Eves E M, Kwon J, Downen M, Tucker M S, Wainer B H, Rosner M R
Ben May Institute, University of Chicago, IL 60637.
Brain Res. 1994 Sep 12;656(2):396-404. doi: 10.1016/0006-8993(94)91484-2.
To determine whether postmitotic neurons can be immortalized by oncogenic transduction, we used two approaches involving conditional expression of a temperature-sensitive SV40 large T antigen (Tts). Initially, Tts was introduced into E17 rat embryonal hippocampal cells that were then cultured at the non-permissive temperature to enrich for postmitotic pyramidal neurons, and subsequently cloned at the permissive temperature. One clonal line (HMR10-3) expressed neuron-specific proteins upon differentiation, was capable of generating action potentials, and formed synapses with primary rat neurons in co-culture. Replating of these postmitotic cells at the permissive temperature resulted in reversible loss of neurofilament expression. Conditionally immortalized cell lines were also generated from the brain of an adult mouse carrying an inducible Tts transgene. These lines proliferated in a T antigen-dependent manner and expressed neuron-specific proteins upon differentiation at the non-permissive temperature. These results suggest that postmitotic neurons can be induced to enter the cell cycle without losing their commitment to a neuronal lineage.
为了确定有丝分裂后的神经元是否可通过致癌转导实现永生化，我们采用了两种方法，涉及温度敏感型SV40大T抗原（Tts）的条件性表达。最初，将Tts导入E17大鼠胚胎海马细胞，然后在非允许温度下培养以富集有丝分裂后的锥体神经元，随后在允许温度下进行克隆。一个克隆系（HMR10-3）在分化时表达神经元特异性蛋白，能够产生动作电位，并在共培养中与原代大鼠神经元形成突触。在允许温度下重新接种这些有丝分裂后的细胞导致神经丝表达可逆性丧失。条件性永生化细胞系也从携带可诱导Tts转基因的成年小鼠大脑中产生。这些细胞系以T抗原依赖的方式增殖，并在非允许温度下分化时表达神经元特异性蛋白。这些结果表明，有丝分裂后的神经元可以被诱导进入细胞周期，而不会失去其对神经谱系的定向。
