心钠素在原代培养大鼠耳蜗螺旋神经元细胞中的表达
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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。
AIM
The purpose of this study to observe whether there was a expression of atrial natriuretic peptides(ANP)in primary cultured spiral ganglion neurons(SGN)from cochlea of rat, and to explain the significance of that.
Methods
Cultured and purified SGN were establish from postnatal day 3～5 Sprague-Dawley rats in vitro. The process of cellular growth and differentiation of SGN were observed by fluorescent inverted/phase contrast microscope. To identify the cultured SGN derived from neurons, the expression of neuron-specific nuclear protein(NeuN)in cultured cells was examined by immunocytochemistry. The expression of ANP in cultured SGN was also examined by immunocytochemistry and reverse-transcription polymerase chain reaction(RT-PCR).
Results
The trypsin dissociated and cultured SGN of SD rats could survive well and had a normal phenotypic differentiation in vitro. The stable neuronal plasticity of SGN existed in the postnatal SD rats under the present experimental conditions. The neuron-specific nuclear protein(NeuN)was positive in cultured SGN, and ANP immunoreactive granules localized around the perinuclear cytoplasm of neurons. Co-expression of ANP and NeuN in SGN was confirmed by immunofluorescence staining, and the expression of ANP-mRNA was aslo detected by RT-PCR.
Conclusion
The results suggested that primary cultured SGN which were derived from neurons could express and synthesize ANP, indicating that the ANP might play a role as a neurotransmitter or neuromodulator in neuromodulation of physiological activity and neurotransmission in SGN of the inner ear.
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。
AIM
The purpose of this study to observe whether there was a expression of atrial natriuretic peptides(ANP)in primary cultured spiral ganglion neurons(SGN)from cochlea of rat, and to explain the significance of that.
Methods
Cultured and purified SGN were establish from postnatal day 3～5 Sprague-Dawley rats in vitro. The process of cellular growth and differentiation of SGN were observed by fluorescent inverted/phase contrast microscope. To identify the cultured SGN derived from neurons, the expression of neuron-specific nuclear protein(NeuN)in cultured cells was examined by immunocytochemistry. The expression of ANP in cultured SGN was also examined by immunocytochemistry and reverse-transcription polymerase chain reaction(RT-PCR).
Results
The trypsin dissociated and cultured SGN of SD rats could survive well and had a normal phenotypic differentiation in vitro. The stable neuronal plasticity of SGN existed in the postnatal SD rats under the present experimental conditions. The neuron-specific nuclear protein(NeuN)was positive in cultured SGN, and ANP immunoreactive granules localized around the perinuclear cytoplasm of neurons. Co-expression of ANP and NeuN in SGN was confirmed by immunofluorescence staining, and the expression of ANP-mRNA was aslo detected by RT-PCR.
Conclusion
The results suggested that primary cultured SGN which were derived from neurons could express and synthesize ANP, indicating that the ANP might play a role as a neurotransmitter or neuromodulator in neuromodulation of physiological activity and neurotransmission in SGN of the inner ear.
引文
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