The aim of the study is to identify the effects and underlying mechanisms of visfatin on inflammation and necroptosis in vascular endothelial cells. Human umbilical vein endothelial cells (HUVECs) were stimulated with visfatin or pretreated with Polyinosinic acid (LOX-1 inhibitor). By using the Western blot, RT-PCR, immunocytochemistry, enzyme-linked immunosorbent assay (ELISA), MTT and flow cytometry technique, the occurrence of inflammation and necroptosis in HUVECs were evaluated. Our results showed that 100 ng/mL visfatin significantly increased the mRNA and protein expression of monocyte chemotactic protein 1 (MCP-1) and LOX-1 after 24 hours’ treatment in HUVECs. However, pretreatment with Polyinosinic acid could significantly reduce the expression of MCP-1 compared with visfatin group. Additionally, 100 ng/mL visfatin could induce the production of necrotic features and increase the mRNA expression of BMF (one of the markers of necroptosis), while pretreating with Polyinosinic acid markedly downregulated the mRNA expression of BMF gene and promoted the cell proliferation. These results indicate that visfatin might induce inflammation and necroptosis via LOX-1 in HUVECs, suggesting that visfatin plays a central role in the development of atherosclerosis.
ObjectiveTo investigate the mechanism of G protein coupled receptor kinase interacting protein 1 (GIT1) affecting angiogenesis by comparing the differentiation of bone marrow mesenchymal stem cells (BMSCs) differentiated into endothelial cells between GIT1 wild type mice and GIT1 gene knockout mice.MethodsMale and female GIT1 heterozygous mice were paired breeding, and the genotypic identification of newborn mice were detected by PCR. The 2nd generation BMSCs isolated from GIT1 wild type mice or GIT1 gene knockout mice were divided into 4 groups, including wild type control group (group A), wild type experimental group (group A1), GIT1 knockout control group (group B), and GIT1 knockout experimental group (group B1). The cells of groups A1 and B1 were cultured with the endothelial induction medium and the cells of groups A and B with normal cluture medium. The expressions of vascular endothelial growth factor receptor 2 (VEGFR-2), VEGFR-3, and phospho-VEGFR-2 (pVEGFR-2), and pVEGFR-3 proteins were detected by Western blot. The endothelial cell markers [von Willebrand factor (vWF), platelet-endothelial cell adhesion molecule 1 (PECAM-1), and vascular endothelial cadherin (VE-Cadherin)] were detected by flow cytometry. The 2nd generation BMSCs of GIT1 wild type mice were divided into 4 groups according to the different culture media: group Ⅰ, primary cell culture medium; group Ⅱ, cell culture medium containing SAR131675 (VEGFR-3 blocker); group Ⅲ, endothelial induction medium; group Ⅳ, endothelial induction medium containing SAR131675. The endothelial cell markers (vWF, PECAM-1, and VE-Cadherin) in 4 groups were also detected by flow cytometry.ResultsWestern blot results showed that there was no obviously difference in protein expressions of VEGFR-2 and pVEGFR-2 between groups; and the expressions of VEGFR-3 and pVEGFR-3 proteins in group A1 were obviously higher than those in groups A, B, and B1. The flow cytometry results showed that the expressions of vWF, PECAM-1, and VE-Cadherin were significantly higher in group A1 than in groups A, B, and B1 (P<0.05), and in group B1 than in groups A and B (P<0.05); but no significant difference was found between groups A and B (P>0.05). In the VEGFR-3 blocked experiment, the flow cytometry results showed that the expressions of vWF, PECAM-1, and VE-Cadherin were significantly higher in group Ⅲ than in groupsⅠ, Ⅱ, and Ⅳ, and in group Ⅳ than in groups Ⅰ and Ⅱ (P<0.05); but no significant difference was found between groups Ⅰ and Ⅱ (P>0.05).ConclusionGIT1 mediates BMSCs of mice differentiation into endothelial cells via VEGFR-3, thereby affecting the angiogenesis.
