Study On The Angiogenesis Effect Of The Con-transplantion Of Bone Marrow Mesenchymal Stem Cells And Umbilical Vein Endothelial Cells Which Were Transfected With VEGF 165 Gene In Vivo

Objective:The preliminary experiments of our research group has proved that bone marrow mesenchymal stem cells co-cultured with umbilical vein endothelial cells which transfected with VEGF 165 gene through adenovirus can strongly promote angiogenesis?proliferation and migration in vitro,at compared with other groups.The groups which were transfected only GFP without VEGF 165 gene can not affect the ability of angiogenesis?proliferation and migration.However,because of the micro environment which has strongly differentation in vivo and in vitro.This experiment committed to observe the role of treatment on ischemic skin flap in back of Sprague-Dawley rat,what was transplanted bone marrow mesenchymal stem cells with umbilical vein endothelial cells which were transfected with VEGF 165 gene.To observe survival status of the ischemic skin flap and angiogenesis,and the same time to detect the VEGF density of peripheral blood.In order to explore the ability of angiogenesis by transplanted bone marrow mesenchymal stem cells with umbilical vein endothelial cells which were transfected with VEGF 165 gene in vivo,to move forward a single step to offer theoretical basis and experimental basis to build vascularized bone tissue engineering which can be used to repair long bone coloboma.Methods:1.We get tibial and femoral bone marrow of SD rat under sterility,and then separating and cultivating bone marrow mesenchymal stem cells in vitro.Cultivating HUVEC after recovering in vitro,and then transfected with VEGF 165 gene to HUVEC through adenovirus.2.Useing blood count plate to count each group cells.Metered volume with 1ml DMEM has 1×10~6 cells according to the countting data in each group.3.We build a ischemic skin flap with 4cm×1.5cm in back of Sprague-Dawley rat,taking each group cells to transplant in corresponding rat and observe survival status of the ischemic skin flap.A group:transplant BMSCS and HUVEC which transfected with VEGF 165 gene.B group:transplant only HUVEC which transfected with VEGF 165 gene.C group:transplant BMSCS and HUVEC which transfected without VEGF 165 gene.D group:transplant only HUVEC which transfected without VEGF 165 gene.E group:blank control group,injection with only DMEM.4.Observing flap state from 1 to 11 day after operration;Detecting VEGF density of peripheral blood in serum with ELISA after operration of 2.4.7.14 day;Using digital camera to take a picture after operration of 11 day,and then counting the flap survival rate with image pro-plus software;Randomly selecting 5 rats in each group to be put to death and taking specimen in living area,which were did HE staining and immunohistochemistry of CD31 to count microvessel density.Results:1.The flap survival quality of A group is overtop other groups.2.VEGF continuously high expression and gradually rise in peripheral blood postoperation in A and B group.The VEGF of A group always exceed B group in different times(P<0.05);The VEGF of C.D and E group have no significant difference in different times(P>0.05);The VEGF of A group always exceed C.D and E group in different times(P<0.05).3.The flap survival rate of each group:A group:86.4%±5.5%;B group:62.5%±4.7%;C group:52.3%±4.2%;D group:45.8%±3.6%;E group:32.4%±1.7%.A.B.D.E group compare which have significant difference with each other(P<0.05);A.C.D.E group compare which have significant difference with each other(P<0.05).4.The results of HE staining and immunohistochemistry of CD31 prove that the MVD of A group is much longer than other groups.Conclusion:Transplanted bone marrow mesenchymal stem cells and umbilical vein endothelial cells which transfected with VEGF 165gene have dramaticlly ability of promoting angiogenesis in vivo.