。方法利用小鼠海马神经元细胞系HT22为研究对象,HT22细胞经氧糖剥夺再灌注损伤及Grasp65过表达干预后,采用MTT法检测细胞存活率;Hoechest33258荧光染色法评估细胞凋亡;并应用细胞免疫荧光技术观察高尔基体的形态;应用Western blot技术检测GM130和GAAP蛋白的表达。结果氧糖剥夺再灌注可导致HT22细胞的活性显著降低(P<0.05),凋亡率显著增高(P<0.05);并可导致高尔基体形态的异常,随着再灌注时间的延长,高尔基体逐渐发生碎裂,尤其以再灌注12 h和24 h最为明显;GM130、GAAP的表达水平在氧糖剥夺再灌注后出现下降,特别是在再灌注12 h、24 h后出现了显著下降(P<0.05)。过表达Grasp65后,HT22细胞在氧糖剥夺再灌注所致高尔基体碎裂出现减少(P<0.05),碎裂程度减轻,同时GM130和GAAP的表达均显著增加(P<0.05),HT22细胞的存活率大大提高(P<0.05),凋亡率显著降低(P<0.05)。结论缺血再灌注损伤的细胞模型中,同样发生了高尔基体的碎裂;过表达Grasp65可以减轻氧糖剥夺再灌注损伤所致的高尔基体碎裂,并可以减少HT22细胞的凋亡,其机制可能与上调GM130和GAAP的表达有关。

Objecive To investigate Morphological alterations of Golgi apparatus after oxygen-glucose deprivation followed by reperfusion （OGD/R） and intervening by over-expressed Grasp65 in HT22 cells and their underlying mecha-nism.Methods Using mouse hippocampal neuronal cell line HT 22 as the research object .After OGD/R and intervening by over-expressed Grasp65,using MTT method to detect cell viability and hoechest 33258 fluorescence staining method to e-valuate cell apoptosis .Morphology of Golgi apparatus were observed with cytochemistry immunofluorescence .The protein ex-pressions of GM130 and GAAP were detected by Western blot .Results HT22 cells activity was significantly decreased （ P〈0.05） via OGD/R induction and apoptosis rate increased significantly （P〈0.05）.OGD/R could also lead to abnormal Golgi morphology .Along with the reperfusion time extended ,the Golgi apparatus gradually fragmented ,it was the most obvi-ous at 12 h and 24 h of reperfusion .The protein expression levels of GM 130 and GAAP were decreased significantly at 12 h and 24 h time points after OGD/R （P〈0.05）.Performing OGD/R treatment after over-expressed Grasp65 in HT22 cells, Golgi fragmentation were significantly decreased （P〈0.05） and the degree of fragmentation alleviated .The protein expres-sions of GM130 and GAAP were significantly increased （P〈0.05）.HT22 cells survival rate was greatly improved （P〈0.05） and apoptosis rate decreased significantly （P〈0.05）.Conclusions In the cell model of ischemia reperfusion inju-ry,the same happened to fragmentation of the Golgi apparatus .Over-expression of Grasp65 could alleviate OGD/R-induced Golgi fragmentation and reduce HT 22 cells apoptosis ,possibly related to the up-regulation of the expression of GM 130 and GAAP.