Transplantation Of Endothelial Progenitor Cells Transferred By Vascular Endothelial Growth Factor And Base Fibroblast Growth Factor Gene For Neovascularization Of Transplanted Tissue

It is a fine application prospect for promoting neovascularization in the areas of plastic surgery, cardiovascular cerebrovascular ischemic disease, limb ischemic disease, et al. The focus of our study is to promote neovascularization for ischemic skin flap and fat graft in the area of plastic surgery.Partial necrosis is a common complication after the surgery of skin flap, which is a tough problem in plastic surgery. The main reason is insufficient blood-supply and injury of ischemia-reperfusion. Revascularization timely is the key method to solve this problem.The survival rate of fat tissue after transplantation is very low, only 30%～70%. The main reason is ischemia. The ability of fat tissue for tolerance of ischemia is very poor. After the fat graft, the revascularization of fat tissue is not timely. So the transplanted fat tissue is necrosis because of ischemia. The adequacy of graft perfusion is considered to be the key to its fate after fat graft.The formation of new blood vessels (neovascularization) occurs through two mechanisms: angiogenesis and vasculogenesis. Angiogenesis is the sprouting of microvessels from a preexisting capillary network, whereas vasculogenesis refers to blood vessel formation from endothelial progenitor cells that differentiate in situ. Then some researchers suggested the two strategies, therapeutic vasculogenesis and therapeutic angiogenesis, for neovascularization. Therapeutic angiogenesis has been applied to the manipulation of various processes that may benefit from increased blood vessel growth. Therapeutic vasculogenesis refers to the augmentation of blood vessel growth using circulating endothelial progenitor cells (as opposed to growth factors) and has generated interest for a variety of ischemic conditions.There are a series of progresses in VEGF gene therapy for angiogenesis. Now studies also demonstrate that bFGF and VEGF have synergetic effect for angiogenesis.In this study, we will use the combined application of therapeutic vasculogenesis of EPC and therapeutic angiogenesis of VEGF and bFGF gene therapy for animal models of fat graft and ischemic skin flap. The object of the study is to build a new strategy for promoting neovascularization of transplanted tissue, and to improve the survival rate and quality of ischemic tissue. This study will lay a foundation for further clinical application.1. Study of cultivate and identification of endothelial progenitor cells from human umbilical cord blood in vitroObjective To study the separation,cultivate,proliferation and identification of endothelial progenitor cells from human umbilical cord blood in vitro. Methods EPC separation was performed by density gradient centrifugation, separated cells were cultured in dishes coated with or without FN, the expression of cell markers CD31,CD34,KDR and CD133 was assessed using immunocytochemistry and immunofluorescence. The microstructure of the cells was observed by transmission electron microscope. The expression of CD133 and the percentage of CD133+ cells in adherent cells were assessed using fluorescence-activated cell sorting (FACS). Results Compared with the cells cultured in dish without FN, number of adherent cells and proliferation of cells cultured in dish with FN was increased. Immunostaining of cell markers CD31, CD34,KDR and CD133 of adherent cells was positive. The percentage of CD133+cells of adherent cells was 1.2%,21.3%,40.2%,1.0% at 0,4,7,10d respectively. Conclusion Density gradient centrifugation is a good method for EPC separation from human umbilical cord blood in vitro, and FN is very useful for culturing EPC in vitro.2 Amplification, extraction, purification and identification of pcDNA3.1(-)/ VEGF165, and VEGF gene transfection on EPC in vitroObjective To amplify, extract, purify and identify the vector of pcDNA3.1(-)/ VEGF165, and investigate the feasible and security of VEGF transfection on EPC mediated by liposome. Methods Eukaryotic expression vector of pcDNA3.1(-)/VEGF165 was amplified, extracted, purified and identified. After transfection the expression of VEGF was assessed using immunofluorescence and ELISA. MTT method was used to detect the effect of transfection by pcDNA3.1(-)/VEGF165 on EPC. Results After amplification, the subsequence of vector was exactly right confirmed by double enzyme digestion and gene sequencing. After transfection the expression of VEGF in EPC was augmented. Expression of VEGF in EPC's medium was 100±15,500±40,450±36,352±35,and 50±10 ng/L at 1,4,7,14,21d respectively. It wasn't found that VEGF transfection had a significantly inhibitory effect on EPC. Conclusion EPC can be the target cell for VEGF transfection, and it provides a novel gene therapeutic strategy for neovascularization which can possibly apply for clinic usage. After transfection, expression of VEGF reached the peak at 4 d.3 Cloning of bFGF gene, construction of pcDNA3.1/bFGF vector, bFGF and VEGF gene transfection on endothelial progenitor cell in vitroObjective To clone bFGF gene, construct pcDNA3.1/bFGF vector, and investigate the feasible and security of bFGF and VEGF gene transfection on EPC mediated by liposome. Methods