Objective To study the effect of vascular endothelial cell growth factor (VEGF) on repair of bone defect with cortical bone allograft. Methods Forty five New Zealand white rabbits, weighted 2.5-3.0 kg, were made bone defect model of 1.5 cm in length in the bilateral radii and then were randomly divided into 3groups. The defect was repaired with only cortical bone allograft in the control group, with the cortical bone allograft and local injection of human recombinantVEGF in the experimental group, and with the cortical bone allograft and abdominal injection of VEGF PAb3 in the antagonist group. Roentgenography, immunohistochemical staining and tetracycline labelling were carried out to evaluate the reparative results 1, 3, 5, 8 and 16 weeks after operation. Results Immunohistochemical staining results showed that a great deal of blood vessels formed in the experimental group, and the number of blood vessels increased gradually with the time and reached the highest value at the 8th week. Tetracyclinelabelling showed the same result.The best results in callus formation, ossification rate and count of microvascular density were shown in the experimental group, while those in the control group were significantly better than those in the antagonist group (Plt;0.05),but there was no significant difference between the experimental group and the control group at the 8th week and the 16th week (Pgt;0.05). Conclusion VEGF can accelerates the bone formation and angiogenesis in the bone allografts, thus it can promote the repair of bone defects.
Objective To develop a new method for a tissue engineered vascular graft by combining endothelial cells and an acelluarized allogenic matrix. Methods Acellularized matrix tubes were obtained by a 0.1% trypsin and 0 02% EDTA solution for 24 hours and 1% Triton X 100 for 176 hours, respectively. Endothelial cells were isolated from alloaorta and expanded in vitro. Finally, the inner surface of acellularized matrix was reseeded with endothelial cells. Acellularity and reseeding were analysed by light microscopy and scanning electron microscopy. Results The acellularization procedure resulted in an almost complete removal of the original cells and the loose three-dimensional (3D) matrix. The acellular matrix could be reseeded with expanded endothelial cells in vitro, and endothelial cells had the potential of spread and proliferation. Conclusion Acellular matrix produces by Tritoon X-100 and trypsin possesses satisfactory biocompatibility for allogenic endothelial cell. Vascular grafts can be generated in vitro by a combination of endothelial cells and allogenic acelluarized matrix.
Objective To observe the effects of Galectin-3 on proliferation of vascular endothelial cells derived from peripheral blood endothelial progenitor cells. Methods The cultured peripheral blood endothelial progenitor cells in vitro were isolated and purified from human peripheral blood, and the cells were differentiated into vascular endothelial cells. Then the cells were cultivated with the galectin-3 of different concentrations, and to observe the proliferation of endothelial cells derived from peripheral blood endothelial progenitor cells. Results The abilities of proliferation of endothelial cells derived from peripheral blood endothelial progenitor cells of 0.1, 1.0, 2.5, 5.0, and 10.0 μg/ml groups were higher than that of 0 μg/ml group, there were not statistic significance of the differences between the 0.1，1.0, 2.5, and 0 μg/ml groups （P＞0.05）. But the abilities of proliferation of 5.0 and 10.0 μg/ml groups were obviously higher than that of 0, 0.1, 1.0, and 2.5 μg/ml groups （P＜0.05）, and the abilities of proliferation of 10.0 μg/ml group was also higher than that of 5.0 μg/ml group （P＜0.05）. Conclusion Galectin-3 can promote the proliferation of endothelial cells derived from peripheral blood endothelial progenitor cell.
Objective
To establish a simple and efficient method to isolate and culture the umbilical vein vascular endothelial cells in canine.
Methods
Twelve umbilical cords [(13.0 ± 1.5) cm in length] were taken from 12 newborn pups of Beagles. And then the vascular endothelial cells were isolated from these umbilical cords digested by 1% collagenase type I for 5, 7, and 10 minutes respectively (4 umbilical cords in each group). After cultured, the vascular endothelial cells were identified by morphology, immunofluorescence, and flow cytometry. And the growth curvature of umbilical vein vascular endothelial cells was detected by MTT assay.
