高温对原代培养神经细胞的影响(英文)

THE EFFECTS OF HYPERTHERMIA ON THE NEUROEPITHELIAL CELLS IN PRIMARY CULTURE

本实验仿照高温致畸动物模型的制作程序处理原代培养的神经细胞 ,取受精后 10 d金黄地鼠胚胎神经管制成细胞悬液 ,进行原代培养。随机分为对照组和高温组 ,高温组于接种后 2 4h置 42℃水浴中持续 2 0 min;对照组置 3 7℃水浴中持续 2 0min,于 3 7℃、5 % CO2 孵箱中继续培养。应用倒置相差显微镜、扫描电镜、透射电镜、MTT测定、凝胶电泳、原位末端标记(TU NEL)、免疫细胞化学反应以及图像分析等技术 ,观察了高温对原代培养神经细胞的影响。结果显示 :高温可使神经细胞的凋亡数增多、存活数减少、神经网络稀疏。高温后细胞内超微结构也发生明显变化 ,表现为核形态不规则 ,染色质浓缩、边聚 ,粗面内质网、高尔基复合体膨大 ,线粒体结构基本正常。MTT测定示细胞内线粒体酶的活性降低 ,DNA电泳可见梯状带纹 ,TUNEL 较DNA电泳更敏感 ,且可在原位显示单个凋亡细胞。高温可诱导 bcl-2和 bax蛋白产物表达出现异常变化。上述结果表明 ,高温可诱导原代培养神经细胞凋亡 ,bcl-2和

The animal experiment studies have demonstrated that hyperthermia is a strong teratogen to many kinds of animal with high incidence of neural tube defects(NTD). Epidermiological investigation showed that hyperthermia was closely related to the acencephaly and excenphaly in human being,but little was known about the distinct mechanism of NTD induced by hyperthermia. In order to study the developmental mechanism of NTD induced by hyperthermia, we observed the effects of hyperthermia on the neuroepithelial cells in primary culture. The neural tubes of the hamster embryos on 10 d after fertilization were obtained, the neuroepithelia were dissociated and then seeded at the density of 1×10 6 cells per well. The cells were divided into experimental and control groups randomly. The experimental groups were exposed to 42℃ for 20 min, whereas groups exposed to 37℃ served as control. After treatment of hyperthermia, the cells were incubated continuously at 37℃ in a 95% air/5% CO 2 humidified incubator, the culture was terminated after different intervals. Phase contrast microscopy, scanning electron microscopy, transmission electron microscopy, MTT assay, agarose gel electrophoresis analysis, TUNEL detection, immunocytochemistry and image analysis were made. The results of the experimental groups indicated that the floating cells and apoptotic cells increased in number, the neurites of the cells were shortened even disappeared, the survival cells decreased in number, the ultrastructure of the cells demonstrated distinct abnormal changes, the function of mitochondria was impaired, the expressions of both bcl 2 and bax were abnormal. The above results suggest that hyperthermia may induce apoptosis of neuroepithelial cells, during which bcl 2 and bax genes may play important regulatory roles.