Total RNA was extracted from MA148, a human ovarian cancer cellular line. After RT-PCR, bFGF was cloned to pGEM-T-Easy. Then the eukaryotic expression vector of pcDNA3.1/bFGF was constructed. After transfection, MTT method was used to detect the effect of transfection by pcDNA3.1/bFGF on EPC. The expression of VEGF and bFGF was assessed using ELISA after cotransfection of VEGF and bFGF gene. Results After amplification, the subsequence of vector was exactly right confirmed by double enzyme digestion and gene sequencing. After transfection the expression of bFGF in EPC increased. Expression of bFGF in EPC's medium was 60±15,360±20,280±17,162±13 and 20±5 ng/L at 1,4,7,14,21d respectively. It wasn't found that bFGF transfection had a significantly inhibitory effect on EPC. After cotransfection of VEGF and bFGF gene, the regulation of VEGF and bFGF expression was the same as single transfection of bFGF gene. Conclusion This study cloned bFGF gene and constructed pcDNA3.1/bFGF vector successfully. EPC can be target cell for VEGF transfection. After cotransfection of VEGF and bFGF gene, expression of VEGF and bFGF reach the peak at 4 d.4. Expression of endogenous VEGF,bFGF and VEGFR-2 in ischemic skin flapObjective To investigate dynamic expression of endogenous VEGF,bFGF and VEGFR-2 in ischemic skin flap for the usage of exogenous VEGF,bFGF gene therapy to ischemic skin flap. Methods The ischemic skin flap model of nude mice was established. The expression of endogenous VEGF,bFGF protein and mRNA at the midpoint and terminatio of the ischemic skin flaps was detected by immunohistostaining and RT-PCR. And the expression of endogenous VEGFR-2 mRNA was detected by RT-PCR. Results The survival rate of ischemic skin flaps is 68.2%. Expression of endogenous VEGF,bFGF and VEGFR-2 mRNA in ischemic skin flap began to increase after the operation. VEGF mRNA at the midpoint and terminatio of skin flaps and bFGF mRNA at the midpoint of skin flaps reached the peak at 24 h after operation. VEGFR-2 mRNA at the midpoint and terminatio of skin flaps and bFGF mRNA at the terminatio of skin flaps reached the peak at 12 h after operation. Conclusion Insufficient expression of endogenous growth factors as VEGF,bFGF at the terminatio of skin flaps may be the reason of partial necrosis. The time in 4d after the operation may be the right time to use exogenous VEGF.5. EPC co-transfected with VEGF165 and bFGF gene for improvinging the survival rate of ischemic flapObjective To investigate the feasibility of EPC co-transfected with VEGF165 and bFGF gene for ischemic flap, increasing neovascularization and augmenting the survival rate. Methods After cotransfection by VEGF165 and bFGF gene, EPCs were transplanted to ischemic flaps of the back of nude mice. CM-DiI was used to trace the transplanted cells. The blood perfusion of flaps was monitored by the laser Doppler, and the capillary density of flaps was detected by CD34 immunohistochemistry. The expression of VEGF and bFGF protein in vitro was assessed using ELISA. Results The flap survival rates of VEGF165 and bFGF gene cotransfection group, VEGF165 gene transfection group, bFGF gene transfection group, EPC group, and the control group were 99.5%±2.9%,97.2%±2.8%,78.5%±2.0%,60.3%±2.1%,34.2%±1.8% respectively. After transfection, the expression of VEGF and bFGF protein increased in VEGF165 and bFGF gene cotransfection group, VEGF165 gene transfection group, bFGF gene transfection group respectively, and reached the peak at 7d. The blood perfusion,capillary density of flaps of study groups were significantly higher than the control group. Conclusion EPC from human cord blood can increase ischemic flaps neovascularization and augmented survival rate, and the ability of promoting neovascularization of EPC co-transfected with VEGF165 and bFGF gene is more powerful than EPC alone.6. EPC co-transfected with VEGF165 and bFGF gene for improving the survival rate of transplanted fat tissue Objective To investigate the feasibility of EPC co-transfected with VEGF165 and bFGF gene for transplanted fat tissue, increasing neovascularization and improving the survival rate. Methods After cotransfection by VEGF165 and bFGF gene, EPCs were transplanted to free transplanted fat tissue at the back of nude micek. The nude mice were divided into 5 groups: VEGF165 and bFGF gene co-transfection group, VEGF165 gene transfection group, bFGF gene transfection group, EPC group, and the control group. CM-DiI was used to trace the transplanted cells. The capillary density of transplanted fat tissue was detected by CD34 immunohistochemistry. Results The survival rates of transplanted fat tissue of 5 groups were 98.9%±9.9%,96.2%±8.6%,88.7%±4.0%,75.3%±6.8%,40.2%±2.5% respectively. After transfection, the expression of VEGF and bFGF protein increased in VEGF165 and bFGF gene co-transfection group, VEGF165 gene transfection group, bFGF gene transfection group respectively, and reached the peak at 7d. The capillary density of transplanted fat tissue of study groups was significantly denser than the control group. Conclusion EPC from human cord blood can augment free transplanted fat tissue neovascularization and increase the survival rate, and the ability of promoting neovascularization of EPC transfected with VEGF165 and bFGF gene is more powerful than EPC alone.