ObjectiveTo explore a simple, efficient method for primary culture in vascular smooth muscle cells of diabetic rat, to establish a long-term stable diabetic vascular smooth muscle cell model in vitro, and lay the foundation for the study of diabetes chronic vascular lesions. MethodsTwenty diabetic Wister rats models were self-made by using streptozotocin intraperitoneal injection plus high fat and sugar feeding, the vascular smooth muscle cells in vitro was cultured by a modified enzymatic digestion. ResultsThe diabetic rat models were successfully established, the vascular smooth muscle cells cultured in vitro by modified enzyme digestion grew fast, the cell survival was 96%, it could meet the requirements for cell experiment in vitro. ConclusionComparing with the traditional method, the modified enzymatic digestion method is simple, economic, high survival rate.
ObjectiveTo investigate the angiogenesis mechanisms of vascular endothelial growth factor (VEGF) combined with basic fibroblast growth factor (bFGF) for arteriosclerosis obliterans of rat hind limb. MethodsThe models of hind limb arteriosclerosis obliterans of 60 male SD rats were established and randomly divided into four groups:normal saline (NS) group, VEGF group, bFGF group, and VEGF+bFGF group. The saline 1 mL and 100μg/L rhVEGF 1 mL were respectively injected into the abdominal cavity on every other day in the NS group and VEGF group. The 100μg/L rhbFGF 1 mL was multiply injected into the hind limb medial vastus muscle in the bFGF group. The 100μg/L rhVEGF 1 mL and 100μg/L rhbFGF 1 mL were respectively injected into the abdominal cavity and the hind limb medial vastus muscle on every other day in the VEGF+bFGF group. The angiogenesis of rat hind limb arteriosclerosis obliterans was observed on day 30 by digital subtraction angiography (DSA). The VEGF and bFGF protein and mRNA expressions in the hind limb medial vastus muscle tissues were tested by the Western blot and RT-PCR methods respectively. Results①The number of new collateral vessel in the VEGF+bFGF group was significantly more than that in the bFGF group (P < 0.05), VEGF group (P < 0.05), and NS group (P < 0.001), which in the VEGF group or bFGF group was significantly more than that in the NS group (P < 0.001), and which had no significant difference between the VEGF group and bFGF group (P > 0.05).②The protein and mRNA expressions of VEGF and bFGF in the VEGF+bFGF group were significantly higher than those in the bFGF group (P < 0.001), VEGF group (P < 0.001), and NS group (P < 0.001), which in the VEGF and bFGF were significantly higher than those in the NS group (P < 0.001), which had no significant difference between the VEGF group and bFGF group (P > 0.05). ConclusionsVEGF and bFGF in combination could increase the expressions of VEGF and bFGF in the rat hind limb ischemia region tissue and promote vascular endothelial cell proliferation and differentiation, and capillary sprouting growth, make angiogenesis of ischemic area, it is provided a new clinical treatment of peripheral arterial disease.