Effect Of Umbilical Cord Mesenchymal Stem Cells Transfected Of Human VEGF-165 Gene On Revascularizition For Diabetic Rats Hind-limb Ischemia

Diabetic peripheral artery disease(PAD) as one of seriously chronic complications of diabetic mellitus(DM) is becming an increasingly significant problem in terms of morbidity and mortality of DM,compared with none diabetic patients.The arteriosclerosis vessels of diabetic PAD are always extensive and segmental diffuse stenosis or occlusion of distal artery,which may lead to gangrene and amputation of diabetic foot(DF),and seriously affect the patients’ life quality.The available treatment options including traditional medication,vascular bypass transplantation and intravascular interventional therapy et al are generally limited in terms of clinical efficiency because most PAD patients are always senior weak with many severe cardiovascular diseases with high surgery risk,So it is very urgent necessary to discover an new clinical treatment.How to promote vessel revascularizition and to establish effective vascular bypass flow become the key therapeutic problems.In recent years stem cells transplantation provide a novel therapeutic treatment on ischemia diseases.Bone marrow mesenchymal stem cells(BMSCs) are the most commen source of MSCs,BMSCs possess the potential for both ex vivo expansion and versatile differentiation which can be induced into bone,cartilage,fat,neural,myocardial,and vascular endothelial cell to participate the repairation of different organization.However very less data is reported the mechanisms underlying the effect for angiogenesis include the stem cells survival rate,differentiation,capacity,homing in vivo et al.Unfortunately,as it is well known,BMSCs invasive procedures,the frequency and differertiation potential significantly decease with old age and declined health condition,meanwhile,due to high glucose and hypoxia-ischemia micro-environments,the survival rate of MSCs transplantation and angiogenesis founction are greatly reduced,which significantly limit the effects of stem cell transplantation treatment. Compared with BMSCs,Umbilical cord mesenchymal stem cells(h UC-MSCs) share most of the characteristics of BMSCs and have many advantages such as their abundant,simple collection,noninvasive accessibility,weak immunogenicity, lack of ethical controversy,also h UC-MSCs have a powerful capacity of differentiation capacity and proliferation in different culture conditions,so h UC-MSCs are considered the ideal candidate of BMSCs.h UC-MSCs are highly suitable for further development of novel cell-based therapeutics.On blood circulation reconstruction,variety growth factors and cell factors can promot vessel regeneration,while vascular endothelial growth factor(VEGF) is the most powerful vascular growth factor to promot endothelial hyperplasia and the vascular regeneration,VEGF gene therapy can not maintain effective concentration in blood after injection because of expensive and short half-life,application VEGF treatment is greatly limited especially in diabetic patients.Therefore,how to improve the survival rate of transplanted MSCs and to increase the secretion of vascular endothelial growth factor are the most important and key factors in the efficacy of transplantation of MSCs.Accordingly,we designed this study to transfer VEGF-EGFP gene with adenovirus vector(Ad) into seed cells h UC-MSCs with gene transfection technique to improve endothelial cell differentiation capacity and to observe the safety and efficacy of gene transfered stem cell transplantation in the treatment of limb ischemia.EGFP gene expression can help to study the h UC-MSCs’ localization and in vitro and in vivo.The changes of transfected h UC-MSCs on the differentiation and proliferation of cells’ morphology and growth conditions were observed.Meanwhile the hind limb ischemia of high-fat diet type 2 DM rats models by femoral artery excision were established.The transfected h UC-MSCs were directly injected into local ischemic limb muscles,angiogenesis and the establishment of collateral circulation were observed to explore the mechanism of gene transfection on vasculogenesis and the blood supply of ischemic region.In this study,application of h UC-MSCs as VEGF gene treatment platform,can not only incease VEGF protein stable therapeutic secretion concentrations,and alse through VEGF of anti-inflammatory,anti-oxidation stress,and anti-apoptosis,vascular micro environment was well improved,which could provide the best survival space for h UC-MSCs’ proliferation,homing and differentiation,reduced h UC-MSCs’ apoptosis,increased the survival rate effectively.Combination use of growth factors by gene engineering can improve the angiogenesis efficiency of h UC-MSCs. Therefore this experiment may provide a new hope for the treatment of diabetic chronic lower extremity ischemic diseases. Part 1 Transfection of adenovirus containing vascular endothelial growth factor gene into umbilical cord mesenchymal stem cells in vitroObjective:To investigate the feasibility of transfection the adenovirus vector vascular endothelial growth factor(VEGF) gene into umbilical cord mesenchymal stem cells(h UC-MSCs) and the effects of VEGF transfection on h UC-MSCs function.Methods:First,to construct VEGF-EGFP recombinant gene adenovirus vectors,isolate and culture h UC-MSCs,then transfected h UC-MSCs with adenovirus vector-EGFP,the efficiency of transfection was detected by fluorescence microscope photography and flow cytometry so to determine the best Multiplicity of Infection(MOI).The recombinant Ad-VEGF adenovirus were marked with the EGFP protein,therefore,the VEGF-EGFP protein expression in h UC-MSC cells was also assessed by fluorescence microscopic photography.Cells were divided into VEGF-EGFP transfection group,the transfection EGFP group and control group.According to the best MOI,to detect the expression of VEGF by Western blot method and RT-PCR,Secreted VEGF from transfected h UC-MSCs cells in the supernatant samples were measured by ELISA,Cells proliferation activity of transfected h UC-MSCs was determined by MTT assays and cells cycle analysis was detected by flow cytometry.Results:Adenovirus mediated VEGF-EGFP gene can successfully transfect h UC-MSCs by fluorescence