| Background:Osteoporosis is a common clinical disease, characterized by decreased bone density, abnormal bone structure, and the risk of fractures. The incidence of osteoporosis is mainly due to an imbalance between bone resorption and bone formation. Moreover, osteoclast dysfunction is an important factor of osteoporosis. Currently, the methods of treating osteoporosis are based on medication, such as bisphosphonates, calcitonin and estrogens, which decrease osteoclast resorption activity. However, these drugs have certain side effects. Finally, osteoclastic resorption paly an important role in removal of cartilage callus and remodeling of hard callus.Therefore, genetically study of osteoclast differentiation and function has a very important significance for clinical treatment of osteoporosis and frcture.Fibroblast Growth Factors/Fibroblast Growth Factor Receptors play an important role in regulating skeletal development. By activating the MAPK, PI3K and PLC-y signaling pathway, FGFs/FGFRs affect cell proliferation, differentiation and apoptosis. FGFR3is an important member of the FGFs/FGFRs family, which isinvolved in the regulation of osteoclast differentiation and bone resorption. However, both FGFR3systemic knockout and Gain-of-function mutation result in enhanced osteoclast activity, the direct regulation of FGFR3on osteoclasts remains unclear.Objective:This research was designed to studythe effects of FGFR3on osteoclasts development, and clarify functionof FGFR3on bone injury healing by regulating osteoclast.Methods:Part Ⅰ:Effects of conditional knock out of FGFR3on bone remodeling1. To measure the height and X-ray results of10d,3m and6mFgfr3f/f;LysCre mice and littermate control mice;2. Mice tibia sections were stained, and the growth plate and trabecular bone was observed;3. Adult mice femoral trabecular bone were scanned by MicroCT to see if there was a change in bone mass of Fgfr3f/f;LysCre mice;4. Decalcification or hard sections of3m’s mice tibia tissue werecounted in morphometry (BV/TV, Tb.Sp, Tb.Th, Tb.N, N.Ob/B.pm, MS/BS, MAR, BFR, N.Oc/B.pm, Oc.S/BS) toevaluate the amount of bone andthe function of osteoblasts and osteoclasts;5. TRAP5b and osteocalcin in serum were measured by Enzyme-linked immunosorbent assay (ELISA);6. Bone marrow mononuclear cells of Fgfr3f/f; LysCre mice and littermate control mice were separated and inducedto osteoclast, then the number of mature osteoclast was counted;7. Osteoclasts in culture were stained in TRAP and FITC-phalloidine respectively;8. Total RNA of osteoclasts in culturewere extracted, the TRAP, CathepsinK, Mmp9expression was measured by quantitative PCR;9. Bone resorption area on bone slices co-cultured with osteoclasts were measured to evaluate the function of osteoclasts;10. The expression of FGFR3protein and the phosphorylation of ERK were analyzed by Western Blot.Part Ⅱ:Effects of conditional knock out of FGFR3on bone fracture healing1. Tibial fracture surgery and femur drilling holewere operated, after7d,14d and21d, X-ray and MicroCT were used to observe the fracture healing;2. Specimens were stained H&E or SO/FG to observe the bone injury healing;3. TRAP staining of tibial and femoral specimens were used to observe the osteoclasts.Results:Part Ⅰ:Effects of conditional knock out of FGFR3on bone remodeling1. By measuring the height of mice and body X-ray examination,we found no difference between Fgfr3f/f; LysCre mice and wild-type mice in the bone development;2. By comparing Fgfr3f/f;LysCremice tibia sections with the control group, we found that the trabecular bone was increased in Fgfr3f/f;lysCre mice;3. MicroCT and morphometry of slices showed that trabecular bone in Fgfr3f/f; LysCre mice was increased and thickened, trabecular bone separation reduced, while bone trabecular connection density increased;4. Differentiation of osteoclast stayed unchanged in FGFR3f/f;lysCre mice by observing TRAP staining, morphology analysis; 5. Osteoclasts in vitro werestained with FITC-phalloidine, there was no difference in actin ring between Fgfr3f/f;LysCre mice and the controls;6. By extracting RNA and RT-PCR reactions, we found that the expression of TRAP, CTSK, and MMP9was decreasedin Fgfr3f/f;LysCre mice. Serum TRAP5b in the mutant mice was reduced. Moreover, bone resorption lacunae area was also significantly decreased inFgfr3f/f;LysCremice;7. By morphometry analysis and serum osteocalcin ELISA testing, we found that the osteoblast number, bone formation rate, mineral apposition rate, the surface area of bone mineralization and serum osteocalcin levels were not changed;8. Western Blot showed that FGFR3protein levels and ERK phosphorylation levels in osteoclasts of Fgfr3f/f;LysCre mice was significantly decreased. After FGF2treatment, ERK phosphorylation levels increased, at the same time,the expression of CTSK, TRAP, MMP9was improved, and the bone resorption area on bone slices were larger.Part II:Effects of conditional knock out of FGFR3on bone fracture healing1. Osteoclasts conditional knock-out of FGFR3delayed the tibia fracture healingFor the resorption of soft callus was decreased and the remodeling of hard callus delayed in the Fgfr3f/f;LysCre mice, it resulted in the Fgfr3f/f;LysCre mice tibia fracture healing delayed.2. Osteoclasts conditional knock-out of FGFR3didn’t affect femur dilling-hole healing, but the removal of newborn trabecular bone near the drilling-hole site was decreased.Conclusions:1. Mice conditional deficient in FGFR3did not affect the bone development, but exhibited increased bone mass;2. Differentiation of osteoclast stayed unchanged, its resorption function was decreased in FGFR3f/f;lysCre mice;3. FGFR3miediatedthe regulation of FGF2on osteoclaststhrough ERK signaling pathway;4. Osteoblastic bone formation was normal in FGFR3f/f;lysCre mice.5. Fracture healing was delayed in FGFR3f/f;lysCre mice;... |
PDF Full Text Request