同型半胱氨酸对海马神经元细胞HT22的毒性作用及其机制的探讨

Neurotoxicity of homocysteine on murine bippocampal neuronal HT22 cells and the mechanisms

目的探讨同型半胱氨酸对海马神经元细胞系HT22细胞的毒性作用及其可能的相关机制。方法使用不同浓度的同型半胱氨酸（0、0．5、1．0、1．5、2．0、2．5mmol／L）作用于分化培养后的HT22细胞，采用MTS分析检测细胞活力的变化，并同时使用N-甲基-D-天冬氨酸（NMDA）受体拮抗剂MK801和美金刚进行干预处理，进一步检测其对细胞活力的影响：使用Hoechst33342和PI染色观察细胞死亡的形态学变化。结果同型半胱氨酸对分化培养后的HT22细胞具有剂量反应关系的毒性作用，半数有效浓度约在1．25mmol／L。Hoechst33342和PI染色结果显示低浓度同型半胱氨酸（1．0mmol／L）主要引起细胞凋亡，而随着同型半胱氨酸浓度的增加（2．0mmol／L），细胞则主要以坏死的形式存在。NMDA受体拮抗剂MK801和美金刚可有效的抑制同型半胱氨酸的神经细胞毒性，分别在10μmol／L浓度时显示最为明显的抑制作用，同单纯同型半胱氨酸作用组相比差异具有统计学意义（P〈0．05）。结论同型半胱氨酸对海马神经元细胞系HT22细胞具有明显的毒性作用，主要导致细胞的凋亡和坏死；其产生的神经毒性在很大程度上是通过激活NMDA受体起作用的。对于同型半胱氨酸-NMDA受体之间的作用的进一步研究将会提供神经元坏死的一个重要的研究方向。

Objective To study the neurotoxicity of homocysteine （HCY） on murine hippocampal neuronal HT22 cells and investigate the mechanisms. Methods Differentiated HT22 cells were treated with different concentrations of HCY （0, 0.5, 1.0, 1.5, 2.0, and 2.5 mmol/L） in the presence or absence of NMDA antagonists MK801 and memantine. The cell viability was tested using MTS cytotoxicity assay. Hoechst 33342 and PI staining were used to observe the morphological changes of the cells. Results HCY induced concentration-dependent toxicity in HT22 cells with the 50% effective concentration （EC50） of about 1.25 mmol/L. Administration of MK801 and memantine significantly increased the cell viability, which reached the highest level after treatment with MK801 and memantine at the concentration of 10 μmol/L （P〈0.05）. Hoechst 33342 and PI staining revealed obvious cell apoptosis at low HCY concentrations （1.0 mmol/L）, while cell necrosis was obvious at a higher concentration （2.0 mmol/L）. Conclusion HCY induced obvious concentration-dependent neurotoxicity in HT22 cells largely through the activation of NMDA receptors, and the interaction between HCY and the NMDA receptors may provides an important pathway for research of neuronal cell loss.