器官型脑片培养 OGD 复氧复糖模型中丁苯酞对于 bcl-2／bax、及 bim 蛋白的调节作用

摘要

目的：观察正丁基苯酞（NBP）对乳大鼠大脑皮质器官型脑片的氧糖剥夺模型（OGD）中细胞凋亡bcl-2、bax及bim蛋白的调节作用。方法用生后7 d的SD乳大鼠制备大脑皮质运动区器官型脑片，将培养2 w的脑片随机分为对照组（ C组）、模型组（ M组）、溶剂对照组（ Sc组）和实验组（ T组），T组在OGD干预30 min后给予NBP 10μM。将脑片制备OGD模型30 min，部分脑片在NBP干预前后不同时间点（0 h、1 h、24 h、72 h）进行PI染色；部分脑片在NBP干预后6 h用戊二醛固定，做石蜡切片，行HE染色及bcl-2、bax、bim免疫组化染色。结果 PI染色可见C组在不同时间点荧光强度无明显变化， M组、Sc组和T组荧光强度均随时间有不同程度增强， M组和Sc组各时间点荧光强度无明显差异，T组荧光强度较前两者降低，随时间延长，降低幅度增大，尤其是72 h时间点。HE染色C组可见皮质运动区特有的大锥体细胞，结构清晰， M组和T组可见细胞肿胀，细胞核溶解， M组相对明显。免疫组化染色可见在3种染色中M组和T组阳性细胞数均较C组增多，其中bcl-2染色中T组阳性细胞增多更明显，bax和bim染色中M组阳性细胞增多更明显，3种染色中C、M、T各组之间差异均有统计学意义。结论NBP对OGD后的细胞损伤具有保护作用，可能是通过对bcl-2、bax以及bim蛋白的调节减少细胞凋亡。%Objective To establish the oxygen and glucose deprivation ( OGD) model of organotypic brain slices of rat cerebral cortex and observe the effects of n-butyl phthalide ( NBP) on bcl-2、bax、bim protein in apoptosis .Method We chose the post-natal 7-day rats(SD) to culture the brain slices on the membrane insert and they were divided into four groups after having been cultured for two weeks:control group (group C),model group (group M),the solvent control group (group Sc) and test group (group T).Group T were given NBP 10μM to brain slices after treating with OGD30 min.Some brain slices were observed by PI dyeing before and after being gived NBP 1 h2,4 h and 72 h.Other brain slices were fixed by glutaral after 6 h treated with NBP and were dying by HE and assessed the positive cells of bcl-2、bax、bim in brain slices of different groups .Results Brain slices showed no different brightness of red fluorescence in group C and showed in-creased brightness according to different time in group M ,group Sc and group T by PI staining .There were no variety in group M and group Sc.But the brightness was dicreased in group T than that of group M especially in 72 h (P<0.05). There were typital giant pyramidal cells in cortical motor area with normal structures .There were much more swelling cells with karyolysis in group M than that in group T .There were more positive cells in group M and group T than that in group C.Among them there were more positive cells of bcl-2 in group T and more positive cells of bax and bim in group M .There were statistic variance in three groups with each dying of bcl-2,bax and bim.Concluison NBP had protective effect on damaged brain slice after OGD.NBP could decrease apoptosis by controlling the protein of bcl -2,bax and bim.