The Study Of Bmscs Transfected With VEGF Gene For Diabetic Foot Ulcer

Background and aims:Diabetic foot ulcer (DFU) is one of the most severe complications of diabetics. It was reported that14%-24%patients with DFU had to eventually undergo amputation. Angiogenesis dysfunction resulting from the imbalance of microenvironment in wound surface is the main reason for protracting course of DFU. Therefore, to promote angiogenesis and to improve microenvironment will be effective in healing of wound surface. Gene therapy with vascular endothelial growth factor (VEGF) has been currently used for lower extremity and myocardial ischemic diseases, and has showed promising results in the experimental study. Furthermore, recombinant adenovirus vector containing VEGF165gene for skin ulcer of diabetic rat has been reported, and the method was demonstrated to have angiogenesis effect and to short the time of healing. However, to our knonledge, there has no study on the effects of VEGF165gene in combination with stem cell therapy for DFU wound. Therefore, the aim of the present study was two fold:Firstly, to construct a recombinant adenovirus vector that containing hVEGF165, and to observe the expression of hVEGF165by transfecting pAdxsi-EGFP(enhanced green fluorescence protein)-hVEGF165into rat bone marrow mesenchymal stem cells (BMSCs) in vitro; Secondly, to transplant this vector in to wound surface of DFU rats (in vivo), and to observe the survival of wound cell and the effects of the therapy in angiogenesis and healing of wound.Methods:hVEGF165was liberated from plasmid and was subcloned into pShuttle-CMV (cytomegalovirus)-EGFP. The pShuttle-CMV-EGFP was then transferred to pAdxsi vector, by which pAdxsi-EGFP-hVEGFi65virus plasmid was obtained and was identified by enzymes restriction analysis and genetic sequencing. The pAdxsi-EGFP-hVEGF165was linearized by digestion with restriction endonuclease PacI, and was then transfected into human embryonic kidney cells HEK293. The retrieved recombinant adenovirus was titrated by using50%tissue culture infective dose (TCID50) assay. Wistar rat BMSCs were cultured and were infected with recombinant adenovirus that containing EGPF (pAdxsi-EGFP). The multiplicities of infection (MOI) of transfection were determined by fluorescence microscope and flow cytometry (FCM), by which the most optimal value of MOI was confirmed and was used for transfecting pAdxsi-EGFP-hVEGF165into BMSCs. The expression of hVEGF165gene was indentified by performing western blot, RT-PCR and enzyme linked immunosorbent assay (ELISA). The effect of transfection on BMSCs proliferation was assessed by MTT. Foot ulcer model was established among120Wistar rats, for which the prepared cells were transplanted into wound surface. Five study groups were establised:arm A, normal ulcer rats injected with PBS; arm B, DFU rats injected with PBS; arm C, gene therapy rats injected with Ad-VEGF165; arm D, stem cell therapy rats injected with BMSCs; arm E, gene transfection therapy rats injected with BMSCs/Ad-VEGF165,24rats were assigned into each of these groups. Survival rates of the transplanted cells labelled with EGFP were reported, and healing effeacy of and pathological changes of wound surface of rats from each of these groups were observed. Anti-CD34immunohistochemisty was performed for estimating regeneration of blood vessels. Western blot and q-PCR were carried out for examining the expression of VEGF.Results:The expression of hVEGF165cDNA in recombinant adenovirus plasmid was indentified by enzymes restriction analysis and genetic sequencing. The titer of virus could be up to1×1010pfu/mL after several rounds of transfection and amplification. The efficiency of transfection on FCM was88%when MOI being150pfu/cell, at which the most optimal of MOI was achieved, as observed on fluorescence. The expression of hVEGF165was detected after48hours of the transfection at both mRNA and protein levels. The results of ELISA showed the expression of hVEGF165peaked on day7, and the production was found even after day20. Furthermore, the expression of hVEGF165protein on day1,3,5,7,9,11,13,15and20in the group transfected with pAdxsi-EGFP-hVEGF165was significantly higher than that of the group transfected with pAdxsi-EGFP and that of untransfected group (P<0.05). The results of MTT demonstrated that there was no significant difference on absorbance between transfected and untransfected BMSCs groups at any specified time point (P>0.05). The DFU model was easily establised in rats. The EGFP labelled BMSCs were observed at the24th hours and on the7th day after transplantation, respectively. Arm E had higher wound healing rates, expression of VEGF at gene and protein levels, and amount of blood capillary compared with arm B, C and D on the day1,3,7,14, respectively. Nevertheless, the results had significat differences as compared with these from arm A (P<0.05).Conclusion:BMSCs was suitable for gene transfection, and hVEGF165gene can be successfully transferred into BMSCs with high efficiency using pAdxsi-EGFP-hVEGF165at a MOI of150pfu/cell. The transfected BMSCs can highly express hVEGF165without having effect on its growth and proliferation. By comparing with other treatments, VEGF gene transfection therapy achieves better results in terms of changes in endogenous VEGF164, the amount of blood vessels and wound healing rates. Gene transfected BMSCs has the effect of promoting angiogenesis and wound healing of DFU.