2002 Fiscal Year Final Research Report Summary
Study of electro-gene therapy on diabetic neuropathy
| Project/Area Number |
13670679
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Neurology
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| Research Institution | KAWASAKI MEDICAL SCHOOL |
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Principal Investigator |
MURAKAMI Tatsufumi KAWASAKI MEDICAL SCHOOL, Medicine ASSOCIATE PROFESSOR, 医学部, 助教授 (30330591)
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| Co-Investigator(Kenkyū-buntansha) |
OOSAWA Yutaka KAWASAKI MEDICAL SCHOOL, Medicine ASSOCIATE PROFESSOR, 医学部, 講師 (80246511)
SUNADA Yoshihide KAWASAKI MEDICAL SCHOOL, Medicine PROFESSOR, 医学部, 教授 (00240713)
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| Project Period (FY) |
2001 – 2002
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| Keywords | diabetic neuropathy / electroporation / gene therapy / VEGF |
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| Research Abstract |
1. Construction of vector which expresses mouse vegfl64 Mouse vegfl64 Cdna which contains singal peptide sequence was synthesized by RT-PCR from mouse muscle total RNA. This DNA fragment was subcloned into Pcaggs vector which has β-actin promoter and strongly expresses in the skeletal muscles. This plasmid (Pcag-Mv3) was transfected in COS7 cells and its expression was confirmed by western blot analysis
2. Gene transfer of mouse vegfl64 into skeletal muscles of normal mice and the effect of vegfl64 on perioheral nerves and organs Fifty ug of Pcag-Mv3 was injected into the anterior tibial muscles of C57B1/6 mice followed by electroporation. After 3 and 9 weeks the sciatic nerves were fixed in glutalaldehyde and stained with toluidine blue. The muscles, liver, spleen and kidney were fixed in 4% paraformaldehyde and stained by hematoxylin and eosin. After 3 weeks the sections of the anterior tiblal muscles showed the marked proliferation of capillary and invasion of monocytes suggesting the local synthesis and secretion of vegfl64. In the liver the extension of sinusoids and the proliferation of endothelial cells were observed probably reflecting the circulation of vegfl64 producted in the muscles by veins. There was no reflecting the circulation of vegfl64 produced in the muscles by veins. There was no morphological differences of spleen and kidney between injected and control mice. After 3 and 9 weeks the sclatic nerves were normal. Though the marked elevation of serum VEGF level was reported in patients with POEM syndrome and it is postulated that VEGF may cause neuropathy in this disease, our data suggest that the short-term elevation of blood VEGF level by our method dose not invoive the peripheral nerves.
We are trying vegfl64 gene transfer into skeletal muscles of diabetic mice with diabetic neuropathy
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