Objective To investigate the protocols of combined culture of human placenta-derived mesenchymal stem cells (HPMSCs) and human umbilical vein endothelial cells (HUVECs) from the same and different individuals on collagen material, to provide the. Methods Under voluntary contributions, HPMSCs were isolated and purified from human full-term placenta using collagenase IV digestion and lymphocyte separation medium, and confirmed by morphology methods and flow cytometry, and then passage 2 cells were cultured under condition of osteogenic induction. HUVECs were isolated from fresh human umbilical vein by collagenase I digestion and subcultured to purification, and cells were confirmed by immunocytochemical staining of von Willebrand factor (vWF). There were 2 groups for experiment. Passage 3 osteoblastic induced HPMSCs were co-cultured with HUVECs (1 ∶ 1) from different individuals in group A and with HUVECs from the same individual in group B on collagen hydrogel. Confocal laser scanning microscope was used to observe the cellular behavior of the cell-collagen composites at 1, 3, 5, and 7 days after culturing. Results Flow cytometry showed that HPMSCs were bly positive for CD90 and CD29, but negative for CD31, CD45, and CD34. After induction, alizarin red, alkaline phosphatase, and collagenase I staining were positive. HUVECs displayed cobble-stone morphology and stained positively for endothelial cell marker vWF. The immunofluorescent staining of CD31 showed that HUVECs in the cell-collagen composite of group B had richer layers, adhered and extended faster and better in three-dimension space than that of group A. At 7 days, the class-like microvessel lengths and the network point numbers were (6.68 ± 0.35) mm/mm2 and (17.10 ± 1.10)/mm2 in group A, and were (8.11 ± 0.62) mm/mm2 and (21.30 ± 1.41)/mm2 in group B, showing significant differences between the 2 groups (t=0.894, P=0.000; t=0.732, P=0.000). Conclusion Composite implant HPMSCs and HUVECs from the same individual on collagen hydrogel is better than HPMSCs and HUVECs from different individuals in integrity and continuity of the network and angiogenesis.
ObjectiveTo compare the different effects of ubiquitin(UB) on human umbilical vein endothelial cells (HUVECs) and macrophages under normal circumstances,and analyze whether UB could protect HUVECs from lipopolysaccharide(LPS) induced injury. MethodsThe morphologic changes of HUVECs in vitro with up-rising concentrations of UB interventions were observed. HUVECs and human macrophages in vitro were divided into 4 groups according to UB concentration (0.01 μg/mL,0.1 μg/mL, 1 μg/mL, and 10 μg/mL). Supernatant and cells of each group were collected in 24 h after UB intervention. The levels of TNF-α and VCAM-1 in supernatant were measured by ELISA while NF-κB protein level in cells was detected by Western blot. HUVECs were divided into a LPS group(LPS 10 μg/mL) and an UB+LPS group(UB 0.1 μg/mL,LPS 10 μg/mL). The supernatant of the two groups were collected in 8,16 and 24 h after LPS and UB intervention. The levels of TNF-α and VCAM-1 in supernatant were measured by ELISA. ResultsThe injury of HUVECs got worse with the ascending concentrations of UB.At the concentration of 50 μg/mL,UB induced HUVECs got ballooned and died massively. With the increase of UB concentration,the levels of TNF-α and VCAM-1 in HUVECs' supernatant ascended firstly and then descended,while those in human macrophages' supernatant ascended gradually. zHowever,the tendency of the NF-κB protein level in the two kinds of cells was similar when the concentration of UB increased.At the consentration of 0.1 μg/mL or 1 μg/mL,ubiquitin induced NF-κB protein level obviously increased.At the concentration of 0.01 μg/mL or 10 μg/mL,UB induced the protein level was similar with those of the control group and even decreased slightly. There was no significant difference in TNF-α or VCAM-1 levels at each time point between the LPS group and the UB+LPS group. ConclusionsUB injuries HUVECs obviously at a low concentration but injuires human macrophages at much higher concentraton. UB can not protect HUVECs from LPS-induced injury in vitro.
