An Experimental Study Of Transfering BMP-2 Gene To Promote The Radial Fracture Union Of The Rabbit By Gene Gun

Objective: Elevating the concentration of the BMP-2 protein at the bone fracture site by tranfering the BMP-2 gene into the tissues around the fracture site has become a reality. But how to improve the efficiency of gene tansfection, shorten the verifying time of a plasmid transfection, find a high efficient and easier method to tansfer a plasmid is still unclear. We develop this study aim to: 1) construct the stable and easily identified pIRES2-EGFP-BMP-2 plasmid. 2)tansfer the pIRES2-EGFP-BMP-2 plasmid into rabbit Bone mesenchymal stem cells (BMSCs) to study the function of the pIRES2-EGFP- BMP-2 plasmid when it was transfered into rabbit BMSCs. 3) study the different result when we use different dose, different time and different frequency to transfer the pIRES2-EGFP-BMP-2 plasmid into the tissue around the radial fracture site of rabbit by using the gene gun. Method: 1) The BMP-2cDNA and pIRES2-EGFP plasmid were amplified and cut with the restriction enzyme EcoR I?BglⅡand then jointed with the joining enzyme T4DNA. Then the jointed product was converted into competence Bacterium coli commune DH5α. Some pIRES2-EGFP-BMP-2 plasmid were cut with the two enzyme EcoRI\ BglⅡand then electrophoresis, and the others were identified and sequenced by shanghai Sangon CO. after it was purified. Then the pIRES2-EGFP-BMP-2 plasmid was introduced into the NIH3T3 cells with the help of transfect agent LipofectamineTM2000 and then incubated in the incubator for 4 weeks and screened by G418. 2) Two New Zealand rabbit were anaesthed and every rabbit get 6ml bone marrow from the left femur and tibia by trochar. The single cell was aquired by centrifugalization and then inoculated and cultured. When it passaged to the 3rd gerneration, it was used for transfection with the aid of LipofectamineTM2000. Some cells were taken out for observation of the expression of GFP after 24h. The rested were continuing cultivated and screened by the the DMEM which contains G418 400mg/L for 2 weeks. When most cells got to dead, the culture solution was changed to DMEM which contains G418 200mg/L to retain the screen until 4 weeks. Then the cells were collected for detection. 3) There are 119 New Zealand rabbits were divided into seven groups randomly with 17 rabbits per group. When the rabbit was anaesthed by the auri-vein injection, the operation site was depilated by razor and sterilized with Iodophors. The operation field was covered by aseptic towl and the dorsi-incision of the left forearm was performed to expose the rabbit radial bone with min-invasive technique. The periosteum was carefully dissected and a transverse fracture was made by a wire saw at the middle part of the radial. Then the different interventions were given according to the group. Group 5μg:5μg pIRES2-EGFP- BMP-2 plasmid was given to transfer into the tissues arounding the radial fracture site by gene gun at the time of operation and fracture made. Group 10μg:10μg pIRES2-EGFP- BMP-2 plasmid was given to transfer into the tissues arounding the radial fracture site by gene gun at the time of operation and fracture made. Group 3 days later 10μg:10μg pIRES2-EGFP-BMP-2 plasmid was given to transfer into the tissues arounding the radial fracture site by gene gun at the time of 3 daye later after the operation time while during the operation we just made the radial frcture and then close the wound. Group 3 days later 0μg:10μg pIRES2-EGFP plasmid was given to tranfect the tissues arounding the radial fracture site by gene gun at the time of 3 daye later after the operation time, while during the operation we just made the radial frcture then close the wound. Group 14 days later another 10μg:10μg pIRES2-EGFP-BMP-2 plasmid was given to tranfect the tissues arounding the radial fracture site by gene gun at the time of operation when the fracture was made, then another time tranfer with the 10μg pIRES2-EGFP-BMP-2 was done at 14 days later to enhance the tranfer by exposing the same field with operation. Group 14 days later another 0μg:10μg pIRES2-EGFP-BMP-2 plasmid was given to transfer into the tissues arounding the radial fracture site by gene gun at the time of operation when the fracture was made, then another time tranfer with 10μg pIRES2-EGFP was done at 14 days later by exposing the same field with operation. Group control(0μg group):10μg pIRES2-EGFP plasmid was given to tranfer the tissues arounding the radial fracture site by gene gun at the time of operation as a control group. 1 week later, two rabbits were randomly selected from every group and were executed. The tissues around the fracture site were got for detection of the expression of BMP-2 protein by Western blot. The X-ray of the fracture site was aquired at the time of 4, 6, 8, 12 weeks after the operation. Two rabbits were randomly selected from every group and executed when the X-ray indicates the fracture unioned. The radial bone was got for observation and HE test was done to observe the mature degree of the new bone tissue. Results: 1) The constructed pIRES2-EGFP-BMP-2 plasmid shows two brands at the 1.2kbp and 5.3kbp after it was enzyme cut by EcoRI\ BglⅡand then electrophoresis. The analysis of the sequenc by the BLAST shows it has the same sequence according to the NM2001200 of gene bank. The GFP is positive when it was observed in the fluorescencet inverted microscope. Western blot shows a brand at the site of relative molecular mass of 30×103. 2) The rabbit BMSCs transfected by pIRES2-EGFP-BMP-2 plasmid shows persistent expression of GFP as a manner of endochylema distribution in the observation of the fluorescencet inverted microscope. Western blot shows a brand at the site of relative molecular mass of 30×103. 3) The pIRES2-EGFP-BMP-2 plasmid can be transfected into tissues arounding the fracture site and can express BMP-2 protein. Group 10μg shows shorter bone uninon time, more quantity of bone callus and the shorter time of bone cavity remodeling than Group5μg and Group 0μg. Group 3 days later 10μg shows shorter bone union time, more quantity of bone callus and shorter bone cavity remodeling time than Group 3 days later 0μg. Group 14 days another 10μg shows no obvious different with Group 14 days another 10μg according to bone union time and the time of bone cavity remodeling, but the quality of the bone callus seems better than other group. The HE tset shows no obvious difference between the intervention group and control group. Conclusion: 1) The pIRES2-EGFP-BMP-2 plasmid was constructed correctedly and can be transdected into NIH3T3 cell. The BMP-2 protein can be expressed in NIH3T3 cell. 2) The pIRES2-EGFP-BMP-2 plasmid can be transfected into rabbit MSCs and can express BMP-2 protein. 3) By the means of gene gun transfection, the pIRES2-EGFP-BMP-2 plasmid can be transfected into the tissues which around the bone fracture site. The BMP-2 protein can be expressed. With the increase of transfecting plasmid dose, the Group 10μg shows shorter bone union time, more bone callus and shorter bone cavity remodeling time than Group 0μg and Group 5μg. Three days later transfection will have no effect on the bone union time, quantity of the bone callus and bone cavity remodelling time than transfect at the fracture time. Another transfection after 14 days later can shorten the bone union time, the bone cavity remodelling time. And it can improve the bone callus quality obviously.