蛋白激酶B信号通路对新生大鼠缺氧缺血后轴突密度影响的实验研究  

The Regulation of Akt Signaling on Axonal Density after Hypoxic-Ischemic Brain Injury in Neonatal Rat

作　　者：熊涛[1] 陈洪菊[1] 屈艺[1] 罗碧如[1] 母得志[1] 

机构地区：[1]四川大学华西第二医院儿科妇儿疾病与出生缺陷教育部重点实验室,成都610041

出　　处：《四川大学学报（医学版）》2013年第2期274-279,共6页Journal of Sichuan University(Medical Sciences)

基　　金：国家自然科学基金(No.30973236;31171020;81172174;81270724);教育部科研基金(No.20110181130002;IRT0935);四川省科技厅基金(No.2010SZ0280)资助

摘　　要：目的探讨新生鼠在体内脑缺氧缺血(hypoxia-ischemia,HI)条件下,脑组织蛋白激酶B(proteinkinase B,Akt)和下游糖原合成酶激酶-3β(glycogen synthase kinase-3β,GSK-3β)活性变化,及其与组织损伤和轴突密度的关系。方法以10日龄新生SD大鼠为研究对象,行右侧颈总动脉结扎手术,8%氧氮混合气缺氧2.5h制作HI模型。Western blot法检测HI后大鼠脑皮层和海马区Akt、GSK-3β总蛋白和磷酸化蛋白表达变化;使用Akt抑制剂渥曼青霉素(wortmannin)和LY294002后,Western blot法检测Akt、GSK-3β在HI后4h,24h改变;HE染色检测渥曼青霉素对HI后脑组织损伤影响,银染法检测轴突密度改变。结果 HI后新生大鼠脑组织中Akt、GSK-3β总蛋白表达无明显变化;p-Akt和p-GSK-3β蛋白表达在0.5h短暂下降,此后2h、4h内呈现逐步上升,8h时恢复到基线水平,24～48h显著下降,72h表达恢复基线水平。渥曼青霉素或LY294002干预未改变Akt、GSK-3β总蛋白表达量,但p-Akt和p-GSK-3β的表达量均降低。HI引起组织损伤和轴突密度降低,渥曼青霉素加重HI后组织损伤和轴突密度降低。结论 Akt信号通路参与调控新生大鼠HI后脑组织损伤和轴突密度。Objective To investigate the activity of protein kinase B （Akt） and its downstream protein, glycogen synthase kinase-3t3 （GSK-3B） under hypoxia-ischemia （HI）, and the possible regulation for axonal density. Methods Postnatal day 10 SD rats were suffered the right common carotid artery ligation and 8% mixture of oxygen and nitrogen hypoxia 2.5 h to produce HI model. The expression of total and phosphorylated Akt and GSK 313 was detected by western blot after HI. After pretreatment of Akt inhibitor, wortmannin or LY294002, Western blot detect the expression of total and phosphorylated of Akt, GSK-3b at 4 h and 24 h after HI. After pretreatment of wortmannin, axonal density was determined by Bielschowsky silver impregnation, and histological injury was evaluated by hematoxylin and eosin （HE） staining. Results The expression of total Akt and GSK 3t3 remained unchanged after HI. p-Akt protein significantly decreased at 0.5 h, increased at 2 h and reached the highest at 4 h, returned to baseline at 8 h, declined at 24 and 48 h after HI, and finally returned to baseline again at 72 h compared with that of sham controls, p-GSK-313 protein decreased at 0. 5 h, increased at 2 h, reached the highest at 4 h, returned to baseline at 8 and decreased at 24 h, reached the lowest at 48 h, and returned to baseline at 72 h. Wortmannin or LY294002 intervention didn＇t change the expression of total Akt and GSK-3~t, while decrease the p- Akt and p-GSK-3t？ expression. HI cause decreased axonal density, and the histological injury of brain. Wortmannin pretreatment could aggravate the histological injury and decrease axonal density after HI. Conclusion The Akt pathway is involved in axonal density and histological brain injury after HI in neonatal rat.

关 键 词：蛋白激酶B 糖原合成酶激酶-3Β 信号通路 缺氧缺血 新生 轴突 

分 类 号：R722.1[医药卫生—儿科]