心钠素在原代培养大鼠耳蜗螺旋神经元细胞中的表达

发布时间：2018-03-09 19:25

本文选题：心钠素　切入点：神经元特异性核蛋白　出处：《第四军医大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的:观察心钠素(atrial natriuretic peptide,ANP)在原代培养大鼠耳蜗螺旋神经元细胞(spiral ganglion neurons,SGN)中是否表达,并探讨其意义。方法:1.对3～5日的新生SD大鼠耳蜗SGN进行原代培养以及纯化,倒置显微镜下进行细胞形态学观察;应用免疫细胞化学方法(免疫酶标以及免疫荧光法)检测原代培养SGN中神经元特异性核蛋白(neuron-specific nuclear protein,NeuN)表达情况,进行培养细胞的神经源性鉴定。 2.应用免疫细胞化学方法检测原代培养的SGN中ANP的表达情况,运用逆转录-聚合酶链式反应(reverse-transcription polymerase chain reaction,RT-PCR)方法检测原代培养的SGN中是否存在编码ANP的mRNA。结果:1.SGN的原代培养:细胞贴壁并生长24 h后,多数SGN呈双极神经元特征形态,胞体两极伸出突起,可达胞体的2～5倍,并附着在成纤维细胞形成的单层细胞层表面生长;有的呈三极神经元形态,神经突向三个方向伸展。48～72 h后,可见细胞突起交织成网状的细胞群,个别细胞突起可达胞体的7～8倍。还可见扁平多角形大胞体的成纤维细胞,以及长梭形双极的雪旺细胞(schwann cells)等非神经细胞。 2. SGN的免疫细胞化学:可见NeuN在SGN胞体和突起中具有阳性表达,而扁平多角形的成纤维细胞与长梭状雪旺细胞NeuN染色阴性;SGN的胞质中含有大量棕黄色ANP免疫反应阳性物质,为分散或成团的颗粒。免疫荧光染色显示原代培养SGN中NeuN、ANP、Hoechst荧光信号的共表达:红色荧光显示NeuN在椭圆形胞体和神经突起着色;绿色荧光显示ANP主要在细胞胞体和突起中着色,在近胞核周围的胞质中分布尤为密集;蓝色荧光为Hoechst衬染胞核。 3.ANP-mRNA在原代培养SGN细胞中的表达:培养5日SGN提取的RNA用RT-PCR方法扩增出编码ANP的单一条带,用凝胶成像及定量扫描仪测得片段大小为269 bp,为目的基因片段。结论:原代培养的SGN NeuN阳性表达,为神经组织来源,而且具有表达与合成ANP的能力,提示ANP可作为一种内源性激素,局部调节并维持内耳微环境稳态平衡;并且可能作为内耳SGN神经调节的一种递质或调质,参与其生理活动和突触传递功能的调节。本实验为进一步研究ANP对SGN神经调节作用的机制奠定了形态学基础。
[Abstract]:Aim: to investigate the expression of atrial natriuretic peptide (ANPs) in spiral neurons of rat cochlea and its significance. Methods 1. The cochlear SGN of newborn SD rats from 3 to 5th was cultured and purified, and the morphology of the cells was observed under inverted microscope. The expression of neuron-specific nuclear protein neuron-specific nuclear protein (Neun) in primary cultured SGN was detected by immunocytochemistry (immunocytochemistry and immunofluorescence), and the neurogenic identification of cultured cells was carried out. 2.Immunocytochemistry was used to detect the expression of ANP in primary cultured SGN. Reverse transcription-polymerase chain reaction (reverse transcription-polymerase chain reaction) was used to detect the existence of mRNA encoding ANP in primary cultured SGN. Results 1. Primary culture of SGN: after 24 h of cell adhesion and growth, most of SGN showed bipolar neuronal features, the two poles of the cell body protruded up to 2 to 5 times of the cell body, and grew on the surface of the monolayer cell layer formed by fibroblasts. Some of them showed tripolar neuronal morphology. After 72 hours of neuronal process stretching in three directions, the neurites were intertwined into a network of cells, and the processes of individual cells could reach 78 times as much as that of the cell body, and the fibroblasts of the flat polygonal large cell body could also be seen. And long fusiform bipolar Schwann cells such as Schwann cells. 2. Immunocytochemistry of SGN: it was found that NeuN was positively expressed in SGN cell bodies and processes, while the cytoplasm of flat polygonal fibroblasts and long fusiform Schwann cells, which were negative for NeuN staining, contained a large amount of brown ANP immunoreactive substances. For dispersed or clustered granules, immunofluorescence staining showed the co-expression of the fluorescence signal of NeuNine ANPA Hoechst in primary culture SGN: red fluorescence showed that NeuN was stained in oval cell body and neurite, and green fluorescence showed that ANP was mainly stained in cell body and process. The distribution of the cytoplasm around the nucleus is particularly dense, and the blue fluorescence is the Hoechst staining of the nucleus. 3. Expression of ANP-mRNA in primary cultured SGN cells: a single band encoding ANP was amplified by RT-PCR from RNA extracted from SGN on 5th. The fragment size was 269bp, which was determined by gel imaging and quantitative scanner. Conclusion: the positive expression of SGN NeuN in primary culture is derived from nerve tissue and has the ability to express and synthesize ANP, suggesting that ANP can be used as an endogenous hormone to regulate and maintain homeostasis in the inner ear microenvironment. As a transmitter or modulator of the inner ear SGN nerve regulation, it may be involved in the regulation of physiological activity and synaptic transmission function. This study provides a morphological basis for the further study of the mechanism of ANP on SGN nerve regulation.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R764

【参考文献】