凝血酶对培养大鼠皮层神经细胞的毒性作用研究

目的通过研究凝血酶对培养神经细胞的毒性作用,探讨脑出血凝血酶致神经细胞损伤的机制.方法采用新生1～2 d大鼠皮层神经元进行原代分散培养,分别在含150 U/ml凝血酶、150 U/ml凝血酶 150 U/ml重组水蛭素以及正常的培养液中孵育3 d后测定下列指标:①甲苯胺蓝染色法测定残存细胞数并计算神经细胞丧失率;②TdT介导的dUTP缺口末端标记法检测TUNEL阳性细胞的表达;③免疫组化二步法测caspase-3免疫反应性(Immune Reactivity,IR)细胞的表达;④培养上清液中乳酸脱氢酶(LDH)活性.结果凝血酶组较水蛭素干预组及对照组神经细胞明显减少(P＜0.05);凝血酶组有较多的TUNEL阳性细胞及caspase-3 IR细胞表达,并明显多于对照组及水蛭素干预组(P＜0.05),凝血酶组TUNEL阳性细胞比率及caspase-3 IR细胞比率明显高于对照组及水蛭素干预组(P＜0.01或P＜0.05);与实验前相比,实验三组各组LDH活性均升高(P＜0.05),三组间两两比较LDH活性无统计学差异(P＞0.05).结论 150 U/ml的凝血酶可导致培养的神经细胞死亡,并可被凝血酶的特异抑制剂水蛭素阻断,细胞死亡的原因可能为细胞凋亡而非坏死,脑出血后凝血酶的释放是脑出血神经细胞凋亡性损伤的机制之一.

Toxic effect of thrombin on cultured cortex neurons in rats

Objective To investigate the mechanisms of neuron injury induced by thrombin. Methods The cortex neurons of the newly born rats were cultured in culture medium containing 150 U/ml thrombin, 150 U/ml thrombin plus 150 U/ml hirudin, and in normal culture medium, respectively. At 3 d after culture, remnant cells were counted by toluidine blue staining and the loss rate of neurons was calculated; TUNEL positive cells were detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labelling; caspase 3 immune reactivity cells were detected by immunohistochemistry; and the LDH activities were measured in the culture medium. Results Neurons in thrombin groups were decreased obviously as compared with those in hirudin groups or the control groups ( P <0 05). There were more TUNEL positive cells and caspase 3 IR cells in thrombin groups than those in hirudin groups or the control groups, so were the TUNEL positive cells rate and the caspase 3 IR cells rate. LDH activities increased in the three groups. However there was no statistical significance between the three groups. Conclusion Thrombin at the dose of 150 U/ml could induce neuron death in culture, which could be blocked by hirudin, a special inhibitor of thrombin. The cause of neuron death is not necrosis but apoptosis.