| Objective:The purpose of this study is to observe the expression of FGFR3 in callus during fracture healing; explore the effect and mechanism of action of FGFR3 in normal fracture, to determine the relationship between FGFR3 and bone absorption; according to PCR detection of tibia tissue of rats, from the perspective of molecular study of fracture occurrence difference expression of FGFR3 in callus tissue under the condition of space-time and the in situ apoptosis; to study whether FGFR3 directly influence the differentiation and function of osteoclasts, and attempts to elucidate the specific mechanism.Methods:purchased 60 adult male SD rats from the experimental animal center of Lanzhou University, SPF grade, weighing about 100-150g, to establish the rat tibia fracture model:normal fracture group and FGFR3 blockers fracture group,30 rats in each group,10% chloral hydrate in trapper it one al injection (3ml/kg) anesthesia, operation according to the corneal reflection to determine the depth of anesthesia, after anesthesia, interlocking intramedullary fixation for fractures of the tibial shaft and then cut the tibia lateral, given to the control group rats were intraperi tonally injected with FGFR3blockers (PD173074), calculated according to the body weight of rats, per kilogram of rats withl mg per day, for 3 days, in 3,5,7 after fracture,14,21, 28 each time point in each group were sacrificed at day 5 rats, DR, CT film examination, from imaging intuitive determine fracture healing, sampling, routine frozen sections, and embedded in paraffin, used for in situ hybridization, in situ apoptosis detection, TRAP staining and image analysis.Results:FGFR3 blockers fracture group X ray results showed that the fracture, the broken end of the fracture line is clearly visible fracture after 7d. at 14d, the fracture lines blurred, ends appear obvious callus calcification shadow fracture callus visible. The twenty-first day, the connection between the broken end of the fracture, callus dilated, and density increased.28d fracture line disappeared completely, the broken end of the fracture and bony connection between the obvious, cortical bone reconstructions. As compared with the normal fracture group, in the middle and late stage ahead of time obviously fracture healing.TRAP staining results visible, normal fracture group fractures after 3-5d fracture were no TRAP positive color clear end in granulation tissue.7d after fracture, bone has the part of osteoclasts color into the end near the membrane fracture. To 14d, osteoclasts bone trabecula round TRAP positive color large amount of new bone callus in the obvious hints of osteoclasts function active. The 28d after fracture, orthoclastic bone scabies still a large number of TRAP positive color. While the osteoclast FGFR3 blocker TRAP positive color at the same time points were higher than normal fracture group (P<0.05).The results of in situ hybridization showed that mRNA positive cells, FGFR3 expression rate in the 3D after fracture at the lower, to 5Dincreased obviously, reached the peak at 7d, then decreased significantly after fracture and 14d, to 28d completely disappeared, FGFR3 blocker fracture group in the same time period is lower than that of the normal display bone fracture group (P<0.05).Under an optical microscope, normal fractures in fracture group 3D after operation, FGFR3 mRNA positive cells were not detected in the broken ends of the granulation tissue, FGFR3 mRNA positive cells were detected in the 5D after operation, mainly appeared in the cartilage callus before the mast chondrocytes,7d after fracture, cartilage cells the continuous differentiation, strong positive expression were FGFR3 mRNA positive cells in the hypertrophic cartilage cells and hypertrophic chondrocytes in front. To fractures after 14d and 28d, the apoptosis of chondrocytes began, cartilage cells into bone cells are replaced, the positive expression of FGFR3 in bony callus cells decreased signal, were not detected in the positive expression of FGFR3 signal. FGFR3 blockers fracture group produce the same results.Under the fluorescence microscope observation shows, after fracture hematoma Area 3D, and the fracture ends in granulation tissue with almost no TUNEL positive apoptotic cells. To the 5D after fracture, the fracture end local to mesenchymal cells dominated the number of TUNEL positive apoptotic cells were increased. Fracture after 7d, located in the cartilage callus endochondral bone sites, callus of TUNEL positive apoptotic cells is very obvious and no new bone trabecula of apoptotic positive cells, The 14d after fracture, bony callus began to replace cartilage callus apoptosis, TUNEL positive cells decreased, occasionally a small amount of new bone in bone cells were apoptosis positive reaction. To the 28d after fracture, occasionally, few osteoclasts apoptosis positive reaction. The control group normal tibia tissue TUNEL positive apoptotic cells in normal fracture group were higher than the same period.Conclusion:according to the experimental results, we can draw the following conclusions:The fibroblast growth factor receptor 3 (FGFR3) plays an important role in the fracture healing process, which is mainly through the regulation of chondrocytes in the late differentiation in regulating osteoclastic bone resorption, induced apoptosis of hypertrophic chondrocytes, inhibition of endochondral bone, so as to affect the healing of fractures. |
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