Objective To construct human recombinant lentiviral expression vector of microRNA-210 (miR-210)and to explore the over-expression of miR-210 on the capillary formation in human umbilical vein endothelial cells 12 (HUVE-12). Methods The recombinant lentiviral expression vector of pGCSIL-green fluorescent protein (GFP)-pre-miR-210 wasconstructed by molecular cloning and transfected to HUVE-12 (LV-miR-210-GFP group), only pGCSIL-GFP was transfectedas control group (LV-GFP group). The miR-210 expression activity was evaluated by GFP reporter through fluorescencedetection and real-time fluorescent quantitative PCR. The ephrinA3 protein expression was measured by flow cytometry. Theconcentration of vascular endothelial growth factor (VEGF) in culture supernatant was determined by ELISA. The cells werecultured in 96-well culture plate coated with Matrigel to assess the abil ity of capillary formation. Results The recombinantplasmid pGCSIL-GFP-pre-miR-210 was confirmed by restriction endonuclease analysis and DNA sequencing. Fluorescencedetection showed that the fluorescence intensity of GFP was highest between 48 and 72 hours after transfection. Real-timefluorescent quantitative PCR showed that the miR-210 expression of LV-miR-210-GFP group was 9.72 times higher than thatin LV-GFP group (t= —11.10，P=0.00). Flow cytometry analysis showed that the positive cell rate of enphrinA3 in LV-miR-210-GFP group (12.52% ± 0.67%) was significantly lower than that in LV-GFP group (73.22% ± 1.45%) (t= —66.12，P=0.00).The concentration of VEGF in supernatant in LV-miR-210-GFP group was significantly higher than that in LV-GFP group[(305.29 ± 16.52) pg/mL vs. (42.52 ± 3.11) pg/mL, t= —27.06，P=0.00]. In vitro capillary-l ike formation assay showed that thenumber of capillaries was significantly larger in LV-miR-210-GFP group than in LV-GFP group (17.33 ± 6.33 vs. 6.33 ± 2.33,t= —2.83，P=0.04). Conclusion The recombinant lentiviral expression vector of miR-210 is constructed successfully andHUVE-12 over-expressing miR-210 can significantly increase the capillary formation, which facil itates further study on themolecular functions of miR-210 in angiogenesis.
In this experiment, bone grafts with or without perios-teum were taken from both ilium, usually a small amont ofmuscle was attached. These two types of grafts were trans-ferred respectively to the subcutaneous layer of the feet andto the defects of the metacarpus. After the operation , thespeciments were under gross and histologic examinations, andvolumetric measurment of the grafts pericdically. Vascularregeneration was found one week after operation, and thosegrafts with periosteum showed vascular regeneration and less absoption more than those with no periosteum. The vascular regeneration of the abundant iliac grafts transfered to the dcfects was more than to the subcutaneous layer.
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.
Tissue-engineered tracheal transplantation has been reported and the technique of decellularized scaffold's preparation is mature. Regeneration of epithelium, cartilage and blood vessel is particularly important during tracheal transplantation. With the increasing improvement on cell acquisition and cell culture, as well as the factor of auxesis and cell differentiation, tissue-engineered technique provided possibility and clinical value for regeneration of epithelium, cartilage and blood vessel. This review focuses on the improvement and prospect of regeneration of epithelium, cartilage and blood vessel during tracheal transplantation.
Objective To observe endothelial progenitor cells (EPCs) participating in the formation of neovascularization in lung adenocarcinoma. Methods EPCs were transfected by recombinant adenovirus carrying LacZ gene in optimal transfection concentration, and then EPCs were injected into animal models of lung adenocarcinoma through the tail vein; afterwards, lung tissues were taken out for pathological examination in the 6th, 7th, 8th week respectively. EPCs were observed to take part in the angiogenesis in the lung adenocarcinoma through X-gal chromogenic dye. Results The optimal multiplicity of infection (MOI) of AD5F35LacZ transfected EPCs was 400. When MOI was 400, maximum transfection efficiency was 97.13±2.08. After 2 weeks, LacZ gene-transfected EPCs began to proliferate in vitro culture, then the EPCs were transplanted into animal models of lung cancer to be involved in the neovascularization formation in the 8th week after transplantation. Conclusion EPCs are involved in the formation of tumor neovascularization after transplantation.
