Relationship between the development of the nerve cell and the expression of the neurotransmitter transporter

在本研究中，我们已经产生了单克隆抗体（单克隆抗体）的多巴胺转运蛋白（DAT）的寡肽。利用抗肽抗体，我们研究了DAT在神经元中的表达，特别是DAT在发育过程中亚细胞定位的变化。本研究以大鼠嗜铬细胞瘤细胞系PC 12细胞为研究对象，用神经生长因子（nerve growth factor，NGF）诱导PC 12细胞分化为神经元样细胞，并分泌多巴胺、肾上腺素和乙酰胆碱。制备了两种单克隆抗体，Mab H-1a和H-1b。Western印迹分析证实，上述抗体识别从大鼠纹状体制备的突触体部分中的~ 85，000 Da蛋白质，但不识别来自小脑的部分中的蛋白质。该抗体对DAT的特异性也较强， ...更多信息 用单克隆抗体H-1a和H-1b标记对照组的PC 12细胞表面，用免疫组织化学方法检测经NGF处理的PC 12细胞表面DAT的表达和亚细胞定位。当细胞用NGF处理时，DAT的表达显着强调首先在细胞质中，然后在生长锥的表面上从轴突生长开始。DAT在PC 12细胞中的活性通过[^3H] -多巴胺的摄取和摄取抑制剂的阻断而得到证实。NGF治疗使多巴胺摄取活性加倍。DAT的特异性抑制剂GBR 12909在10 μ M浓度下阻断[^3H] -多巴胺<-7>。用逆转录聚合酶链反应（RT-PCR）检测PC 12细胞中DAT和去甲肾上腺素转运体（NET）mRNA的表达。在NGF处理的细胞中，DAT mRNA在孵育7天后显著增加，而NET mRNA显著降低。少

In the present study we have produced monoclonal antibodies (MAbs) against oligopeptides of a dopamine transporter (DAT) protein. Using the antipeptide antibody, we have examined the expression of DAT in the neuron, especially the changes in subcellular localization of DAT during development. In this study we used PC12 cells, which are a clonal line of rat pheochromecytoma cells that secrete dopamine, epinephrine and acetylcholine and develop as neuron-like cells after treatment with nerve growth factor (NGF).An attempt to produce antipeptide antibodies against the oligopeptides specific in amino acid sequence to a rat dopamine transporter was made with an in vitro immunization method. Two monoclonal antibodies, Mabs H-1a and H-1b, were produced. Western blot analysis confirmed that the above antibodies recognized a -85,000 Da protein in a synaptosomal fraction prepared from the rat striatum but none in the fraction from the cerebellum. The specificity of the antibody to DAT was also c … More onfirmed by an antibody absorption test using two kinds of synthetic oligopeptides, one of which is specific only to DAT.These results have confirmed the specificity of the present antibody to DAT.The expression and subcellular localization of DAT were immunohistochemically examined with Mabs H-1a and H-1b in PC12 cells treated with NGF.The antibody labeled the surface of PC12 cells in control. When the cells were treated with NGF,the expression of DAT was significantly emphasized first in the cytoplasm and then on the surface of growth cones from the beginning of neurite outgrowth. The activity of DAT in the PC12 cells was pharmacologically confirmed by the uptake of [^3H] -dopamine and blockade by uptake inhibitors. The NGF treatment doubled the dopamine uptake activity. GBR12909, a specific inhibitor of DAT,blocked the [^3H] -dopamine at a concentration of 10^<-7> M.The expression of DAT and norepinephrine transporter (NET) mRNA in the PC12 cells was examined by reverse transcriptase-polmerase chain reaction (RT-PCR). DAT mRNA significantly increased in the NGF-treated cells after 7 days of incubation, whereas NET mRNA markedly decreased. Less