microscope photography and flow cytometry,24 hours after transfection the secreted VEGF of cell culture supernate can be detected by ELISA and can keep stable expression for at least 96 h,the VEGF expression of transfected VEGF-EGFP group was significantly higher than control group and EGFP group by Western blot method and RT-PCR.The ability of h UC-MSCs’ anti-apoptosis and survival got improved,MTT assay and flow cytometry results showed that VEGF gene transfection can increase the proliferation and differentiation of h UC-MSCs.Conclusions:All results suggested that Ad-VEGF infection increased the expression of VEGF effectively in h UC-MSC cells.Adenovirus mediated VEGF-EGFP gene can successfully transfect h UC-MSCs,and improve h UC-MSCs proliferation and differentiation,and significantly efficient stable expression of VEGF.VEGF gene transfection on h UC-MSCs therapy can provide better theoretical basis for the revascularizition of diabetic ischemia limb disease. Part2 High-fat diet type 2 diabetes lower limb ischemia model in ratsObjective:To assess how to establish the high-fat diet type 2 diabetic lower limb ischemia model based on the traditional inguinal femoral artery method.Methods:The SPF 20 male SD rats, DM group(10) were fed with high-fat diet for at least 6 months, intraperitoneal injection of STZ induced diabetes model(35mg/kg),the control group(10) were fed with common food,the skin in the middle of the right lower limb after anesthesia longitudinally,under inguinal femoral artery was isolated,ranging from the inguinal ligament until the knee joint,to cut off the femoral artery under the inguinal ligament, proximal ligation,then to the distal sharp dissection to the knee,ligation of femoral artery in all branches,cause the right lower limb ischemia model, to observe postoperative rat limb activity,body color,skin temperature,etc,and postoperative 1d,3d,14 d and 28 d after general anesthesia,maintain a relatively constant temperature,light,Laser Doppler Perfusion Imaging(LDPI) was used to evaluate the perfusion of both the left(ischemic) and right(non-ischemic) rats’ hindlimbs.Postoperative 28 d after anesthesia,isolated abdominal aorta,anticoagulant heparin saline water,the contrast at a rate of about 2ml/s about 1.5ml CT angiography of lower limbs. And then the auadriceps and gastrocnemius of contralateral and ipsilateral(ischemic side) were tested by HE staining and CD31 Immuno histochemistry and VEGF level with western blot method.Results:Postoperative 1d after ligation of femoral artery,the right lower limb ischemia for both groups were significantly reduced evaluated by Laser doppler perfusion imaging(LDPI) and CT angiography,which improved the successful lower limb ischemia model established,Postoperative 7d and 14 d the blood flow of both groups had a tendency to recover gradually,but on postoperative 28 d the blood flow of DM group had obvious relatively slow on surgical sides than those of the control group(P<0.05),CT angiography showed that DM group proximal femoral artery ligation had only a small amount of compensatory increased in blood vessels, no obvious blood flow in distal vessel. Histopathologic and Immuno histochemical staining showed that: Postoperative 28 d the ischemic muscle tissue of DM group had some cell lesions,inflammatory cell infiltration,and micro capillary density significantly lower than the health side,the expression VEGF of DM ischemic muscle tissue was greatly higher compared with that of the control group(P<0.05).Conclusions:Long-term high fat diet diabetic rats,through the femoral artery ligation can simulate the diabetic lower limb ischemia model,which can further help to explore and pathologic mechanism of the stem cells therapy on angiogenesis research of PAD. Part 3 Effect of umbilical cord mesenchymal stem cells transfected with human VEGF gene for diabetic limb ischemic of ratsObjective:To investigate effectiveness of VEGF over-expressed h UC-MSC cells transplantation on revascularizition in high-fat diet diabetic rats hind-limb ischemia.Methods:Lower limb ischemia models of high-fat diet type 2 diabetic rats were established by femoral artery ligation,which were divided into VEGF-EGFP+h UC-MSCs group,EGFP+h UC-MSCs group,h UC-MSCs group and PBS group randomly,then the VEGF over-expressed h UC-MSCs cells or control cells were used to transplant skeletal muscle tissues of lower limb ischemia model in type 2 diabetic rats.Fluorescent microscope was used to observe the transplanted cells survival rate and location in ischemia limb muscles.And on 3d,2or 4 weeks after transplantation the blood perfusion in ischemic skeletal muscle by LDPI detection and rat ischemia limb activity,body color,skin temperature,etc were observed,on 4weeks after transplantation the micro capillary density and quantity of ischemia limbs were further assessed by HE staining,CD31 Immune histochemistry,CT angiography,the expression levels of several proliferation and migration related genes such as ERK,AKt,MMP2,MMP9,TIMP1 and TIMP2 were also determined by western blot and quantitative RT-PCR assays.Results:On 1,2 or 4weeks after injection by fluorescent microscope VEGF gene transfected h UC-MSCs were observed in ischemia limb of rats which showed that VEGF-EGFP expressed and survived at high level throughout 4 weeks.And on 2,4weeks after injection blood perfusion of VEGF over-expressed h UC-MSCs transplanted limb were significantly improved compared with those of h UC-MSCs transplantation or control groups. Meanwhile on 4 weeks after injection,the new blood capillaries and vascular proliferation of VEGF over-expressed h UC-MSCs transplanted limb were significantly increased compared with those of simply h UC-MSCs transplantation by HE staining,Immune histochemistry,CT angiography.The m RNA and protein expression levels of ERK,AKT,MMP2 and MMP9 in VEGF over-expressed h UC-MSCs transplantation group increased dramatically compared with control group,while the expression of TIMP1 and TIMP2 had no significant change.Conclusions:VEGF over-expressed h UC-MSCs transplantation was more effective to stimulate angiogenesis and to increase blood perfusion than that of simply h UC-MSCs transplantation,as maybe a new choice to improve the lower limb vascular lesions of diabetics.