| Budget Amount *help |
¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 1999: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1998: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
Materials and Methods In the LSR animals, deformities naturally appeared at 4-6 weeks of age and developed until 16 weeks. Most of them demonstrated lordosis, which was generally seen at the T7-10 levels, and was often accompanied by slight scoliosis.
LSRs were killed at 6-14 weeks old under general anesthesia. The thoracic spine was dissected out and fixed for 5 days at 4゜C, in 4% fresh made paraformaldehyde in 0.1M phosphate buffer. Then, they were dehydrated in ethanol, cleared in chloroform, and decalcified with formic acid. Decalcified tissues were dehydrated and embedded in paraffin. Sagittal sections of 6μm thick were cut and mounted on 3-aminopropyl-triethoxysilane-coated slides.
Bone samples dissected out from fetus of Japanese White Rabbit fetuses 1 week before birth were frozen in liquid nitrogen. Messenger RNA was extracted from the specimens, reverse transcribed and amplified by polymerase chain reaction using primers for type I collagen, osteopontin(Osp) and bone morphogene
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tic protein-2(BMP-2). PCR products were subcloned into the multicloning sites of plasmid, and Digoxigenin(DIG)-labeled single-stranded antisense and sense RNA probes were prepared using a DIG RNA labeling kit(Boehringer Mannheim Gmbh, Biochemica, Mannheim, Germany)according to the manufacturer's instructions.
Hybridization was carried out according to the manufacturer's protocol(Boehringr Mannheim Yamanouchi, Tokyo, Japan) with minor modifications(2).
Results In the vertebral body in the lordotic thoracic spine, stronger positive signals for type I collagen mRNA were detected in osteoblasts in the endosteum in the ventral than in the dorsal of vertebral body. In the normal vertebral body, there were no differences from ventral to dorsal. Positive signals for BMP-2 mRNA showed a similar pattern. In the vertebral body in the lordotic thoracic spine, positive signals for Osp mRNA were detected in osteoclasts in Howship's lacunae and osteocytes around these pits in the dorsal endosteal portion. No such signals were detected in the ventral endosteal portion of the same vertebral body. In the normal vertebral body, there were no differences in Osp mRNA expression pattern ventral and dorsal portions.
Discussion In the ventral endosteum of thoracic lordotic spine, osteogenesis. Osp mRNA-positive osteoclasts in Howship's lacunae and osteocytes around the pits were detected in the dorsal endosteal portion of the same vertebral body, indicating bone resorption(3). The vertebral bones showed a specific mode of remodeling in response to the force producing lordosis. Less
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