Objective To study the biological behavior of osteoblast and vascular endothelial cell culture. Methods The osteoblasts and vascular endothelial cells were obtained from calvarial bone and renal cortox of 2-week rabbits respectively. The experiment were divided into group A (osteoblasts), group B (vascular endothelial cells) and group C(co-cultured osteoblasts and vascular endothelial cells). The cells were identified with cytoimmunochemical staining. The cellular biological behavior and compatibilitywere observed under inverted phase contrast microscope and with histological staining. The cells viability and alkaline phosphatase(ALP) activity were measured. Results The cytoimmunochemical staining showed that the cultured cells were osteoblasts and vascular endothelial cells .The cellular compatibility of osteoblasts and vascular endothelial cells was good. The ALP activity was higher in group C than in group A and group B(P＜0.01), and it was higher in group A than in group B(P＜0.05). In group C, the cellproliferation were increased slowly early, but fast later. Conclusion Thecellular compatibility of osteoblasts and vascular endothelial cells were good. The vascular endothelial cells can significantly increased the osteoblast viability and ALP activity,and the combined cultured cells have greater proliferation ability.
【摘要】 目的 通过比较两种原代人脐静脉内皮细胞的分离培养方法并对细胞特异性抗原进行鉴定,探索提高原代内皮细胞体外培养存活率及纯化率的方法。 方法 采用一次性无菌注射器向人脐静脉灌注消化液,消化液的浓度和消化时间分别025%(质量体积比)胰蛋白酶,10 min和01%(质量体积比)胶原酶Ⅱ,15 min。通过在倒置显微镜下观察细胞的形态特点和用免疫荧光染色的方法对细胞进行鉴定,比较两种消化方法的优劣。 结果 01%胶原酶Ⅱ,15 min的消化方法较025%胰蛋白酶,10 min对原代人脐静脉内皮细胞有更好的分离效果,活细胞数量多且细胞纯度较高。免疫荧光染色结果表明细胞内有Ⅷ因子相关抗原表达。结论 胶原酶Ⅱ可以有效分离脐静脉内皮细胞,最佳消化条件是01%胶原酶Ⅱ,37℃,15 min。【Abstract】 Objective To explore the optimal method for primary culture of human umbilical vein endothelial cells (HUVECs). Methods HUVECs were prepared from human umbilical cords by 01% collagenase Ⅱ digestion for 15 minutes and 025 trypsinase digestion for 10 minutes,respectively. HUVECs were observed under inverted microscope and identified by immunofluorescence.The two methods of digestion were compared. Results More HUVECs were harvested through the method of 01% collagenase Ⅱ for 15 minutes,which expressed Ⅷ related antigen. Conclusion The method of 0.1% collagenase Ⅱ digestion for 15 minutes is a better choice to isolate HUVECs.
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.
Objective To study the differenation of adult marrow mesenchymal stem cells(MSCs) into vascular endothelial cells in vitro and to explore inducing conditions. Methods MSCs were isolated from adult marrow mononuclear cells by attaching growth. MSCs were divided into 4 groups to induce: the cells seeded at a density of 5×103/cm2 in 2% and 15% FCS LDMEM respectively (group1 and group 2)， at a density of 5×104/cm2 in 2% and 15% FCS LDMEM respectively (group 3 and group 4); vascular endothelial growth factor(VEGF) supplemented with Bovine pituitary extract was used to induce the cell differentiation. The differentiated cells were identified by measuring surfacemarks (CD34, VEGFR2, CD31 and vWF ) on the 14th day and 21st day and performed angiogenesis in vitroon the 21st day.The cell proliferation index（PI）of different inducing conditions were measured. Results After induced in VEGF supplemented with Bovine pituitary extract, the cells of group 3 expressed the surface marks CD34, VEGFR-2, CD31 and vWF on the 14th day, the positive rates were 8.5%, 12.0%, 40.0% and 30.0% respectively, and on the 21st day the positive ratesof CD34 and VEGFR2 increased to 15.5％ and 20.0％, while the other groups did not express these marks; the induced cells of group 3 showed low proliferating state(PI was 10.4%) and formed capillary-like structure in semisolid medium. Conclusion Adult MSCs can differentiate into vascular endothelial cellsafter induced by VEGF and Bovine pituitary extract at high cell densities and low proliferatingconditions，suggesting that adult MSCs will be ideal seed cells forthe therapeutic neovascularization and tissue engineering.
