实验性大鼠脑出血细胞凋亡与Bcl-2和Bax表达关系的研究

Apoptosis and Expressions of Bcl-2 and Bax after Experimental Intracerebral Hemorrhage in Rats

目的研究大鼠脑出血(ICH)后血肿周围细胞凋亡及其与凋亡相关蛋白Bcl-2和Bax表达的变化规律和关系。方法90只SD大鼠随机分为正常对照组(n=10),假手术组(n=40),ICH组(n=40)。假手术组和ICH组又依术后12h、1d、2d、3d和7d各分为5个亚组。用胶原酶配合肝素诱导大鼠脑尾状核出血,末端脱氧核苷酸转移酶(TdT)介导的dUTP缺口末端标记法(TdT-mediated-dUTP nick end labeling,TUNEL)观察不同时间点细胞凋亡的变化,SABC法观察Bcl-2和Bax蛋白在各时点表达的变化,免疫印迹(Western-blot)技术检测Bcl-2和Bax蛋白相对光密度比值,电镜观察细胞超微结构改变。结果①ICH组TUNEL阳性细胞12h可检测到,3d达高峰,7d仍有表达(P<0.01),阳性细胞主要见于血肿周围,多见神经元,有少量神经胶质细胞和血管内皮细胞;②ICH组Bcl-2和Bax蛋白免疫反应阳性细胞分别于出血2、3d达高峰,7d仍有表达,主要表达于血肿区及血肿周围、同侧及对侧皮层;多见神经元,少数为神经胶质细胞、吞噬细胞和中性粒细胞;③Western-blot正常对照组和假手术组均可检测到Bcl-2、Bax蛋白,但相对光密度比值较小;ICH组Bcl-2相对光密度比值2d达最大值,Bax则3d达最大值,在7d仍明显高于正常对照组(P<0.05)。结论ICH后存在细胞凋亡;Bcl-2和Bax参与了ICH细胞凋亡;ICH组TUNEL阳性细胞7d仍可检测到,提示ICH后7d内进行抗凋亡治疗可以不同程度地阻止细胞凋亡的发生。

Objective To investigate the relationship and disciplinarian between apoptosis and expressions of Bcl- 2 and Bax protein after intracerebral hemorrhage （ICH） in rats. Methods Ninety SD rats were randomly divided into normal control group （NCG, n = 10）; sham-control group （SCG, n = 40）; intracerebral hemorrhage group （ICHG, n =40）. Acorrding to time after operation, SCG and ICHG were divided intol2h, ld, 2d, 3d and 7d 5 sub - group. ICH model was induced by injection of V~ bacterial collagenase mixed with heparin into the caudate nucleus. The expression of TUNEL positive cells was observed by terminal deoxynucleotidyl transferase mediated dUTP nick end - labelling. The expression of Bcl - 2 immune reactivity （IR） cells and Bax IR cells was viewed by SABC method . The relative optical density of Bcl- 2 and Bax protein was measured by Western- Blot. The changes of fme structure in cells were observed under electron microscope. Results ① TUNEL positive cells appeared in the peri - hematoma at 12h and peaked on 3d, was till higher on 7d（ P 〈 0.01）. Most positive cells appeared aroud hematoma to be neurons, whereas some appeared to be glia and endothelial cells. ②Bcl- 2 and Bax IR positive cells mainly appeared in the area of the focus and around the focus, homonymy and opposite side. Bcl - 2 IR cells peaked on 2d and Bax IR on 3d, was fill higher on 7d. The positive cells mostly were neurons, some were glias, phagocytes and neutrophil. ③Westem blot analysis of the Bcl - 2 and Bax protein showed that the optical density ratios of NCG and SCG were lower than ICHG in ipsilateral perilesional in any time （ P 〈 0.01 ）, and the max of Bcl - 2 was on 2d whereas Bax on 3d in ICHG. They were very higher on 7d than that in NCG. Conclusion Apoptotic injury appears in ICH. Bcl- 2 and Bax are involved in apoptosis caused by ICH; TUNEL positive cells can be observed till 7d, so anti- apoptosis can be achieved in 7d after ICH.