[L-DOPA-producing primary fibroblasts genetically modified with a retrovirus vector system].
Although intracerebral grafting has become a new strategy for the treatment of Parkinson's disease, many problems related to the grafts remain. We focused on primary skin fibroblasts as grafts. Rat primary skin fibroblasts were transfected with a retrovirus vector containing the cDNA of human tyrosine hydroxylase (TH) (pLTHSNL) or cytomegalovirus promoter (pCTHSNL) as a foreign promoter, and catecholamine production and release by these genetically modified fibroblasts, were analyzed in vitro immunocytochemically and by high-performance liquid chromatography with electrochemical detection (HPLC-ECD). The cells were supplemented with biopterin (BH4; (6R)-L-erythro-tetrahydrobiopterin), a cofactor required for TH activity, and they produced and released L-DOPA into the culture medium. When exposed to the combination of a foreign promoter and BH4, L-DOPA production increased in a time-dependent manner, and was unaffected by the number of cell-passages or the duration of liquid-nitrogen freezing. This suggests that the foreign gene (THcDNA)-containing retrovirus vector had integrated into the chromosomal DNA of the target cells (fibroblasts). Primary fibroblasts can be easily obtained and cultured. Thus, genetically modified primary skin fibroblasts transfected with THcDNA using this retrovirus vector system appear to be a promising graft for transplantation and gene therapy of Parkinson's disease in the future.
Ishida
A
A
Department of Neurosurgery, University of the Ryukyus School of Medicine, Okinawa, Japan.
Mukawa
J
J
D000818:Animals; D017690:Cell Transplantation; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D005347:Fibroblasts / Q000378:metabolism* / Q000637:transplantation; D005822:Genetic Vectors; D007150:Immunohistochemistry; D007980:Levodopa / Q000096:biosynthesis*; D051381:Rats; D017208:Rats, Wistar; D012190:Retroviridae / Q000235:genetics*; D012867:Skin / Q000166:cytology; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics
D018076:DNA, Complementary; D007980:Levodopa; D014446:Tyrosine 3-Monooxygenase