| Part Ⅰ Separation and culturing of SD rat BMSCs in vitro and the transfection of gene VEGF-165&BMP-2 and the identification of gene expression of transfected cellsObjective(1)To explore the method of separation and culturing of bone marrow stromal cells(BMSCs)in SD rats via density gradient centrifugation and adherence method.(2)To explore the method of transfection of double genes VEGF-165 and BMP-2 into bone marrow stromal cells in SD rats by liposome mediating.(3)Identification of the expression of genes and identification of the ability of osteogenesis in vitro after BMSCs transfection in SD rats.Methods(1)Experimental group: Group A(double gene transfection): p IRES-h BMP2-h VEGF165 transfection;Group B(single gene transfection):p IRES-h BMP2 transfection;Group C(single gene transfection): p IRES-h VEGF165 transfection;Group D: empty plasmid transfection(blank control)(2)Separate BMSCs via density gradient centrifugation after extraction of bone marrow in SD rats,then collect the middle cell layer,culture cells via adherence method.(3)Detect surface molecule of BMSCs in SD rats by flow cytometry.(4)Transfect gene VEGF-165 and BMP-2 into BMSCs of SD rat by liposome mediating(Lipofect-amine2000)and plot the growth curve of BMSCs after transfection via MMT method.(5)Determinate the expression of gene VEGF-165 and BMP-2 by enzyme-linked immunosorbent assay,and observe the staining expression of alkaline phosphatase,bone collagen and osteocalcin.Results:(1)Morphologic changes of BMSCs in rats:BMSCs adherent growth occured 12 hours after inoculation,BMSCs took a stripe shape,adherent cells increased with the passage of culture time,unadherent cells were removed gradually by fluid exchange.About a week after culturing,the colony began to form and become bigger and more integrated gradually,finally monolayers that adhere to the wall formed.After passage,the cells were oval or spherical and its adherent time were earlier than that before passage,proliferation rate became faster.To the third generation,cell proliferation rate reached peak value and its form were stability and cells distributed on the bottom of culture bottle.To the 5th generation,cell proliferation rate reduced,partial cells apoptosis.(2)BMSCs surface molecular detection:CD44 and CD90 were positive,and CD34 and CD45 were negative.It is confirmed that this experiment was successful in isolating and obtaining rat BMSCs.(3)Observe BMSCs after transfection marked by immunofluorescence via fluorescence microscopy,except for blank control group.Green fluorescence could be seen after 24 hours of transfection.Fluorescence intensity was enhanced obviously after 3 days of transfection.(4)Changes of growth curve of BMSCs after transfection:the OD value of bone marrow stromal cells was highest in 3 days.There was no significant difference in OD peak time and peak value between single gene transfection group and blank control group untransfected BMSCs.(5)One week after gene transfection,the contents of BMP-2 protein and VEGF-165 protein were the highest in the double gene transfection group,which were significantly higher than those of the two single gene transfection group.The two proteins were the lowest in the blank control group(P<0.05).(6)one week after gene transfection,the expression of alkaline phosphatase,bone collagen and osteocalcin in the double gene transfection group was significantly higher than that in the single gene transfection group and blank control group observed by staining method,analysed by histochemical score,differences were statistically significant(P<0.05).ConclusⅠion:(1)BMSCs of SD rats can be isolated from rat bone marrow and can be amplified in vitro.(2)Liposome-mediated gene transfection does not affect the mitotic proliferation of BMSCs after transfection.(3)The expression of BMSCs target gene in the double gene transfection group was good,the expression level of BMP-2 and VEGF-165 was significantly higher than that of single gene transfection group.Co-transfection of BMSCs with two genes promotes gene expression synergistically.(4)Co-transfection of BMSCs of SD rat with two genes can obtain better osteogenic ability in vitro than single gene transfection.Part Ⅱ Construction of double gene modified BMSCs tissue engineered bone and experimental study of repairing bone defect in ratsObjective(1)The bone defect of tibia in SD rats was made to provide an experimental model of bone defect for the treatment with double genes modified tissue engineering bone.(2)To verify the osteogenic effect of p IRES-h BMP2-h VEGF165 gene modified BMSCs combined with artificial inactivation of allogeneic bone in animal model of bone defect.Methods(1)Establishment of tibial bone defect model in SD rats: select SD rats,then amputate a 5 mm bone segment including periosteum by wire saw in the middle tibia of hind leg,cauterize bone stump and close medullary cavity by bone wax,finally fix fracture end via penetrating the medullary cavity with 0.8mm Kirschner’s needle.Confirm bone defect by X-ray examination 8 weeks later.(2)Co-culture Gene modified BMSCs and artificial inactivation of allogeneic bone for 2 weeks to construct four kinds of tissue engineering bone as Group A :double gene transfection,Group B: BMP-2 single gene transfection,Group C: VEGF-165 single gene transfection,Group D :blank group.(3)After successful modeling,tissue engineering bone was implanted into SD rat model of bone defect by operation.The osteogenic effect in vivo was observed by histologic observation,X-ray examination,bone mineral density and biomechanical measurement after 4 and 12 weeks of operation.Results:(1)On the day after operation,all SD rats recovered from anaesthesia and were unwilling to eat and exercise,and their gait was drunk.After feeding for 3 days,the rats recovered basically to the preoperative condition.(2)The results of X-ray examination: after 4 weeks of bone grafting,callus formation was seen at the fracture end of the double gene transfection group,and its fracture line was blurred.After 12 weeks of bone grafting,a plastic platelike bone junction could be seen at the fracture end of double gene transfection group,whose bone defect were repaired and medullary cavity were connected.Single gene transfection had a poor callus formation could not find obvious platelike bone junction.Os integumentale sclerosis could be found at at the fracture end of blank group,whose medullary cavity were closed.(3)The results of bone mineral density and biomechanical measurement: after 12 weeks of bone grafting,the bone mineral density(BMD)and maximum load of newborn bone tissue of double gene transfection group were significantly higher than those of the other three groups,and the difference was statistically significant(P<0.05).(4)Histological staining results: the histological score of newborn bone tissue of double gene transfection group was significantly higher than those of the other three groups,and the difference was statistically significant.HE staining of newborn bone at 4 weeks after treatment,bone-like tissue and a small amount of bone trabeculae and osteoblastomas were found in bone defect area of double gene transfection group,and only a few bone trabeculae and chondrocytes could be found in bone defect area of single gene transfection group,and only a few fibroblasts were found in bone defect area of blank group.HE staining of newborn bone at 12 weeks after treatment,a large number of mature trabeculae and continuous platelike bone formation could be found in bone defect area of double gene transfection group,and only a small amount of cartilage matrix were found in bone defect area of single gene transfection group,and only fibrous tissue were found in bone defect area of blank group(P<0.05).(5)Immunohistochemical staining results: the expression of BMP2 and VEGF protein in double gene transfection group was significantly higher than that in the other three groups,the difference is statistically significant(P<0.05).Conclusion:(1)The model of SD rats bone defect can be established objectively,and the method is simple,practical and reliable.(3)Tissue engineering bone constructed by p IRES-h BMP2-h VEGF165 double gene modified BMSCs combined with artificial inactivation of allogeneic bone has remarkable effect on osteogenesis in animal model of bone defect. |
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