| Budget Amount *help |
¥9,600,000 (Direct Cost: ¥9,600,000)
Fiscal Year 2003: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2002: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2001: ¥7,600,000 (Direct Cost: ¥7,600,000)
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| Research Abstract |
cDNA encoding the NH2-terminal domain of human and mouse sonic hedgehog(Shh) was amplified by PCR from the full length human and mouse Shh cDNA. It was inserted into the multiple cloning site of the pCR3.1(+) expression vector. The complete coding resion of human and mouse BMP-2 was inserted into the multiple cloning site of the pCR3.1(+) expression vector as well as BMP-4. Human and mouse fibroblasts derived from oral tissue were transfected with human and mouse BMP-2, BMP-4, and Shh cDNA plasmid vector each by calcium phosphate precipitation. Selected storongly positive clones were mouse fibroblast-clones by transfected with mouse BMP-2, BMP-4, and Shh cDNA plasmid vector each. Selected positive clone was grown to confluence in large scale flasks and cells were enclosed in gelatin capsules and implanted into the thigh muscles of nude mice. 14days after implantation, a radiopaque area was observed in cells-implanted area on the soft X-ray films and light microscopic examination disclo
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sed new bone formation by endochondral ossification. with toluidine bulue staining.
Vascularized muscle flaps in mice cervical, shoulder and limbs were elevated to examine longterm-viability and the following results were obtained that ectopectoralis flap, superficial gluteal muscle flap and biceps femoris muscle flap were useful for gene-transfer and cells-implant experiments.
In the A9 cells, C3H10T1/2 cells, KMS-6 cells and fibroblasts derived from mouse oral tissues transfected with mouse BMP-2, BMP-4 and Shh cDNA plasmid vector each, BMP and Shh-positive cells were detected in C3H10T1/2 cells and fibroblasts derived from mouse oral tissues. Though BMP and Shh protein secretion was stable by 12 passages, these protein secretions and gene expressions were not detected soon and therefore, these gene expressions were transient as we, had expected.
Storongly positive cells transfected with mouse BMP-2, BMP4, and Shh cDNA plasmid vector each were enclosed in gelatin capsules and implanted into ectopectoralis flap, superficial gluteal muscle flap and biceps femoris muscle flap and a radiopaque area was observed in only cells-implanted area on the soft X-ray films and light microscopic examination disclosed new bone formation.
Optison solution(200μl) containing mouse BMP-2, BMP-4 and Shh cDNA plasmid vector each were injected into ectopectoralis flap, superficial gluteal muscles flap and biceps femoris muscle flap with ultrasound for 2 minutes(2.5 W/cm2). 14days after injection, a small radiopaque area was observed in only solution-injected area on the soft X-ray films and light microscopic examination disclosed new bone formation. Less
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