Experimental Study On Effect Of BMP-7 Gene In Vivo Transfer On Old Rats Fracture Healing Mediated By Polyethylenimine

There is a high incidence of delayed union, ununion and failure of inter-fixation after fracture in the elderly patients. It's common that the elderly patients have some age-related diseases when the fracture occurs including angiocardiopathy, cerebralvascular diseases, diabetes, CNS diseases and so on, which make the patients'condition more complicated and dangerous. Besides, the elderly patients can not stand the long time immobilization, usually have more complications of facture, needs special nursing. All these make the outcome of fracture in the age far from ideal. Not only have the patients to suffer much physical agony, but there is heavy economic burden on the patients'family and the society. In our country, as senile people number increases rapidly, the incidence of fracture of the elderly people increase dramatically. It's an important topic that how to treat the senile patients with fracture better.What is the most important reason why the therapy results is not ideal in the elderly people with fracture? It's recognized that the weakened reparation capability of fracture in the old body should be emphasized first. Many animal experiments have showed that the fracture healing process is much slower in the old rats than in the younger. When the old rats fractured, the callus appears late and the mineralization of the callus becomes slower comparing with the younger rats. Meantime the old one can not reach the normal range as to biomechanical quality and BMD. Researchers have take many experiments from different aspects to make clear what induce the healing process different in the old. Through morphology research it is found that the main abnormal during the fracture healing process in old rats includes as follows: delays of the reaction of periosteum and differentiation of osteoblast progenitor, decline of angiopoiesis and ossification of enchondral callus. Through morphometrology techniches it is found that the ossification area reduce with aging, but the enchondral callus forming is normal. At the cytology level, the number of MSCs decrease as age advancing, and these MSCs differentiate to lipocyte instead of osteoblast when stimulated by the environment changes. The biological significance of a mitogenic decrement for mesenchymal progenitor cells could lead to a quantitative deficiency and impaired healing in the elderly. At the molecular level,the expression of the BMP-2.4.7 is lower or shorter in the old rat fracture than in the younger. From above it is easily recognized that the healing of the old is different with what we know about the fracture healing process.The abnormal of fracture healing in the elderly may not be explained totally by osteoporosis. Not all aged fracture patients have osteoporosis, and meantime many osteoporosis patients are not old. It's found that there is a high morbidity of fracture in the old people even their BMD are normal, this may because the collagen part of the bone tissue becomes turbulence and the extend of mineralization still keep normal. The normality of collagen number and structure is the most important factor to maintain bone's stiffnity. The fracture healing may be unimpaired in the osteoporosis animal model. Bone marrow stroll cells could appear normal both in number and differentiation potential in a osteoporosis donor, this is different from the aged MSCs. After treatment with medication the fracture risk decrease in the osteoporosis, but their fracture reparation capacity is still abnormal, which means the fracture healing may delay. In conclusion, osteoporosis is not the main reason that the fracture healing process delays with aging.Gene therapy has been developed to improve the fracture healing. Through the gene vector's transfer, the therapy gene is expressed at the fracture location, the osteoinductive cytokines promote the healing reaction. Gene therapy has been approved to be a effective method for improving fracture healing. Virus vectors are currently used in most gene therapy experiment. Even virus vectors have high transfer capacity, because of safety consideration, the clinical research are limited. Last century 1990'Bossif found that polyehtylenimine(PEI) could act as a gene vector, later research show PEI has many advantages over other non-viral gene vector. PEI has been found to be effective, safe, easy modified, cheap and simply manipulated, so it is the fastest developing vector among the non-viral vectors in past ten years.Under the background as above, we designed this research. The purpose of this experiment