摘要
目的 研究溶血磷脂酸 (LPA)对原代培养大鼠小脑颗粒细胞的细胞毒性作用及其损伤机制。方法 将原代培养的大鼠小脑颗粒细胞暴露于不同剂量LPA中 ,测定噻唑蓝 (MTT) ;应用流式细胞仪、透射电镜、激光共聚焦显微镜等技术观察细胞的凋亡率、凋亡细胞的核形态及细胞质和线粒体内活性氧 (ROS)的形成。结果 LPA对小脑颗粒细胞的损伤呈剂量依赖效应。经 5 0 μmol/LLPA作用后细胞生存率为正常细胞的 (5 4 .8± 11.5 ) % ,细胞凋亡率达 (40 .5± 2 .3) % ,细胞染色质发生浓缩和形成凋亡小体 ,细胞质和线粒体内ROS形成增加 ,而经四甲基吡嗪 (TMP)预处理后细胞生存率升至 (84 .7± 8.8) % ,细胞凋亡率减至 (16 .7±5 .8) % ,细胞核形态无明显改变 ,细胞质和线粒体内ROS形成被抑制。结论 LPA具有神经毒性作用。通过增加线粒体内ROS形成 ,继而诱导神经元凋亡可能是其损伤机制之一。能减少内源性ROS形成的抗氧化剂可对抗LPA诱导的神经元凋亡。
Objective To study the injurious mechanism and the cytotoxic effect of lysophosphatidic acid(LPA) on primary rat cerebellar granule cells. Methods The primary rat cerebellar granule cells were exposed to different dose of LPA and cell viability were assessed by the MTT assay. The apoptosis rate, nuclear morphology of apoptotic cells, and the formation of cytosolic and intramitochondrial reactive oxygen species(ROS) during the apoptosis process were observed by using flow cytometry, transmission electron microscopy and laser confocal scanning microscopy(LCSM). Results LPA showed potent dose dependent neurotoxicity. When cerebellar granule neurons were exposed to 50 μmol of LPA for 24 h, the cell viability and apoptosis rate respectively averaged ( 54.8 ± 11.5 )% and ( 40.5 ± 2.3 )%, and the nuclear morphology was characterized by chromatin condensation and the formation of apoptotic bodies. Upon exposure to LPA for 2 h, the intramitochondrial ROS increased significantly. The cytosolic ROS also increased markedly 4 h after the treatment with LPA. With pertreatment of tetramethylpyrazine (TMP), cell viability and apoptosis rate respectively averaged ( 84.7 ± 8.8 )% and ( 16.7 ± 5.8 )%. No apparent morphological alteration occurred and the cytosolic and intramitochondrial ROS formation was supressed significantly in cell pretreated with TMP. Conclusions The present investigation showed that LPA is neurotoxic, and proved that ROS are key mediators of LPA induced neuronal apoptosis. Accordingly, some antioxidant that can diminish LPA induced endogenous ROS could also reduce neuronal apoptosis induced by LPA.
出处
《卒中与神经疾病》
2004年第6期321-324,共4页
Stroke and Nervous Diseases
关键词
小脑颗粒神经元
细胞凋亡
溶血磷脂酸
线粒体
活性氧
Cerebellar granule neurons Apoptosis Lysophosphatidic acid Mitochondria Reactive oxygen species