| Budget Amount *help |
¥4,900,000 (Direct Cost: ¥4,900,000)
Fiscal Year 2001: ¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 2000: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
Efficacy of liposome and hydrogel-polymer as vector for gene transfer to cerebral arteries by intrathecal administration was investigated.
1. Liposome vector
Cationic lipid, N,N,N',N^1-bis(2-hydroxyethyl)-2,3,-dioleoyloxy-1,4-butanediammonium iodide / L-dioleoyl phosphatidylethanolamine, was used.
1) in vitro evaluation
Safety of cationic lipid was evaluated using cultured rat smooth muscle cells (SMC). Cationic lipid was dissolved in cultured solution at a concentration of 10, 20, 40, 80, 160μM, and SMC was induced in the solution for 24 hours. Cytotoxicity was evaluated by microculture tetrazolium method. At a concentration of 80 and 160μM, cytotoxicity was seen, and a concentration of 40μM was used in the following experiment. β galactosidase control vector was mixed with cationic lipid and solved in the cultured solution at a concentration of 40μM, in which SMC was incubated for 48 hours. β galactosidase assay revealed expression of β galactosidase in the SMC incubated in the solution
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with β galactosidase mixed with cationic lipid was seen about eight times as those incubated in the solution with β galactosidase alone.
2) in vivo evaluation
β galactosidase mixed with cationic lipid was injected into cisterna magna of SD rat, and expression of β galactosidase at the basilar artery was serially evaluated. The basilar artery was taken, homogenized, and lysed, and expression of β galactosidase was measured using β galactosidase assay. Until 8 days after the injection, expression of β galactosidase was seen, however, on 14 days after the injection, it was markedly decreased.
The results indicated that cationic lipid was useful for gene transfer in the acute cerebrovascular disease such as cerebral vasospasm after subarachnoid hemorrhage.
2. Hydrogel polymer
1) Evaluation of safety
Rabbit middle cerebral artery was exposed under operative microscope after craniotomy and it was coated with hydrogel polymer. Two weeks to 3 months after, the brain was taken and histopathological change was examined under light microscopy. No abnormal histological changes were seen.
2) gene transfer
Rabbit middle cerebral artery was coated with hydrogel polymer mixed with Luciferase gene in the same manner as described above, and the middle cerebral artery was serially taken 2 weeks to 3 months after the surgery. The middle cerebral artery was homogenized and lysed to examine Luciferase activity in the arterial wall using Luciferase assay system. Two weeks and 1 month after Luciferase activity was markedly seen, however, it decreased 2 months after, and it was not detected 3 months after.
The results indicated that hydrogel polymer can offer a long and slow transfer of gene into cerebral artery. Less
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