细胞周期抑制剂对糖氧剥夺诱导神经元凋亡的保护机制研究

Protective Effects of Cell Cycle Inhibitor on Neuronal Apoptosis Induced by Oxygen-glucose Deprivation in vitro

目的:探讨细胞周期抑制剂Roscovitine(Ros)对糖氧剥夺(OGD)诱导的鼠大脑皮质神经元凋亡的保护作用及可能机制。方法:体外培养大鼠皮质神经元,随机分为对照组、OGD1h后恢复糖氧供给(OGD/R)3h、6h、12h、24h组及Ros(100μM)组。Western Blot检测各组神经元磷酸化视网膜母细胞瘤蛋白(p-Rb)和E2F1的表达情况;免疫荧光细胞化学染色观察OGD/R12h组及Ros组神经元p-Rb表达;TUNEL法检测OGD/R12h组及Ros组神经元凋亡情况。结果:OGD/R各组神经元p-Rb及E2F1的表达均较对照组增高(P<0.05),12h达最高;Ros组p-Rb及E2F1的表达减少,少于OGD/R12h组(P<0.05);Ros组p-Rb和TUNEL阳性细胞率均低于OGD/R 12h组(P<0.01),两组中大部分TUNEL阳性细胞与p-Rb表达共定位。结论:Ros可能通过抑制Rb磷酸化及E2F1介导的凋亡机制来减少缺血缺氧后的神经元凋亡。

Objective： To explore the protective effect and potential mechanism of roscovi- tine on rat cortical neuronal apoptosis induced by oxygen-glucose deprivation （OGD） in vitro. Methods： Primary rat cortical neuronal cultures were randomly divided into 6 groups： control group, the groups of 3 h, 6 h, 12 h, 24 h reoxygenation after 1 h of OGD and roscovitine （100 μM） treated group. The expressions of phospho-Rb （p-Rb） and E2F1 were detected by western blot. Immunofluorescence staining was used to detect percentage of neurons with p-Rb- positive and TUNEL-positive, and the correlation between them was analyzed. Results： When compared with the control group, the expressions of p-Rb and E2F1 in each group after OGD were increased （P〈0. 05） and peaked at 12 h after OGD, which were decreased in the roscovitine treated group （P〈0.05）. Roscovitine significantly reduced the expression of p-Rb and decreased neuronal apoptosis at 12 h after OGD （P〈0.01）. A majority of apoptotic neurons of TUNEL staining had a co-localization with p-Rb staining. Conclusion： Roscovitine could reduce neuronal apoptosis, perhaps by way of inhibiting phosphorylation of Rb and transcription factor E2F1 after an ischemia/hypoxia injury in vitro.