ObjectiveTo investigate the heterotopic osteogenesis of tissue engineered bone using the co-culture system of vascular endothelial cells (VECs) and adipose-derived stem cells (ADSCs) as seed cells.MethodsThe partially deproteinized biological bone (PDPBB) was prepared by fibronectin combined with partially deproteinized bone (PDPB). The ADSCs of 18-week-old Sprague Dawley (SD) rats and VECs of cord blood of full-term pregnant SD rats were isolated and cultured. Three kinds of tissue engineered bone were constructed in vitro: PDPBB+VECs (group A), PDPBB+ADSCs (group B), PDPBB+co-cultured cells (VECs∶ADSCs was 1∶1, group C), and PDPBB was used as control group (group D). Scanning electron microscopy was performed at 10 days after cell transplantation to observe cell adhesion on scaffolds. Forty-eight 18-week-old SD rats were randomly divided into groups A, B, C, and D, with 12 rats in each group. Four kinds of scaffolds, A, B, C, and D, were implanted into the femoral muscle bags of rats in corresponding groups. The animals were killed at 2, 4, 8, and 12 weeks after operation for gross observation, HE staining and Masson staining histological observation, and the amount of bone collagen was measured quantitatively by Masson staining section.ResultsScanning electron microscopy showed that the pores were interconnected in PDPB materials, and a large number of lamellar protein crystals on the surface of PDPBB modified by fibronection were loosely attached to the surface of the scaffold. After 10 days of co-culture PDPBB and cells, a large number of cells attached to PDPBB and piled up with each other to form cell clusters in group C. Polygonal cells and spindle cells were mixed and distributed, and some cells grew along bone trabeculae to form cell layers. Gross observation showed that the granulation tissue began to grow into the material pore at 2 weeks after operation. In group C, a large number of white cartilage-like substances were gradually produced on the surface of the material after 4 weeks, and the surface of the material was uneven. At 12 weeks, the amount of blood vessels on the surface of group A increased, and the material showed consolidation; there was a little white cartilage-like material on the surface of group B, but the pore size of the material did not decrease significantly; in group D, the pore size of the material did not decrease significantly. Histological observation showed that there was no significant difference in the amount of bone collagen between groups at 2 weeks after operation (F=2.551, P=0.088); at 4, 8, and 12 weeks after operation, the amount of bone collagen in group C was significantly higher than that in other 3 groups, and that in group B was higher than that in group D (P<0.05); there was no significant difference between group A and groups B, D (P>0.05).ConclusionThe ability of heterotopic osteogenesis of tissue engineered bone constructed by co-culture VECs and ADSCs was the strongest.
To study the potential molecular mechanism of tumor angiogenesis in its microenvironment, we investigated the effects of HepG2 conditioned medium on the proliferation of vascular endothelial cell and vascular angiogenesis in our laboratory. Human umbilical vein endothelial EA.hy926 cells were co-cultured with HepG2 conditioned medium in vitro. The proliferation and the tubulogenesis of EA.hy926 cells were detected by teramethylazo salt azole (MTT) and tube formation assay, respectively. The results showed that the survival rate of the EA.hy926 cells was significantly increased under the co-culture condition. HepG2 conditioned medium also enhanced the angiogenesis ability of EA.hy926 cells. In addition, the expressions of intracellular VEGF and extracellular VEGFR (Flk-1) were regulated upward in a time-dependent manner. In conclusion, the proliferation of vascular endothelial cells and Vascula angiogenesis were improved under the condition of indirect co-culture.