亚低温对大鼠局灶性脑缺血神经元细胞凋亡及调控基因Bcl-2和Bax表达的影响

目的研究不同时程的亚低温治疗对大鼠大脑中动脉闭塞(MCAO)模型的神经元细胞凋亡及Bcl-2,Bax基因表达的影响。方法健康雄性SD大鼠共40只,月龄两三个月。按抽签法随机分为5组,每组8只(n=8),分别为:假手术组(A)、对照组(B)、亚低温0.5h组(C)、亚低温1.0h组(D)、亚低温3.0h组(E)。采用Longa线栓法制作MCAO模型,缺血3.0h后再灌注。缺血后即刻分别进行不同时程(0.5,1.0,3.0h)的亚低温治疗,再灌注后24h断头取脑,连续切片作TUNEL染色及Bcl-2,Bax免疫组化染色。结果凋亡神经元细胞(TUNEL阳性细胞)主要见于梗死灶周围的纹状体及额顶部皮层。Bcl-2及Bax阳性细胞也主要见于梗死灶周围的纹状体及额顶部皮层,与该区凋亡细胞的集中分布相一致。与对照组的Bcl-2细胞阳性率相比,亚低温3.0h组增加犤(23.0±3.8)%,P<0.05)犦;与对照组的Bax细胞阳性率相比,亚低温1.0,3.0h组均降低犤分别为(45.9±4.1)%,(32.4±3.7)%,P<0.05,P<0.01犦;凋亡细胞阳性率与Bcl-2/Bax的比值呈负相关关系(r=-0.9759,P<0.01)。结论亚低温1.0h以上有抑制细胞凋亡作用;Bcl-2/Bax的比值能影响细胞凋亡的发生。

Effects of mild hypothermia on Bcl-2 and Bax gene expression in ischemic brain of rats

AIM:To investigate the effects of mild hypothermia of different durations on neuronal apoptosis and expressions of Bcl-2 and Bax genes in rat models of middle cerebral artery occlusion (MCAO). METHODS:Forty Sprague Dawley rats were divided randomly into 5 equal groups,namely sham- operation group (group A),normothermic ischemic controls (group B),30- min mild hypothermia group (group C),1- hour mild hypothermia group (group D),and 3- hour mild hypothermia group (group E).MCAO models were established by Longa's method,and reperfusion was allowed after 3- hour occlusion.Mild hypothermia for 0.5,1.0 and 3.0 hours respectively were initiated at the start of occlusion,and at the 24th hour of subsequent reperfusion,the rat brain were removed and continuously sectioned for TUNEL staining,Bcl- 2 and Bax immunohistochemical staining respectively. RESULTS:Apoptotic neurons (TUNEL- positive) were identified primarily in the cortex of the frontal and parietal lobes and peri infarct striatum, where Bcl- 2- and Bax positive cells were also spotted,in similar distribution pattern to that of TUNEL positive cells.Compared with the control group, the percentage of Bcl- 2 positive cells was significantly higher in group E (23.0± 3.8,P< 0.05), while the percentage of Bax positive cells was lowered in both groups D and E (45.9± 4.1, P< 0.05; 32.4± 3.7,P< 0.01, respectively).Inverse correlation was noted between the apoptotic rate and the Bcl- 2 to Bax protein ratio (r=- 0.975 9,P< 0.01). CONCLUSION:Mild hypothermia lasting more than 1.0 hour inhibit cellular apoptosis, the occurrence of apoptosis is determined by Bcl- 2 to Bax protein ratio.