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目的通过研究新生大鼠脑缺血时bcl-2基因在海马区的表达特点,探讨bcl-2基因在神经元凋亡中的作用。方法54只7日龄新生SD大鼠随机分为1个对照组和8个实验组。给动物吸入含有92%氮气和8%氧气的混合气体建立新生大鼠缺血缺氧脑损伤动物模型,分别在脑损伤后不同时间点(0.5、1、3、6、12、24、48、72 h等)断头处死动物,取海马组织,用免疫组织化学方法检测海马脑神经细胞凋亡及bcl-2基因表达情况。结果各组海马脑区阳性凋亡细胞的病理变化是染色质固缩边集、细胞质浓缩,胞浆内出现浓染颗粒,核膜也可出现,质膜分隔胞质,可形成凋亡小体。TUNEL染色的阳性细胞数与bcl-2蛋白阳性的细胞数在HIBD后的不同时间点出现分别有显著差异(FTUNEL=334.0,PTUNEL<0.01;Fbcl-2=42.7,Pbcl-2<0.01)。凋亡阳性细胞出现与bcl-2蛋白表达在12 h呈负相关(γ=-0.79,P<0.05)。HIBD后0.5~12 h,bcl-2蛋白表达越多,阳性凋亡细胞越少。海马区bc1-2蛋白阳性细胞在HIBD后立即出现,6 h达到高峰,之后渐下降。结论bc1-2基因强表达于海马缺血区和缺血周边区的神经元,与神经元的存活或死亡有一定联系。
Objective To investigate the expression of bcl-2 gene in hippocampus of neonatal rats with cerebral ischemia and to explore the role of bcl-2 gene in neuronal apoptosis. Methods Fifty-four newborn SD rats of 7 days old were randomly divided into 1 control group and 8 experimental groups. Animal models of hypoxic-ischemic brain damage in neonatal rats were established by inhalation of a mixed gas containing 92% nitrogen and 8% oxygen. The rats were sacrificed at different time points (0.5,1,3,6,12,24,48, 72 h) were sacrificed and the hippocampus was removed. The apoptosis of hippocampal neurons and the expression of bcl-2 gene were detected by immunohistochemistry. Results The pathological changes of positive apoptotic cells in hippocampal brain regions of rats in each group were chromatin condensation edge sets. Concentration of cytoplasm was concentrated, dense particles appeared in the cytoplasm, and nuclear membrane could also appear. The plasma membrane separated the cytoplasm and formed apoptotic bodies . The numbers of TUNEL-positive cells and bcl-2-positive cells were significantly different at different time points after HIBD (FTUNEL = 334.0, PTUNEL <0.01; Fbcl-2 = 42.7, Pbcl-2 <0.01). There was a negative correlation between the appearance of apoptotic cells and the expression of bcl-2 protein at 12 h (γ = -0.79, P <0.05). After 0.5 to 12 h after HIBD, the more bcl-2 protein expressed, the fewer positive apoptotic cells. Bcl-2 protein positive cells in hippocampus appeared immediately after HIBD, peaked at 6 h, then gradually decreased. Conclusion The bc1-2 gene is strongly expressed in neurons in the hippocampus ischemic and ischemic peripheral areas, and has some connection with the survival or death of neurons.