Establishment of cerebellar Purkinje cell lines by somatic cell fusion

为了进一步阐明小脑神经退行性疾病的发病机制，我们用体细胞融合的方法建立了小鼠小脑神经元杂交瘤细胞系。用抗GABA抗体进行免疫组织化学染色，可鉴定出几株体外生长良好且表达GABA的神经性杂交瘤细胞系。然而，我们无法检测到已知的小脑浦肯野细胞的特异性标志物。在无血清条件下培养后，部分细胞也表现出对酪氨酸羟化酶的免疫反应。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自于亲代小脑神经元表达的那些，并且这些杂交瘤的神经元特性在培养条件下可以改变。由于我们现在意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退行性变的小鼠模型(如PCD)中生产小脑神经元杂交瘤，并将这些杂交瘤与从正常胎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的和正常的小脑中建立了cDNA文库，现在正试图用差异杂交的方法来鉴定它们之间表达差异的基因。

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.