Sestrin2参与调控新生鼠缺氧缺血性脑损伤后细胞自噬机制  

Mechanisms of Sestrin2 regulating cell autophagy after hypoxic-ischemic brain damage in neonatal rats

作　　者：马慧顺 陈洪菊 唐军 Ma Huishun;Chen Hongju;Tang Jun(Department of Pediatrics,Key Laboratory of Birth Defects and Related Diseases of Women and Children(Sichuan University),Ministry of Education,West China Second University Hospital,Sichuan University,Chengdu 610041,Sichuan Province,China)

机构地区：[1]四川大学华西第二医院儿科、出生缺陷与相关妇儿疾病教育部重点实验室,成都610041

出　　处：《中华妇幼临床医学杂志（电子版）》2020年第1期32-41,共10页Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition)

基　　金：国家自然科学基金项目(81501304,81330016,81630038,81771634,81842011);国家临床重点专业基金项目(1311200003303);四川省青年科技创新团队基金项目(2016TD0002)。

摘　　要：目的探讨应激诱导蛋白Sestrin2,对新生SD大鼠缺氧缺血性脑损伤(HIBD)后神经细胞自噬作用及其机制。方法选择80只新生10日龄SD大鼠(受试鼠)为研究对象。对其中60只建立HIBD模型,并根据这60只受试鼠缺氧缺血(HI)后的处死时间点,将其分别纳入HI后4、8、12、24、72 h组(除HI后24 h组为20只受试鼠外,其余均为10只)。对剩余的20只受试鼠仅分离右颈总动脉,既不结扎右颈总动脉,亦不进行缺氧处理,处死时间对应于HI后24 h组,纳入假手术组(n=20)。①对HI后4、8、12、24、72 h组及假手术组(各组受试鼠均为10只),获取海马组织标本后,采用蛋白质印迹法,检测Sestrin2、肝酶B1(LKB1)、磷酸化LKB1(p-LKB1)、AMP活化蛋白激酶(AMPK)、磷酸化AMPK(p-AMPK)、哺乳动物雷帕霉素靶蛋白(mTOR)、磷酸化mTOR(p-mTOR)、自噬相关蛋白Beclin1、微管相关蛋白1轻链(LC)3及凋亡相关蛋白活化型天冬氨酸特异性半胱氨酸蛋白酶(CC)3在受试鼠海马组织中的相对表达量,并采用方差分析及最小显著性差异(LSD)-t法,对上述指标进行6组之间,或HI后4、8、12、24、72 h组分别与假手术组的组间比较。②对于HI后24 h组与假手术组各组剩余的10只受试鼠,获取大脑标本后,采用免疫组织化学SP法,检测海马组织中p-AMPK、LC3及CC3表达水平,包括积分吸光度(IA)值测定及染色结果,并采用t检验,对这3项指标的IA值进行2组比较。本研究通过四川大学动物实验伦理委员会批准[审批文号:SYXK(川)2013-185]。结果①蛋白质印迹法检测结果显示,HI后4、8 h组受试鼠海马组织中p-mTOR及CC3水平,均分别高于假手术组;HI后12、24 h组受试鼠海马组织中Sestrin2、p-LKB1、p-AMPK、p-mTOR、Beclin1、LC3及CC3水平,均分别高于假手术组;HI后72 h组受试鼠海马组织中Sestrin2及CC3水平,均高于假手术组;并且这些差异均有统计学意义(P<0.05)。其中,HI后8 h组受试鼠海马组织中p-mTOR水平最高,HI�Objective To investigate the role and mechanism of stress-inducing protein Sestrin2 in the regulation of nerve cell autophagy in neonatal rats after hypoxic-ischemic brain damage(HIBD).Methods Eighty postnatal 10 days newborn Sprague-Dawley rats(test rats)were selected as research subjects.For the 60 test rats,the HIBD model was established;then according to the sacrifice time of 4,8,12,24,72 h after hypoxia-ischemia(HI),60 test rats were included into 4,8,12,24,72 h after HI group,which was 20 cases in 24 h after HI group and the rest were 10 cases in each group.For the remaining 20 test rats,the right common carotid artery was separated but not ligated,no hypoxia treatment was performed and the sacrifice time corresponded to 24 h after HI group,and they were included into sham operation group(n=20).①After obtained hippocampal tissue samples of rats in 4,8,12,24,72 h after HI group and sham operation group(10 cases in each group),western blotting was used to detect relative expression levels of Sestrin2,liver kinase B1(LKB1),phosphorylated LKB1(p-LKB1),adenosine monophosphate-activated protein kinase(AMPK),phosphorylated AMPK(p-AMPK),mammalian target of rapamycin(mTOR),phosphorylated mTOR(p-mTOR),autophagy-related protein Beclin1,microtubule-associated protein 1 light chain(LC)3,and apoptosis-related protein cleaved-caspase 3(CC3)in hippocampus tissue of rats,and analysis of variance and least significant difference(LSD)-t methods were used to compare the above indicators among 6 groups or between 4,8,12,24,72 h after HI groups and sham operation group,respectively.②After obtained cerebrum samples of remaining rats in 24 h after HI group and sham operation group(10 cases in each group),immunohistochemistry SP method was used to detect the expression levels of p-AMPK,LC3 and CC3,including integrated absorbance(IA)values and staining results in rats′hippocampus tissue.And using t test to compare the IA values of the three indicators between two groups.This study was approved by the Animal Experimental Ethics

关 键 词：Sestrin 2蛋白 大鼠 信号传导 缺氧缺血 脑 自噬 细胞凋亡 印迹法 蛋白质 大鼠 Sprague-Dawley 动物 新生 

分 类 号：R73[医药卫生—肿瘤]