| Objectives:The aim of this study was to evaluate the feasibility and potential applications of human vascular endothelial growth factor expressing adenovirus vector(Ad-VEGF) transfected rat bone marrow derived-mesenchymal stem cells(BM-MSCs) by local intramuscular injection in the hindlimbs ischemia of diabetic rats.Methods:1)Isolation, culture and immunophenotypic identification of rat BM-MSCs:2to3weeks old SD rats were sacrificed by cervical dislocation. The ferums and tibias were isolated and the adherent soft tissues on ferums and tibias were removed thoroughly. Thereafter, the bone marrow cells dwelled in the marrow cavity were collected by D-PBS flushing, then the bone marrow cells’pellet were prepared by centrifugation. For BM-MSCs primary culture, the prepared bone marrow cells were inoculated in LG-DMEM containing10%FBS and10ng/ml bFGF and cultured in a humidified atmosphere of95%air and5%CO2at37℃. The non-adherent cells were removed by changing the medium every2days. When these primary cultures reached60%～80%confluence, subculture was performed with a cell density of2～5×105/ml. Phenotypic properties of passage3BM-MSCs including CD45, CD29and CD90were measured by flow cytometry, and labelled by BrdU in vitro.2)Ad-VEGF amplification and titer analysis:HEK293cells were inoculated in LG-DMEM containing10%FBS and cultured in a humidified atmosphere of95%air and5%CO2at37℃. Ad-VEGF were used to directly transfect into HEK293cells which grown to50%～80%confluency.48hours later, HEK293cells were harvested, then freezed and thawed three times at-80℃and37℃. Supernatant virus solution were diluted as10-fold gradient which were collected from HEK293cells harvested by centrifugation, and used to directly transfect HEK293cells growing in96-well culture plate at a density of1×104cells per well.18hours later, fluorescent positive cells were counted and virus titers were calculated under the fluorescence microscope.3)Rats BM-MSCs transfected by Ad-VEGF:when passage3BM-MSCs grown to50%～80%confluency, discarded the culture medium, then cells were transfected with different multiplicity of infection (MOI) of Ad-VEGF and incubated in LG-DMEM containing10ng/ml bFGF and2%FBS.48hours later, the transfected BM-MSCs were observed under fluorescence microscope and the transfection efficacy were analyzed by flow cytometry. Meanwhile, the appropriate MOI value were identified through combination with the cells growth inhibition effects by virus infection.4)Preparation of rats type2diabetes hindlimb ischemia model:adult male SD rats were fed high fat chow on weight380～450g for1month, and then the blood from rat tails vein was used to measured the serum lipids level. The type2diabetic rat model was induced by a single intraperitoneal injection of30mg/kg streptozotocin(STZ). The blood glucose level was measured at1,7, and14days after STZ injection, and diabetes was confirmed when the blood glucose level was>16.7mmol/L. Diabetic rats that had been anesthetized with an intraperitoneal injection of0.33ml/100g,10%chloralhydrate were prepared by ligation of bilateral femoral arteries and its branches proximal to the inguinal ligaments for producing a hindlimb ischemia model.5)Cells transplantation:the successful type2diabetes hindlimb ischemia model rats were randomly assigned into a Ad-VEGF transfected BM-MSCs transplantation group (n=13), a Ad-GFP transfected BM-MSCs transplantation group (n=14) and a BM-MSCs transplantation control group(n=15). BrdU-labeled passage3Ad-VEGF transfected BM-MSCs, Ad-GFP transfected BM-MSCs, non-transfected BM-MSCs were resuspended with400μl normal saline, then the single cell suspension liquid(containing cells in the total number of2×106) were injected into10points of the left hindlimb ischemic muscles including rectus femoris and gastrocnemius at beginning of ligation site,the same parts of the right hindlimb were injected with a equivalent normal saline as control.6)Evaluation of angiogenesis after transplantion:angiography of hindlimbs arteries in the model rats was performed after2,6, and10weeks