AN EXPERIMENTAL-STUDY ON RAT MODEL OF PARKINSONISM BY GENE-THERAPY

Objective To induce significant improvement of motor abnormalities and striatal dopamine (DA) levels in rat model of Parkinson's disease (PD ), by intracerebral grafting of the genetically modified muscle cells expressing tyrosine hydroxylase (TH). Methods Primary myoblasts and my otubes from the rat were prepared by cell culture and a plasmid, pCMVT H, containing TH gene and a promoter of cytomegalovirus (CMV) was cons tructed by DNA recombination technique. The primary muscle cells were transfected with newly constructed pCMVTH DNA vector, by using lipofec tion. These genetically modified muscle cells were grafted into the ca udate-putamen of 6-OHDA-lesioned rats, representing PD models. Before and after grafting, the rotational behaviour and the striatal levels o f DA and its metabolities were tested at different postoperative survi val times. In addition, the immunocytochemistry for showing TH activit y was done both in vitro and in vivo. Results The newly constrcuted pl asmid, pCMVTH was proved to contain TH gene and have correct direction of insertion. The cultured primary myoblasts and myotubes lipofected with pCMVTH were immunocytochemically shown to express TH activity in vitro. After grafting, these TH-expressing muscle cells showed to have a long-term survival cells in vivo and induced a marked decrease in a bnormal locomotion and a increase in striatal DA levels for PD rat mod el. Conclusions In experimental gene therapy for PD, the pCMVTH is a u seful vector for carrying TH gene. The lipofection is a practical tech nique for transferring a target gene into eukaryotes and primary cultu red muscle cells should be a good vehicle for DNA transfer and intrace rebral grafting.