缺氧缺血性脑损伤新生大鼠早期缺氧诱导因子-1α表达的意义

摘要

目的 研究缺氧诱导因子-1α(HIF-1 α)在新生大鼠缺氧缺血性脑损伤(HIBD)早期mRNA和蛋白水平的表达变化及其作用.方法 1.实验1:36只7日龄SD大鼠按随机数字表法分为假手术组(Sham组,6只)和模型组(HIBD组,30只),模型组根据HIBD后处死大鼠时间的不同又分为5个亚组(6h、12 h、24h、48 h、72 h,每组各6只).实时荧光定量PCR(qPCR)、Western blot分别检测HIF-1α mRNA和蛋白表达水平.2.实验2:将45只7日龄SD大鼠按随机数字表法分为3组:Sham组、HIBD组、2-甲氧基雌二醇组(2ME2组),每组各15只.依据实验1选取HIF-1αmRNA及蛋白表达水平最高的时间点处死取脑,免疫荧光检测HIF-1α蛋白的分布与表达,HE染色观察脑组织病理形态学变化,干湿重法测定脑组织含水量,原位末端标记法(TUNEL)检测细胞凋亡.结果 HIBD早期HIF-1αmRNA和蛋白表达水平均呈先升高后下降趋势,并于HIBD后24h其mRNA表达水平(3.38±o.21)和蛋白表达水平(2.81±0.36)最高.Sham组HIF-1α蛋白主要在细胞质中表达,HIBD组其主要在细胞核中表达.Sham组、HIBD组和2ME2组3组间在HIF-1α染色阳性细胞数、脑组织含水量、细胞凋亡率的比较差异均有统计学意义(均P＜0.05),且2ME2组均显著低于HIBD组(均P＜0.05),脑组织病理形态学改变也较HIBD组轻.结论 HIBD后24 h HIF-1α mRNA和蛋白表达水平最高,抑制HIF-1α的表达,可减轻缺氧缺血所致的脑损伤,推测HIF-1α在新生大鼠HIBD早期可能起损伤作用.%Objective To study the expression of hypoxia-inducible factor-1a(HIF-1α) at mRNA and protein levels in the early stage of hypoxic-ischemic brain damage (HIBD) in neonatal rats and its role.Methods (1) Experiment 1:thirty-six postnatal 7-day SD rats were divided into Sham group (n =6) and model group (HIBD,n =30) according to the random table method,then the rats in the model group were divided into 5 subgroups according to the time of sacrifice after HIBD(6 h,12 h,24 h,48 h,72 h,n =6).The expression levels of HIF-1cα mRNA and protein were detected by quantitative Real-time PCR(qPCR) and Western blot,respectively.(2) Experiment 2:forty-five postnatal 7-day SD rats were randomized into 3 groups:Sham group (n =15),HIBD group (n =15) and 2-methoxyestradiol(2ME2) group(n =15).According to the experiment 1,at the time point of the highest expression levels of HIF-1 α mRNA and protein,rats were killed and the brains were collected.The location and expression of HIF-1 α protein were detected by immunofluorescence,histopathological changes of brain were observed by HE staining,brain water content was measured by dry-wet method,cell apoptosis was detected by nick end labeling(TUNEL) method.Results At the early stage of HIBD,the expression levels of HIF-1 α mRNA and protein increased at first and then decreased,and the mRNA expression level (3.38 ± 0.21) and protein expression level (2.81 ± 0.36) were the highest at 24 h after HIBD.In Sham group,HIF-1 α protein was mainly expressed in the cytoplasm,while in HIBD group it was mainly expressed in the nucleus.The number of HIF-1α staining positive cells,brain water content and apoptosis rate were significantly different among Sham group,HIBD group and 2ME2 group (all P ＜ 0.05),and which were significantly lower in 2ME2 group than those in HIBD group (all P ＜ 0.05),and the pathological changes were also less serious than those in HIBD group.Conclusions The mRNA and protein levels of HIF-1 α are the highest at 24 h after HIBD.Inhibiting the expression of HIF-1 α can ameliorate the brain damage of neonatal rats induced by hypoxia-ischemia.Therefore,it is hypothesized that HIF-1α may cause injury in the early stage of HIBD in neonatal rats.