cDNA cloning of cell membrane surface antigens involved in the neuronal differentiation and identification of the antigen positive cells in mouse cerebrum

P19胚胎癌细胞是分析神经元分化的最简单系统之一。为了确定参与小鼠早期神经元分化的神经细胞上的膜相关分子，我们用神经元连接的P19 EC细胞的膜部分作为抗原免疫大鼠，产生了两种单克隆抗体Sky-1和Sky-2。Sky-1和Sky-2分别识别90 kDa蛋白质的碳水化合物部分(RANDAM-1)和40 kDa蛋白质的多肽核心(RANDAM-2)。在P19 EC细胞中，RANDAM-1表达共存于部分Nestin阳性细胞，而RANDAM-2表达仅见于大部分Nestin阳性细胞和β-Ⅲ-微管蛋白阳性神经元。在胚胎和成年小鼠脑中，RANDAM-1在胚胎8.5天(E8.5天)表达，抗原定位于神经上皮细胞和脉络丛。RANDAM-2在E6.0开始表达，抗原w…更多的As不仅分布于胚胎脑神经上皮细胞，而且分布于成年脑神经元。综上所述，RANDAM-1是在神经干细胞上表达的阶段特异性抗原，RANDAM-2在小鼠中枢神经系统(CNS)的神经干细胞和分化的神经细胞上都有结构性表达。对RANDAM-2进行的DNA克隆表明，其核苷酸序列与PA2.26抗原完全匹配，PA2.26抗原是一种以前在致瘤角质形成细胞中发现的唾液粘蛋白样跨膜糖蛋白。从E6.5可以检测到RANDAM-2转录本，然后在剩余的胚胎阶段和成年期持续表达，定位于中枢神经系统。在生长因子诱导的神经球和成人大脑中，RANDAM-2表达的细胞不仅与巢蛋白阳性的细胞吻合良好，而且与谷氨酸阳性的神经元吻合良好，但与γ-氨基丁酸阳性的细胞不吻合。这些结果表明，RANDAM-2是小鼠神经发生过程中表达于未分化神经元细胞和谷氨酸能神经元上的I型膜表面抗原之一。较少

P19 embryonic carcinoma (EC) cells are one of the simplest systems for analyzing the neuronal differentiation. To identify the membrane-associated molecules on the neuronal cells involved in the early neuronal differentiation in mice, we generated two monoclonal antibodies, SKY-1 and SKY-2,by immunizing rats with a membrane fraction of the neuronally committed P19 EC cells as an antigen. SKY-1 and SKY-2 recognized the carbohydrate moiety of a 90 kDa protein (RANDAM-1) and the polypeptide core of a 40 kDa protein (RANDAM-2), respectively. In the P19 EC cells, the expression of RANDAM-1 was colocalized to a part of Nestin-positive cells, whereas that of RANDAM-2 was observed in most nestin-positive cells as well as β-III-tubulin positive neurons. In the embryonic and adult brain of mice, RANDAM-1 was expressed at embryonic day 8.5 (E8.5), and the localization of antigen was restricted on the neuroepithelium and choroids plexus. The RANDAM-2 expression commenced at E6.0, and the antigen w … More as distributed not only on the neuroepithelium of embryonic brain but on the neurons of adult brain. Collectively, it was concluded that RANDAM-1 is a stage specific antigen to express on the neural stem cells, and RANDAM-2 is constitutively expressed on both the neural stem cells and differentiated neuronal cells in the mouse central nervous system (CNS).Complementary DNA cloning of RANDAM-2 indicated that its nucleotide sequence completely matched that of PA2.26 antigen, a sialomucin-like transmembrane glycoprotein previously found on tumorigenic keratinocytes. RANDAM-2 transcripts were detectable from E6.5,and then the expression continued throughout the remaining embryonic stages and adulthood, with a localization restricted to the CNS. In growth factor-induced neurospheres and adult cerebrum, RANDAM-2-expressing cells coincided well not only with nestin-positive cells but also with glutamate-positive neurons, but not with γ-aminobutyric acid-positive ones. These results indicate that RANDAM-2 is one of the type I membrane surface antigens constitutively expressed on undifferentiated neuronal cells and the glutamatergic neuronal cells during mouse neurogenesis. Less