低氧预处理对大鼠脑缺血再灌注B细胞淋巴瘤2、半胱氨酸天冬氨酸蛋白酶3 mRNA表达的影响

Effects of hypoxic preconditioning on the expression of Bcl-2 and caspase-3 mRNA in rats after cerebral ischemiareperfusion

目的:观察低氧预处理对大鼠大脑中动脉缺血再灌注模型B细胞淋巴瘤2、半胱氨酸天冬氨酸蛋白酶3mRNA表达的影响,探讨其脑保护作用及可能机制。方法:①实验于2004-10/2005-06在苏州大学附属第一医院神经内科完成。选用清洁级成年SD雄性大鼠55只,随机分为3组:假手术组,对照组(缺血再灌注组)和低氧预处理组。②采用线栓法制备大鼠脑缺血再灌注模型,假手术组只手术不插线。低氧预处理组在低氧预处理后12h制备脑缺血再灌注模型。③假手术组在缺血3h再灌注24h、对照组和低氧预处理组在缺血3h再灌注3,6,12,24,48h不同时间点,进行脑切片的苏木精-伊红染色、原位末端标记法检测细胞凋亡,免疫组化法检测B细胞淋巴瘤2水平、原位杂交染法检测半胱氨酸天冬氨酸蛋白酶3mRNA表达。结果:55只大鼠均进入结果分析。①假手术组无凋亡细胞,低氧预处理组的凋亡细胞较对照组明显减少(P<0.05)。②低氧预处理组大鼠缺血再灌注3h可见B细胞淋巴瘤2阳性细胞数开始表达,再灌注24h达高峰,缺血再灌注6,12,24,48h均较对照组增高(101.40±2.78,68.20±1.45;137.92±2.41,81.34±2.28;172.21±2.89,103.12±2.61;105.68±12.67,60.12±1.47;P<0.05)。③低氧预处理组和对照组缺血再灌注3h半胱氨酸天冬氨酸蛋白酶3mRNA阳性细胞数的表达逐渐增加,至24h达高峰;低氧预处理组缺血再灌注12,48h阳性细胞数的表达均较对照组减少(45.96±5.02,85.48±7.88;33.84±4.67,56.31±7.02;P<0.05)。结论:低氧预处理的脑保护作用与其抑制神经细胞凋亡有关;而上调B细胞淋巴瘤2及下调半胱氨酸天冬氨酸蛋白酶3mRNA的表达,可能是实现保护作用、抑制凋亡的机制之一。

AIM： To study effects of hypoxic preconditioning on the expression of Bcl- 2, caspase-3 mRNA in cerebral ischemic-reperfusion of middle cerebral artery of rats, and explore the cerebra protection and possible mechanism. METHODS： ① The experiment was carried out in the Department of Neurology, First Affiliated Hospital of Soochow University from October 2004 to June 2005. A total of 55 adult male clean degree SD rats were selected and randomly divided into sham operation group, ischemiareperfusion group （control group）, and hypoxic preconditioning group. ② The model was established by inserting the thread to middle cerebral artery occlusion, the sham-operation group was performed by the same way except for inserting the thread. The preconditioning group was performed at the 12^th hour before ischemia by placing rats ③ The sham-operation group after 24 in the hypoxic chamber of oxygen. hours of cerebral ischemia for 3 hours , and the others after 3 hours, 6 hours, 12 hours, 24 hours and 48 hours of cerebral ischemia for 3 hours and rcperfusion, the rat brain sections were applied with hematoxylin-eosin （HE） staining. Cell apoptosis was detected with in situ end-labeling. The level of Bcl-2 was measured with immunohistochemical method. The expression of caspase-3 mRNA was tested With in situ hybridization. RESULTS： Totally 55 rats were involved in the result analysis. ① There was no apoptosis neuron of the sham-operation group, and less apoptosis neuron was found in the hypoxic preconditioning group than that in the control group （P 〈 0.05）. ②The expression of Bcl-2 in the hypoxic preconditioning group from the 3^rd hour after reperfusion, at the 24^th hour reached its peak, and significantly increased as compared with the control group from the 6^th hour to the 48^th hour （101.40±2.78,68.20±1.45; 137.92 ±2.41,81.34 ±2.28; 172.21 ±2.89,103.12 ±2.61;105.68 ±12.67, 60.12±1.47;P 〈 0.02）. ③The expression of positive cell of caspase-3 mRNA in the hypoxic preconditioning group and the control group significantly increased at the 3^rd hour, and reached the peak at the 24^th hour. The expression in the hypoxic preconditioning group decreased at the 12^th hour and 48^th hour as compared with the control group （45.96±5.02, 85.48±7.88 ; 33.84±4.67,56.31 ±7.02; P 〈 0.05 ）. CONCLUSION： The cerebral protective effect of hypoxic preconditioning is related with the inhibition of neural apoptosis. The increase of Bcl-2 and decrease of caspase-3 mRNA may be one of the mechanisms of protective effect and inhibiting apoptosis.