凝血酶诱导大鼠海马神经细胞凋亡及其与Bcl-2及Bax表达变化关系的实验研究

目的研究不同浓度凝血酶诱导海马神经元凋亡的作用及其机制.方法将原代培养新生大鼠海马神经元分为对照组,凝血酶组(1U/ml,10U/ml,20U/ml,40U/ml),凝血酶受体激活肽组.应用TUNEL及流式细胞仪检测凋亡细胞数及凋亡百分率,免疫细胞化学方法检测Bcl-2,Bax蛋白表达.结果低浓度凝血酶组(1U/ml)凋亡细胞数和凋亡率与对照组无差异,Bcl-2表达增加;随凝血酶浓度增加,TUNEL阳性细胞数及凋亡率明显增多,Bcl-2表达下调,Bax表达上调,Bcl-2/Bax比值降低.凝血酶受体激活肽的作用与大剂量凝血酶类似.结论凝血酶可能通过激活PAR-1受体诱导凋亡,凋亡呈剂量依赖性.Bcl-2的表达减少,Bax的表达增加,Bcl-2/Bax降低可能为高浓度凝血酶诱导凋亡的机制之一.

EFFECT OF THROMBIN ON THE APOPTOSIS AND THE CHANGE OF BCL-2 AND BAX EXPRESSION IN RAT HIPPOCAMPAL NEURONS

Objective To investigate the mechanism of apoptosis of primary cultured hippocampal neurons induced by different concentrations of thrombin. Methods Primary cultured hippocampal neurons were divided into a control group, 4 groups with different doses of thrombin (1U/ml, 10U/ml, 20U/ml, 40U/ml) and a group with thrombin receptor activating peptides (TRAP). Apoptotic cells were counted and apoptotic rate of neurons measured by terminal deoxynucleotidyl transferase (TdT) mediated~ dUTP-biotin nick end-labeling (TUNEL) method and flow cytometry. Bcl-2 and Bax protein expressions were determined by immunocytochemical method. Results There was no significant difference in apoptotic cell number and apoptotic rate between the low-dose thrombin and the control group, and Bcl-2 expression was increased in low-dose thrombin. With the increased concentration of thrombin, the TUNEL-positive cell number and apoptotic rate became larger. Bcl-2 expression was down-regulated, Bax expression up-regulated and the ratio of Bcl-2/Bax decreased. The effect of TRAPwas similar to that of high-dose thrombin. Conclusion Thrombin induces neuron apoptosis in a dose-dependent manner, probably by activating PAR-1. That Bcl-2 expression decreased, Bax expression increased and Bcl-2/Bax ratio decreased may be one of the mechanisms for hippocampal neuron apoptosis induced by high-concentration thrombin.