Objective To explore the effect of natural hirudin on proliferation of human microvascular endothelial cells (HMVECs) and its preliminary mechanism of promoting angiogenesis. Methods Three-dimensional culture models of HMVECs were established in vitro and observed by inverted phase contrast microscopy after 24 hours of culturing. Then, the three-dimensional culture models of HMVECs were treated with different concentrations (1, 4, and 7 ATU/mL) of the natural hirudin, respectively, and Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum as control. The cell proliferations of 4 groups were detected by cell counting kit 8 (CCK-8) method at 24, 48, and 72 hours; the angiogenesis of 4 groups were observed by tube formation assay at 24 hours; the expressions of vascular endothelial growth factor (VEGF) and Notch1 of HMVECs in 4 groups were observed by immunofluorescence staining at 24 hours. Results The observation of cells in three-dimensional culture models showed that HMVECs attached to Matrigel well, and the cells formed tube structure completely after 24 hours. The results of CCK-8 test showed that the absorbance (A) value of 1 and 4 ATU/mL groups were higher than that of control group at each time point (P<0.05), andA value of 4 ATU/mL group was the highest. The A value of 7 ATU/mL group was significantly lower than those of 1 and 4 ATU/mL groups and control group (P<0.05). The tube formation assay showed that the tube structure was more in 1 and 4 ATU/mL groups than in 7 ATU/mL group and control group, and in 4 ATU/mL group than in 1 ATU/mL group, showing significant differences (P<0.05). There was no significant difference between 7 ATU/mL group and control group (P>0.05). The results of immunofluorescence staining showed that compared with control group, the Notch1 expression was higher in 1 and 4 ATU/mL groups and lower in 7 ATU/mL group; and there was significant difference between 4 and 7 ATU/mL groups and control group (P<0.05). The VEGF expression was higher in 1, 4, and 7 ATU/mL groups than in control group, in 4 ATU/mL group than in 1 and 7 ATU/mL groups, showing significant differences (P<0.05). Conclusion Natural hirudin can promote angiogenesis at low and medium concentrations, but suppress angiogenesis at high concentrations. Its mechanism may be related to the VEGF-Notch signal pathway.
ObjectiveTo investigate the effect of natural hirudin on revascularization of ischemic skin flap in rats using Micro-CT and three-dimensional (3D) reconstruction.MethodsThirty-two Sprague Dawley rats were prepared a ischemic skin flap (8.0 cm×1.8 cm) model on the back and randomly divided into hirudin group and control group (16 rats in each group). At immediate and within 3 days after operation, the rats were treated with hypodermic injection of natural hirudin 0.3 mL (including natural hirudin 6 ATU) every day in hirudin group and the equal amount of normal saline in control group. At 6 days after operation, the survival rate of skin flap was evaluated, histological changes were observed by HE staining, and the volemia, length of blood vessels, and number of blood vessels were analyzed with Micro-CT 3D reconstruction.ResultsBoth groups of rats survived to the end of the experiment without infection. Different degrees of necrosis occurred in the distal part of the skin flaps in both groups at 6 days after operation, but the flap survival rate of the hirudin group (72.11%±8.97%) was significantly higher than that of control group (58.94%±4.02%) (t=3.280, P=0.008). Histological observation showed that the histological hierarchy of the hirudin group was clearer than that of the control group, with more microangiogenesis and less inflammatory response and inflammatory cell infiltration. Micro-CT 3D reconstruction showed that the flap vessels in the hirudin group were more and denser, and the volemia, length of blood vessels, and number of blood vessels were significantly higher than those in the control group (P<0.05).ConclusionNatural hirudin can reduce the inflammation of tissue, promote the regeneration and recanalization of blood vessels in ischemic skin flap, so as to improve the survival rate of the flap.