Zumsteg Valérie, Boison Detlev
Institute of Pharmacology and Toxicology, University of Zurich, CH-8057, Zurich, Switzerland.
J Neurosci Methods. 2002 Oct 15;120(1):85-94. doi: 10.1016/s0165-0270(02)00186-3.
Adenosine is an efficient inhibitor of neuronal activity with the ability to suppress seizure activity in various animal models of epilepsy. In the present study adenosine-releasing neuronal cells were generated as a potential source for therapeutically active grafts. Mice with a genetic disruption of the gene encoding adenosine kinase (Adk(-/-))-the major adenosine metabolizing enzyme-were used as a source for the derivation of adenosine releasing neuronal cells. Since homozygous Adk(-/-) mice constitute a lethal phenotype, embryonic neuroectoderm was derived from intercrosses of Adk(+/-)-mice. Therefore, a rapid genotyping procedure had to be developed using a fluorescent 5'-exonuclease (TaqMan) assay, which permitted the genotyping of embryonic cell material within 3 h. During this time period the cells to be grafted displayed an unaltered viability. Cultured neuroectodermal Adk(-/-) cells released up to 2 micro g adenosine per mg protein per hour. Adk(-/-) neuroectoderm grafted into the lateral brain ventricle of adult mice was found to survive for at least 6 weeks. The method described here suggests the feasibility to graft adenosine releasing neuroectodermal cells as a potential therapeutic approach for the treatment of pharmacoresistant epilepsy.
腺苷是一种有效的神经元活动抑制剂，能够在各种癫痫动物模型中抑制癫痫发作活动。在本研究中，产生了释放腺苷的神经元细胞，作为具有治疗活性移植物的潜在来源。将编码腺苷激酶（Adk(-/-)）（主要的腺苷代谢酶）的基因发生遗传破坏的小鼠用作衍生释放腺苷的神经元细胞的来源。由于纯合Adk(-/-)小鼠具有致死表型，胚胎神经外胚层来自Adk(+/-)小鼠的杂交。因此，必须开发一种使用荧光5'-核酸外切酶（TaqMan）测定的快速基因分型程序，该程序可在3小时内对胚胎细胞材料进行基因分型。在此时间段内，待移植的细胞显示出未改变的活力。培养的神经外胚层Adk(-/-)细胞每毫克蛋白质每小时释放多达2微克腺苷。发现移植到成年小鼠侧脑室的Adk(-/-)神经外胚层至少存活6周。这里描述的方法表明移植释放腺苷的神经外胚层细胞作为治疗药物难治性癫痫的潜在治疗方法是可行的。
