Deissler H, Blass-Kampmann S, Kindler-Röhrborn A, Meyer H E, Rajewsky M F
Institute of Cell Biology, University of Essen Medical School, Germany.
J Neurosci Res. 1996 Mar 15;43(6):664-74. doi: 10.1002/(SICI)1097-4547(19960315)43:6<664::AID-JNR3>3.0.CO;2-C.
As part of investigations on ethylnitrosourea (EtNU)-induced neuro-oncogenesis in the rat, we have produced monoclonal antibodies (Mabs) specific for neural cell surface antigens (NCAs) by immunization with cells of the clonal tumorigenic neural rat cell line BT4Ca. Mabs designated as anti-NCA (alpha NCA1, alpha NCA2, alpha NCA3, alpha NCA4, and alpha NCA5) recognize proteins of 25 kDa and 23 kDa, as shown by immunoprecipitation and Western blot. The predominant 25-kDa protein was purified from BT4Ca cells by immunoaffinity chromatography with immobilized Mab alpha NCA1 and identified by N-terminal sequencing as the rat homologue of the CD9 antigen. Identification of proline as N-terminal amino acid of the purified protein suggests post-translational modification of CD9 in the rat central nervous system. The NCA/CD9 protein was localized in distinct regions of fetal and adult rat brain by immunofluorescence staining of frozen sections. Flow cytometric analyses of isolated fetal rat brain cells (FBC) showed that the proportion and number of NCA/CD9-expressing cells increased during prenatal development. Immunoreactivity of approximately 40% of brain cells isolated 13 days post conception (p.c.) indicated that NCA/CD9 is expressed by neuronal precursors at this stage of development. In primary cultures of rat FBC isolated 18 days p.c., the NCA/CD9 antigen was expressed by all premature and mature astrocytes, oligodendrocytes, ependymal cells, and microglial cells, but not by E-N-CAM-expressing neuronal progenitor cells and neurons. Furthermore, eight out of ten EtNU-induced malignant neural rat cell lines as well as EtNU-induced tumors of the central and peripheral nervous system exhibited intermediate or strong immunoreactivity with Mab alpha NCA1. Expression of the NCA/CD9 protein is, therefore, characteristic of both normal glial precursor cells and their malignant counterparts in the rat.
作为对大鼠中乙基亚硝基脲（EtNU）诱导的神经肿瘤发生进行研究的一部分，我们通过用克隆致瘤性神经大鼠细胞系BT4Ca的细胞免疫，制备了针对神经细胞表面抗原（NCA）的单克隆抗体（Mab）。标记为抗NCA的单克隆抗体（αNCA1、αNCA2、αNCA3、αNCA4和αNCA5）识别25 kDa和23 kDa的蛋白质，免疫沉淀和蛋白质印迹结果表明了这一点。通过用固定化的单克隆抗体αNCA1进行免疫亲和层析从BT4Ca细胞中纯化出主要的25 kDa蛋白质，并通过N端测序鉴定为CD9抗原的大鼠同源物。纯化蛋白质的N端氨基酸为脯氨酸，这表明大鼠中枢神经系统中CD9发生了翻译后修饰。通过对冰冻切片进行免疫荧光染色，将NCA/CD9蛋白定位在胎鼠和成年大鼠大脑的不同区域。对分离的胎鼠脑细胞（FBC）进行流式细胞术分析表明，在产前发育过程中，表达NCA/CD9的细胞比例和数量增加。受孕后13天分离的约40%的脑细胞具有免疫反应性，这表明在发育的这个阶段，神经前体细胞表达NCA/CD9。在受孕后18天分离的大鼠FBC原代培养物中，所有未成熟和成熟的星形胶质细胞、少突胶质细胞、室管膜细胞和小胶质细胞均表达NCA/CD9抗原，但表达E-N-CAM的神经祖细胞和神经元不表达。此外，十分之八的EtNU诱导的恶性神经大鼠细胞系以及EtNU诱导的中枢和外周神经系统肿瘤与单克隆抗体αNCA1表现出中度或强免疫反应性。因此，NCA/CD9蛋白的表达是大鼠正常神经胶质前体细胞及其恶性对应细胞的特征。
