Establishment of cerebellar Purkinje cell lines by somatic cell fusion

为了获得阐明小脑神经退行性疾病发病机制的线索，我们采用体细胞与神经母细胞瘤细胞系融合的方法建立了小鼠小脑神经元杂交瘤。几种体外生长良好且表达GABA的神经杂交瘤细胞系可通过抗GABA抗体免疫染色进行鉴定。然而，我们无法检测到已知的小脑浦肯野细胞特异性标记。在无血清条件下培养后，部分细胞也表达了对酪氨酸羟化酶的免疫反应性。这表明在这些神经元杂交瘤中表达的神经元特性并不总是来源于亲代小脑神经元中表达的神经元特性，并且这些杂交瘤的神经元特性可以在培养条件下改变。由于我们现在认识到神经元杂交瘤不足以研究神经元特性的表达，我们放弃了从人类小脑变性（如pcd）的小鼠模型中产生小脑神经元杂交瘤，并将其与正常窝鼠建立的神经元杂交瘤进行比较。相反，我们已经从这些突变小脑和正常小脑中建立了cDNA文库，现在正试图用差分杂交方法鉴定它们之间表达不同的基因。

In order to obtain clues for elucidating pathogenetic mechanism of cerebellar neurodegenerative disorders, we established murine cerebellar neuronal hybridomas by somatic cell fusion with a neuroblastoma cell line. Several neuronal hybridoma cell lines growing well in vitro and expressing GABA could be identified by immunostaining with anti-GABA antibody. However, we could not detect markers known to be specific for cerebellar Purkinje cells. After culturing these cells under serum-free condition, a part of them also expressed immunoreactivity against tyrosine hydroxylase. This indicates that neuronal properties expressed in these neuronal hybridomas are not always derived from those expressed in parental cerebellar neurons, and that neuronal properties of these hybridomas can change under culture conditions. Since we now realize that neuronal hybridomas are inadequate for studying the expression of neuronal properties, we gave up producing cerebellar neuronal hybridomas from murine models of human cerebellar degenerations, such as pcd, and comparing those with neuronal hybridomas established from normal littermates. Instead, we have made cDNA libraries from these mutant and normal cerebella, and are now trying to identify genes, the expression of which is deferent between them, using differential hybridization method.