| ObjectiveTransfecting Ad-BMP-2-IRES-HIF-1αmu whith was structured by ourresearch group into bone marrow-derived endothelial progenitor cells thatcultured in vitro,then to transplant them into the site of rabbitavascularnecrosis of the femoral head, in order to test the effect that promotenecrotic zone vascularization and osteanagenesis.Method1. Abtaining mononuclear cells by Lymphocytes layere fluid.2. Inducing the cells in M199medium into endothelial progenitor cells3. Testing endothelial progenitor cells by three different means.4. Transfecting Ad-BMP-2-IRES-HIF-1αmu into endothelial progenitor cells atoptimum index5. Making the modles of avascularnecrosis of the femoral head6. Three group：Group A （6）: Transfecting Ad-BMP-2-IRES-HIF-1αmu intoendothelial progenitor cells; Group（6）: endothelial progenitor cellssuspension; Group C（6）:only the culture fluid7. Transplant them into the site of rabbit avascularnecrosis of the femoralhead8. Measurement(1) The determination of animal model：x ray and HE staining of histologicalsection(2) The determination of BMP-2expressions (3) The results of revascularization detection：Immunohistochemicalexamination of CD34and observation for transparent slice of Ink perfusionand quantitative image analysis about blood vessel area(4) Histological detection on the site of avascularnecrosis of the femoral head9. Statistical analysisResults1. Identification of endothelial progenitor cells(1) Cellular morphologyThe anchorage-dependent cells showed typical crazy-paving patternunder the inverted microscope at15days after induction culture(2) EPCs specific surface markers were determined by the flow cytometrymethodThe positive rates of endothelial progenitor cells specific surface markersdetermined by the flow cytometry method were as follow:CD34(56.61±6.24)%, CD133(49.36±5.16)%, VEGFR-2(54.70±4.28)%(3) The endothelial progenitor cells ability of combining FITC-UEA-1andDiI-ac-LDLLThe cells which showed two fluorescence colors under the fluorescencemicroscope were endothelial progenitor cells, the positive rates of which wasabout50%.2. Transfection result was observed by the immunofluorescence microscopyThe value of MOI in the Group A was200,Transfection result wasobserved by the immunofluorescence microscopy were no obvious cytopathiceffect.3. The determination of animal model indexThe result of X-ray: after6weeks,there were low density imagealterations in the site of femoral head;proving that models has beensuccessful. After6weeks,histologic examination showed the tissue cells suchas osteoblasts to become less, bones become thinner and fracture, bonetrabecular had been destroyed, appeared a large number of empty hollows. 4. The determination of BMP-2expressionsUnder inverted microscope observation, cells number which pexpressedBMP-2were (13±1.41) in Group A, and there was g expression of BMP-2positive cells which were discovered in Group B.5. The results of revascularization detectionImmunohistochemical results of CD34showed (200times vision),2w、4wand8w after transplantation,there was statistical significance between GroupA and Group B、Group C (p <0.01); After2weeks of transplantation,statisticalcomparison between Group B and Group C was p <0.05;4weeks and8weeks after transplantation, statistical comparison between group B andgroup C were p <0.01, there were all statistically significant.Angiographic ink perfusion for transparent section morphology testshowed: Group A appeared a lot of angiogenesis、Group B had a smallangiogenesis、GroupC had not been seen obvious angiogenesis. analysisedInk perfusion of blood vessels area of image,2weeks,4weeks and8weeksafter transplantation, comparison between Group A and Group B、Group Cwere all tatistical significance (p <0.01); For every period of time aftertransplant, there was statistical significance between Group B and Group C (p<0.01).6. Histology detection of femoral head necrosis after transplantation6weeks after transplantation, HE staining of tissue section showed:Group A: new bone trabecular, capillary and osteoblast cell components, suchas continuous trabecular bone were discovered and bone empty hollows hadsignificantly reduced; Group B: angiogenesis, osteoblast and other tissuecells were less, the trabecular bone appeared fracture phenomenon, therewere still some bone empty hollows; Group C: a lot of trabecular bonefracture were discovered, and more bone empty hollows existed, there werenot new trabecular bone and vascular tissue appearing,and no obvious change compared with transplantationbefore.Conclusion1. Endothelial progenitor cells can induced by marrow-derived mononuclearcells.2. Endothelial progenitor cells can promote strangly vasculogenesis.3. It can promote strangly vasculogenesis and increase BMP-2factor by theway of transplanting EPCs transfected with Ad-BMP-2-IRES-HIF-1αmu. |
PDF Full Text Request