Kang U J, Fisher L J, Joh T H, O'Malley K L, Gage F H
Department of Neurosciences, University of California at San Diego, La Jolla 60638-0627.
J Neurosci. 1993 Dec;13(12):5203-11. doi: 10.1523/JNEUROSCI.13-12-05203.1993.
Primary skin fibroblasts were genetically modified with catecholamine-synthesizing enzyme genes and studied as potential syngeneic donor cells to supply catecholamines in animal models of Parkinson's disease. Primary skin fibroblasts obtained from inbred Fischer 344 rats were transduced with tyrosine hydroxylase (TH) or aromatic L-amino acid decarboxylase (AADC) cDNAs using retroviral vector system. The transduced cells were characterized in vitro by enzymatic assay, immunocytochemistry, and HPLC analysis of catecholamine production and release. Accumulation of high levels of dopamine was detected in the media in a time-dependent manner. Secretion of dopamine and its metabolites appeared to be constitutive without significant storage capacity in vesicles or regulation at the level of secretion. The feasibility of regulating the final dopamine production by the AADC-transduced cells was explored in two ways. First, administration of various doses of the precursor, L-dopa, resulted in a controlled production of dopamine by these cells. Second, coculturing AADC-transduced cells with TH-transduced cells in various proportions allowed control of dopamine production. TH-transduced cells served as an endogenous source of precursor. We propose the use of these cells to study the role of AADC in restoring the dopamine-deficient behavior and to compare the effect of dopamine-producing cells with L-dopa-producing cells either by cografting TH-transduced cells with AADC-transduced cells or by grafting TH-transduced cells alone. The role of AADC in vivo will be assessed in future experiments involving animal models of Parkinson's disease.
原代表皮成纤维细胞用儿茶酚胺合成酶基因进行基因改造，并作为潜在的同基因供体细胞进行研究，以在帕金森病动物模型中提供儿茶酚胺。使用逆转录病毒载体系统，将从近交系Fischer 344大鼠获得的原代表皮成纤维细胞用酪氨酸羟化酶（TH）或芳香族L-氨基酸脱羧酶（AADC）cDNA进行转导。通过酶分析、免疫细胞化学以及儿茶酚胺产生和释放的高效液相色谱分析，在体外对转导细胞进行表征。在培养基中以时间依赖性方式检测到高水平多巴胺的积累。多巴胺及其代谢产物的分泌似乎是组成性的，在囊泡中没有明显的储存能力，在分泌水平也没有调节作用。通过两种方式探索了调节AADC转导细胞最终多巴胺产生的可行性。首先，给予不同剂量的前体L-多巴，可使这些细胞可控地产生多巴胺。其次，将AADC转导细胞与TH转导细胞按不同比例共培养可控制多巴胺的产生。TH转导细胞作为前体的内源性来源。我们建议使用这些细胞来研究AADC在恢复多巴胺缺乏行为中的作用，并通过将TH转导细胞与AADC转导细胞共移植或单独移植TH转导细胞，比较产生多巴胺的细胞与产生L-多巴的细胞的效果。AADC在体内的作用将在未来涉及帕金森病动物模型的实验中进行评估。