transplantation by digital subtraction angiography(DSA) through antegrade abdominal aorta catheterization followed by injection of4ml,30%iohexol with a velocity of1ml/s and pressure of111kPa. Thereafter, paraffin sections of bilateral rectus femoris and gastrocnemius were sliced(5μm thickness), and H&E staining of the sections was used to observe and calculate the small vessel density by the ratio of the vessels to muscle fibers number. Moreover, immunofluorescence target to BrdU was applied to trace the survival and turnover of transplanted Ad-VEGF transfected BM-MSCs, Ad-GFP transfected BM-MSCs, and non-transfected BM-MSCs.Results:1)The isolated and purified BM-MSCs expressed higher level of CD90and CD29but negative for CD45, which were consistent with the typical phenotypes belonging to mesenchymal stem cells. The frequency of in vitro BrdU-labelling BM-MSCs was more than75%.2)The titer of Ad-VEGF successfully amplified in HEK293was reach up to8×108pfu/ml.3)The most efficiently transfection efficiency of BM-MSCs was53%by Ad-VEGF when MOI value was50. However, the vitality of BM-MSCs was obviously inhibited when MOI was higher than50.4)High fat chow group weight(404.4±18.41g), blood triglycerides(TG)(1.46±0.29mmol/L), total cholesterol(TC)(2.39±0.39mmol/L), were all significantly higher than that of normal chow group(P<0.05); The glucose levels in rats peripheral blood in high fat chow group were5.8±0.60mmol/L,20.52±4.01mmol/L,25.63±4.74mmol/L,24.12±3.35mmol/L before and after STZ injection at1,7,14days, respectively,which showed higher than the standard criteria of diabetes(>16.7mmol/L); the high index of weight, blood lipids level, blood glucose level was basically in line with type2diabetes standard. After ligation of femoral arteries and its branches, hindlimb muscles displayed acute ischemia signs and dark purple colour.5)DSA imaging analysis showed that the femoral arteries of the model rats disrupted near the inguinal ligaments, vasculature of bilateral legs in model rats could be observed at2w,6w,10w post-transplantation, and were abundant gradually with time elapse. Among them, the vasculature of Ad-VEGF transfected BM-MSCs group were always more abundant than Ad-GFP transfected BM-MSC, and non-transfected BM-MSCs not noly left hindlimbs but also right hindlimbs. However, there were no obviously different display of vessels between left and right muscles within the same group.6)Capillary densities of hindlimb muscles with H&E staining:the capillary densities of bilateral rectus femoris and gastrocnemius in Ad-VEGF transfected BM-MSCs group were all significantly richer than that of corresponding muscles in non-transfected BM-MSCs group, and Ad-GFP transfected BM-MSCs group at2w,6w,10w post-transplantation, respectively(P<0.05). At2weeks after transplantation, capillary densities of rectus femoris and gastrocnemius in left leg in each group were higher than that of right leg(P<0.05). At6weeks post-transplantation, capillary densities of gastrocnemius in left leg in Ad-GFP transfectered BM-MSCs, and non-transfected BM-MSCs were higher than that of right leg. While at the same time the capillary densities of rectus femoris and gastrocnemius in left leg in Ad-VEGF transfected BM-MSCs were higher than that of right leg and this property lasted to the10weeks post-transplantion(P<0.05).7)Immunofluorescence detected that some implanted BrdU labbled Ad-VEGF transfected BM-MSCs, Ad-GFP transfected BM-MSCs, non-transfected BM-MSCs were stained positively with BrdU in injection sites after2weeks transplantation.Conclusions:In vitro modified rat BM-MSCs successfully by transfecting of human VEGF contained vector Ad-VEGF. Transplantation of VEGF gene modifed BM-MSCs by local injection could implant in situ and improve blood supply and contribute to angiogenesis in hindlimb ischemia of diabetic rats indicating that VEGF gene modifed BM-MSCs implantation may be the feasibility for inducing angiogenesis and could be used in diabetic foot therapy. |
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