CAJ | 학술논문

目的：探讨围生期脑缺血再灌注损伤后,不同发育期胎鼠胚脑 Wnt 信号通路关键分子糖原合成酶激酶(GSK)-3β及β-连环蛋白(catenin)mRNA 表达水平变化。方法采用完全随机设计,将40只成功受孕的雌性 SD 大鼠分为实验组及对照组,每组各20只。两组分别于孕14,16,18及20 d(分别计为E14,16,18及20)时,各取5只孕鼠予相关处理：实验组钳夹双侧子宫动脉30 min,建立围生期脑缺血再灌注模型,并于再灌注24 h 后留取胎鼠胚脑标本；对照组则不钳夹子宫动脉,仅在与实验组对应时间点留取胎鼠胚脑标本。采用实时定量(RT)-PCR 检测胎鼠胚脑 Wnt 信号通路关键分子 GSK-3β和β-catenin mRNA 表达水平；采用尼氏染色和 TUNEL 标记法,检测两组胎鼠胚脑神经细胞凋亡情况。统计学比较两组胎鼠在相同发育期及同组胎鼠在不同发育期胚脑 GSK-3β及β-catenin mRNA 相对表达量,以及胚脑神经细胞凋亡率差异。结果①两组胎鼠在相同发育期(E14,16,18或20)胚脑 GSK-3β及β-catenin mRNA相对表达量比较,以及同组胎鼠胚脑 GSK-3β或β-catenin mRNA 相对表达量在不同发育期(E14,16,18及20)比较,差异均无统计学意义(P >0.05)。②实验组胎鼠胚脑神经细胞凋亡率,在相同发育期均较对照组高,且差异有统计学意义[(0.254±0.001)vs (0.027±0.004),t =46.547；(0.256±0.022)vs (0.029±0.003),t=45.917；(0.263±0.011)vs (0.029±0.002),t =34.355；(0.265±0.031)vs (0.030±0.003), t=91.108；均为 P <0.001]。不同发育期(E14,16,18及20)同组胎鼠胚脑神经细胞凋亡率比较,差异均无统计学意义(P >0.05)。③胎鼠胚脑尼氏染色结果显示,实验组神经细胞的细胞质嗜碱性增强,细胞核固缩浓染。结论Wnt 信号通路和发育期宫内缺血缺氧性脑损伤的关系尚待进一步研究。
목적：탐토위생기뇌결혈재관주손상후,불동발육기태서배뇌 Wnt 신호통로관건분자당원합성매격매(GSK)-3β급β-련배단백(catenin)mRNA 표체수평변화。방법채용완전수궤설계,장40지성공수잉적자성 SD 대서분위실험조급대조조,매조각20지。량조분별우잉14,16,18급20 d(분별계위E14,16,18급20)시,각취5지잉서여상관처리：실험조겸협쌍측자궁동맥30 min,건립위생기뇌결혈재관주모형,병우재관주24 h 후류취태서배뇌표본；대조조칙불겸협자궁동맥,부재여실험조대응시간점류취태서배뇌표본。채용실시정량(RT)-PCR 검측태서배뇌 Wnt 신호통로관건분자 GSK-3β화β-catenin mRNA 표체수평；채용니씨염색화 TUNEL 표기법,검측량조태서배뇌신경세포조망정황。통계학비교량조태서재상동발육기급동조태서재불동발육기배뇌 GSK-3β급β-catenin mRNA 상대표체량,이급배뇌신경세포조망솔차이。결과①량조태서재상동발육기(E14,16,18혹20)배뇌 GSK-3β급β-catenin mRNA상대표체량비교,이급동조태서배뇌 GSK-3β혹β-catenin mRNA 상대표체량재불동발육기(E14,16,18급20)비교,차이균무통계학의의(P >0.05)。②실험조태서배뇌신경세포조망솔,재상동발육기균교대조조고,차차이유통계학의의[(0.254±0.001)vs (0.027±0.004),t =46.547；(0.256±0.022)vs (0.029±0.003),t=45.917；(0.263±0.011)vs (0.029±0.002),t =34.355；(0.265±0.031)vs (0.030±0.003), t=91.108；균위 P <0.001]。불동발육기(E14,16,18급20)동조태서배뇌신경세포조망솔비교,차이균무통계학의의(P >0.05)。③태서배뇌니씨염색결과현시,실험조신경세포적세포질기감성증강,세포핵고축농염。결론Wnt 신호통로화발육기궁내결혈결양성뇌손상적관계상대진일보연구。
Objective To study the expression level changes of glycogen synthase kinase(GSK)-3βand β-catenin mRNA of Wnt signaling pathway in embryo brain at different developmental stages after intrauterine hypoxia-ischemia brain damage and reperfusion.Methods A total of 40 cases of pregnant SD rats were divided into experimental group and control group through completely random design,and each group contains 20 cases.In different gestational stages,such as 14 days(E14),1 6 days(E1 6),18 days(E18) and 20 days(E20 ),5 cases of pregnant SD rats were taken out and deal with follows:for experimental group,the gestational rat′s bilateral uterine arteries were occluded for 30 min to establish transient intrauterine ischemia model,then followed by reperfusion,and embryo brain samples were collected at 24 h after reperfusion.For control group,pregnant SD rats were subjected to same surgical procedures without occlusion,and collected embryo brain samples at same time point with experimental group.Real-time polymerase chain reaction (RT-PCR)was used to detect the expression levels of GSK-3β and β-Catenin mRNA of Wnt signaling pathway in embryo brain.The apoptosis of fetal brain cells in two groups of rats were detected by methods of TUNEL and Nissle staining.Differences of the expression levels of GSK-3β,β-Catenin mRNA and the apoptosis rates in embryo brain were compared statistically between two groups of fetal rats during the same developmental stage and in the same group of fetal rats during different developmental stages.Results ① There were no significant differences between two groups of fetal rats which at same development stages(E14 ,1 6 ,18 or 20)and among fetal rats at same group but in different development stages(E14,1 6,18 and 20)in expression levels of GSK-3βandβ-catenin mRNA in embryo brain (P >0.05 ).②The apoptosis rates of embryonic brain nerve cells in experimental group were higher than those of control group at the same developmental stages,(0.254±0.001)vs (0.027 ±0.004),t =46.547;(0.256±0.022)vs (0.029±0.003),t=45.91 7;(0.263±0.01 1)vs (0.029±0.002),t =34.355;(0.265 ± 0.031)vs (0.030 ±0.003),t = 91.108;P <0.001,respectively.There were no significant differences of apoptosis rates of embryonic brain nerve cells in different developmental stages(E14,1 6,18 and 20)in the same groups (P > 0.05 ).③ The results of Nissle staining of embryo brain showed that the cytoplasm of neurons in the experimental group was basophilic increased,and nuclear condensation.Conclusions The relationship between Wnt signaling pathway and intrauterine hypoxia-ischemia brain damage in the development stages need further study.