| Objectives The aim of this study was to establish a standard procedure for isolation and culture of rats bone marrow derived mesenchymal stem cells(rBMMSCs), and to investigate the angiogenesis evidence in hindlimb ischemia of diabetic rats after intramuscular injection of BrdU labeled rBMMSCs. Methods 1)Isolation, culture and phenotypic identification of rBMMSCs:bilateral intact femur of 2 to 3 weeks old SD rats were cut off aseptically and 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 rBMMSCs primary culture, the prepared bone marrow cells were inoculated in LG-DMEM containing 10%FBS and cultured in a humidified atmosphere of 95% air and 5% CO2 at 37℃, fresh complete medium was changed every 2 days, whereas the nonadherent blood cells were eluted at the same time. When the adherent cells grown to 60%～70% confluency, subculture was performed with a cell density of 2～5×105/ml. Phenotypic properties of passage 3 rBMMSCs including CD45, CD29 and CD90 were measured by flow cytometry, and passage 3 rBMMSCs were labelled by BrdU in vitro. 2)Preparation of rat diabetic hindlimb ischemia model:male SD rats weighting 180～250 g were fed normal chow for 1 week. The diabetic rat model was induced by a single intraperitoneal injection of 55 mg/kg streptozotocin (STZ). The blood glucose level was measured at 1,7, and 14 days after STZ injection, and diabetes was confirmed when the blood glucose level was> 16.7 mmol/L. Diabetic rats that had been anesthetized with an intraperitoneal injection of 0.35 g/kg,10% chloral hydrate were prepared by ligation of bilateral femoral arteries and its branches proximal to the inguinal ligaments for producing a hindlimb ischemia model.3)rBMMSCs transplantation:BrdU-labeled passage 3 rBMMSCs were resuspended with 250μl normal saline, then the single cell suspension liquid(containing rBMMSCs in the total number of 2～5×106) were injected into 10 points 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.4)Evaluation of angiogenesis after transplantion:angiography of hindlimbs arteries in the model rats was performed after 2,6, and 10 weeks transplantation by digital subtraction angiography(DSA) through antegrade abdominal aorta catheterization followed by injection of 4 ml,30% iohexol with a velocity of 1 ml/s and 111 kPa pressure. Thereafter, paraffin sections of bilateral rectus femoris and gastrocnemius were sliced(5μm thickness), and HE 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, immunohistochemistry was applied to trace the survival and turnover of transplanted rBMMSCs. Results 1)The isolated and purified rBMMSCs expressed higher level of CD90 and CD29 but negative for CD45, which were consistent with the typical phenotypes belonging to mesenchmal stem cells. The frequency of in vitro BrdU-labelling BMMSCs was 82±3%.2) The glucose levels in rats peripheral blood were 5.4±0.5 mmol/L,27.3±1.6 mmol/L,27.3±2.2 mmol/L, and 25.4±2.3 mmol/L before and after STZ injection at 1,7,14 days, which showed higher than the standard criteria of diabetes(>16.7mmol/L). After ligation of femoral arteries and its branches, hindlimb muscles displayed acute ischemia signs and dark purple colour in the model rats.3)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 at 2 weeks post-transplantation, and was obviously increased after implantation of rBMMSCs at 6 and 10 weeks, the vasculature of left leg was always higher than the right.4)After 2 weeks transplantation, capillary densities of rectus femoris and gastrocnemius in left leg were 0.296±0.10 and 0.357±0.15, which were higher than right leg that were 0.132±0.06 and 0.225±0.08, respectively(p<0.05).5)Immunohistochemistry detected that some implanted rBMMSCs were stained positively with BrdU in vascular wall, which might have been incorporated into the vasculature. Conclusions The rBMMSCs with mesenchymal stem cells phenotypic properties were successfully isolated and cultured in this study. Transplantation of rBMMSCs by local injection could improve blood supply and contribute to angiogenesis in hindlimb ischemia of diabetic rats indicating that rBMMSCs implantation may be a novel method for inducing angiogenesis. |
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