is to find a practical gene therapy method to enhance fracture healing in the old rats. PEI is a reasonable choice as a gene vector. We choose BMP-7 gene as the target gene because it is the most potential osteoinductive among cytokines. This research has 4 stages as described below:PartⅠ: In vitro research of gene transfer by PEI1 Identification of PEI cytotoxicity. A different concentration of PEI was cultured with NIH3T3 cells, then the survive rate of cells was observed through MTT test. The result is PEI has no obvious cytotoxity on the NIH3T3 cell lines.2 Investigate the particle's physical characters of PEI and PEI-DNA complex. Observe the particles'micro-shape through TEM. When dispersed in solution PEI particles show a high-branched micro shape, the complex formed by PEI and DNA appears symmetrically spherical, The particles'radius is respectively 70nm in PEI and about 119nm in complex tested by dynamic light scatter method.3 Investigation of PEI's capacity to combine DNA molecule. Mix PEI and DNA according to different volume/mass ratio, test the complex running velocity in the gel electrophoresis. Result: When the ration is 2 or over, PEI can combine all DNA particles.4 Investigation of the gene-transfer efficiency. Under different ration transfer the EGFP gene into NIH3T3 cells by PEI, comparing the results with Lipo200-midiated same gene transfer. Result: The best transfer outcome appears when the ration is 3,and there is no statistical difference between the gene-transfer capacity of PEI and Lipo2000.Conclusion: PEI (MW13KDa,branched) has very low cytotoxicity in vitro experiment. It can condense the DNA molecular into nanometer-sized particles, which means it is a kind of nano-gene-vector. The ratio of PEI and DNA is the most important factor affecting PEI's gene transfer capacity. The best transfer efficiency can be acquired when the ratio is 3, and the result has no statistical difference from Lipo2000's. PEI is a safe gene vector with low cytotoxicity.PartⅡ: BMP-7 gene transfer into NIH3T3 cell mediated by PEI1 Clone the full length cDNA sequence of BMP-7, and construct a eucaryon expression vector. Total mRNA was extracted from cell line U2 OS. The full length of BMP-7 cDAN was obtained by a reverse transcript-polymerase chain reaction (PT-PCR). The product was ligated with the expression plasmid vector pcDNA3.1. Using restricted enzyme digestion assay to identify the length of the BMP-7 segment, sequence analysis was taken to make sure the DNA is correct. Result: The sequence analysis show we acquired a intact and correct BMP-7 cDNA segment.2 Using PEI transferring reagent to transfer BMP-7 gene into fibroblast. NIH3T3 cells were cultured by routine ways previously. Mix PEI and BMP-7 gene in accordance with the ideal ratio, then add the complex into well. 48 hour after transfect, detect the quantity of BMP-7 protein through Western-blot test .Result: Western-blot test showed the synthesis of BMP-7 protein was obviously increased. Conclusion: Obtain full length BMP-7 cDNA segment through RT-PCR, and construct a plasmid expression vector pcDNA3.1-BMP-7 successfully. Using PEI to transfer BMP-7 gene in to NIH3T3 cell is a valid way to increase BMP-7 protein synthesis.PartⅢ: EGFP gene transfection into mice muscle tissue in vivo by PEI. Inject the PEI/pEGFP-N1 complex percutaneously into muscle of mice thigh. At different time point after transfection obtain the muscle specimen, prepair freeze slice and observe the slice under confocal microscope. Calculate the green fluorescence positive cells and transfection efficiency. Result: The third day after transfection the positive cells become most, and then decline until the 5 day hardly observe one.Conclusion: PEI can successfully transfer the DNA in vivo.PartⅣ: The effect of BMP-7 gene transfection mediated by PEI on the fracture healing of old ratEstablish old rat femur fracture mold. Mix the BMP-7 plasmid 50μg and PEI whose volume is 3 fold over the mass of plasmid. Inject the transfect complex into the femur broken location.As contrast, BMP-7 plasmid 50μg without PEI was injected into fracture area. To observe the healing process, take X-rays, histological slices,Ⅰtype collagen immunohistochemical stains after fracture. At eighth week after fracture, take BMD and biomechanical examination. Result: The healing process was enhanced obviously treated by PEI-BMP-7 transfection, the quality of callus are improved through BMD and biomechanical test. Conclusion: BMP-7 gene therapy mediated by PEI is a valid way to enhance the healing of old rat . In vivo gene therapy mediated by PEI is a promising technique in fracture reparation research area.