摘要
目的研究缺氧诱导因子-1α（HIF—1α）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、12h、24h、48h、72h，每组各6只）。实时荧光定量PCR（qPCR）、Western blot分别检测HIF-1αmRNA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HIF1α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±0．21）和蛋白表达水平（2．814-0．36）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24hHIF-1αmRNA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 -1α（HIF-1α）） at mRNA and pro- tein levels in the early stage of hypoxic - ischemic brain damage （HIBD） in neonatal rats and its role. Methods （ 1 ） Ex- periment 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 - let 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 -1α mRNA and protein, rats were killed and the brains were collected. The location and expression of HIF -1α protein were detected by immunofluorescence, histopathological changes of brain were observed by HE stai- ning, 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 -1α 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 -1α 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 -1α are the highest at 24 h after HIBD. Inhibiting the expression of HIF -1α 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.
出处
《中华实用儿科临床杂志》
CSCD
北大核心
2017年第17期1321-1325,共5页
Chinese Journal of Applied Clinical Pediatrics
基金
河南省科技攻关计划项目（142102310334）
