本文選題：心鈉素　切入點：神經(jīng)元特異性核蛋白　出處：《第四軍醫(yī)大學》2010年碩士論文　論文類型：學位論文

【摘要】： 目的:觀察心鈉素(atrial natriuretic peptide,ANP)在原代培養(yǎng)大鼠耳蝸螺旋神經(jīng)元細胞(spiral ganglion neurons,SGN)中是否表達,并探討其意義。 方法:1.對3～5日的新生SD大鼠耳蝸SGN進行原代培養(yǎng)以及純化,倒置顯微鏡下進行細胞形態(tài)學觀察;應用免疫細胞化學方法(免疫酶標以及免疫熒光法)檢測原代培養(yǎng)SGN中神經(jīng)元特異性核蛋白(neuron-specific nuclear protein,NeuN)表達情況,進行培養(yǎng)細胞的神經(jīng)源性鑒定。 2.應用免疫細胞化學方法檢測原代培養(yǎng)的SGN中ANP的表達情況,運用逆轉(zhuǎn)錄-聚合酶鏈式反應(reverse-transcription polymerase chain reaction,RT-PCR)方法檢測原代培養(yǎng)的SGN中是否存在編碼ANP的mRNA。 結(jié)果:1.SGN的原代培養(yǎng):細胞貼壁并生長24 h后,多數(shù)SGN呈雙極神經(jīng)元特征形態(tài),胞體兩極伸出突起,可達胞體的2～5倍,并附著在成纖維細胞形成的單層細胞層表面生長;有的呈三極神經(jīng)元形態(tài),神經(jīng)突向三個方向伸展。48～72 h后,可見細胞突起交織成網(wǎng)狀的細胞群,個別細胞突起可達胞體的7～8倍。還可見扁平多角形大胞體的成纖維細胞,以及長梭形雙極的雪旺細胞(schwann cells)等非神經(jīng)細胞。 2. SGN的免疫細胞化學:可見NeuN在SGN胞體和突起中具有陽性表達,而扁平多角形的成纖維細胞與長梭狀雪旺細胞NeuN染色陰性;SGN的胞質(zhì)中含有大量棕黃色ANP免疫反應陽性物質(zhì),為分散或成團的顆粒。免疫熒光染色顯示原代培養(yǎng)SGN中NeuN、ANP、Hoechst熒光信號的共表達:紅色熒光顯示NeuN在橢圓形胞體和神經(jīng)突起著色;綠色熒光顯示ANP主要在細胞胞體和突起中著色,在近胞核周圍的胞質(zhì)中分布尤為密集;藍色熒光為Hoechst襯染胞核。 3.ANP-mRNA在原代培養(yǎng)SGN細胞中的表達:培養(yǎng)5日SGN提取的RNA用RT-PCR方法擴增出編碼ANP的單一條帶,用凝膠成像及定量掃描儀測得片段大小為269 bp,為目的基因片段。 結(jié)論:原代培養(yǎng)的SGN NeuN陽性表達,為神經(jīng)組織來源,而且具有表達與合成ANP的能力,提示ANP可作為一種內(nèi)源性激素,局部調(diào)節(jié)并維持內(nèi)耳微環(huán)境穩(wěn)態(tài)平衡;并且可能作為內(nèi)耳SGN神經(jīng)調(diào)節(jié)的一種遞質(zhì)或調(diào)質(zhì),參與其生理活動和突觸傳遞功能的調(diào)節(jié)。本實驗為進一步研究ANP對SGN神經(jīng)調(diào)節(jié)作用的機制奠定了形態(tài)學基礎(chǔ)。
[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.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R764

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