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

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

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。

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.