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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 bax在高温诱导凋亡过程中发挥重要调节作用
In this experiment, the primary cultured neurons were processed according to the preparation procedure of the animal model of hyperthermia teratogenicity. The embryonic neural tube of the golden hamster was made into the cell suspension on day 10 after fertilization, and the primary culture was carried out. The rats were randomly divided into control group and high temperature group. The high temperature group was kept in 42 ℃ water bath for 24 h after inoculation, while the control group was kept in water bath at 37 ℃ for 20 min, and then continued in 37 ℃ and 5% CO2 incubator to cultivate. The effects of high temperature on primary cultured neurons were observed by inverted phase contrast microscope, scanning electron microscopy, transmission electron microscopy, MTT assay, gel electrophoresis, TU NEL, immunocytochemistry and image analysis. The results showed that: high temperature can increase the number of apoptotic nerve cells, survival decreased, sparse neural network. After hyperthermia, the intracellular ultrastructural changes also occurred, which showed irregular nuclear morphology, chromatin condensation, edge aggregation, rough endoplasmic reticulum, Golgi complex enlargement and mitochondrial structure were normal. MTT assay showed that mitochondrial enzyme activity decreased, DNA ladder electrophoresis showed that TUNEL was more sensitive than DNA electrophoresis, and in situ display of a single apoptotic cells. High temperature can induce abnormal expression of bcl-2 and bax protein products. The above results show that high temperature can induce primary cultured neuronal apoptosis, bcl-2 and bax play an important regulatory role in the process of apoptosis induced by high temperature