Results
Few vascular endothelial cells were collected at 5 and 10 minutes after digestion; many vascular endothelial cells were seen at 7 minutes, and became cobblestone with culture time, with a large nucleus; after passage, cell morphology had no obvious change. Fluorescence microscope results showed that positive von Willebrand factor (vWF) and CD31 cells were observed in most of cells. The flow cytometry test displayed that the positive cell rates of vWF and CD31 were 99.0% ± 0.7% and 98.0% ± 1.2%, respectively. The above results indicated that cultured cells were vascular endothelial cells. MTT assay showed that vascular endothelial cells proliferation increased significantly with culture time.
Conclusion
Enzyme digestion is a convenient method to isolate vascular endothelial cells from canine umbilical vein, and a large number of cells and high purity of cells can be obtained by the method.
ObjectiveTo investigate the role of protease-activated receptor-2 (PAR-2) activation on the expression of vascular endothelial growth factor (VEGF) in MKN28 gastric cancer cells.
Methods①MKN28 cells were treated with increased concentrations of trypsin (0, 0.1, 1.0, 10.0, and 100.0 nmol/L respectively) for 6 hours, or treated with 10.0 nmol/L trypsin for 3, 6, 12, and 24 hours (blank control group was treated with PBS) respectively, then the expression levels of VEGF mRNA and its protein in MKN28 cells were detected by real-time reverse transcription polymerase chain reaction (qRT-PCR) and Western bolt method, with the concentration of VEGF protein in broth was detected by enzyme linked immunosorbent assay (ELISA) method.②MKN28 cells were divided into blank control group (treated with PBS), trypsin group, trypsin+PD98059 group, trypsin+SB203580 group, PD98059 group, and SB203580 group, then the expression levels of VEGF mRNA and its protein in MKN28 cells were detected by qRT-PCR method and Western bolt method respectively.
Results①The effect of different concentration of trypsin. Compared with blank control group, the expression levels of VEGF mRNA and its protein in 0.1, 1.0, 10.0, and 100.0 nmol/L group were higher (P < 0.05); compared with 0.1 nmol/L group, the expression levels of VEGF mRNA and its protein in 1.0, 10.0, and 100.0 nmol/L group were higher (P < 0.05); compared with 1.0 nmol/L group, the expression levels of VEGF mRNA and its protein in 10.0 and 100.0 nmol/L group were higher (P < 0.05); but there was no significant difference between 10.0 nmol/L and 100.0 nmol/L group (P > 0.05). The broth concentration of VEGF protein in blank control group, 0.1, 1.0, 10.0, and 100.0 nmol/L group crept upward (P < 0.05).②The effect of different treated time of 10.0 nmol/L trypsin. The expression levels of VEGF mRNA in blank control group, 3, 6, 12, and 24 hours group crept upward, and there was significant difference between any 2 groups (P < 0.05). But the expression of VEGF protein was not similar with VEGF mRNA. Compared with blank control group, the expression levels of VEGF protein in 3, 6, 12, and 24 hours group were higher (P < 0.05); compared with 3 hours group, the expression levels of VEGF protein in 6, 12, and 24 hours group were higher (P < 0.05); but there was no significant difference among 6, 12, and 24 hours group (P > 0.05). The broth concentration of VEGF protein in blank control group, 3, 6, 12, and 24 hours group crept upward, and there was significant difference between any 2 groups (P < 0.05).③The effect of extracellular regulated protein kinase (ERK) inhibitor (PD98059) and p38 inhibitor (SB203580). The expression levels of VEGF mRNA and its protein in trypsin group were all higher than corresponding indexes of blank control group, trypsin+PD98059 group, trypsin+SB203580 group, PD98059 group, and SB203580 group (P < 0.01), but there was no significant difference among blank control group, trypsin+PD98059 group, trypsin+SB203580 group, PD98059 group, and SB203580 group (P > 0.05).
ConclusionActivation of PAR-2 can induce the expressions of VEGF mRNA and its protein in MKN28 gastric cancer cells, that is mediated by ERK1/2-and p38-dependent pathway.
Objective To investigate the role of vascular endothelial growth factor-C (VEGF-C) and its receptors in the formation of lymphatic vessels and lymphatic metastasis in gastric cancer. Methods By the domestic and overseas literatures review, the expressions of VEGF-C and its receptors in gastric cancer, their role in tumor lymphatic metastasis and prospect in treatment of gastric cancer were summarized.Results There was a significant correlation between VEGF-C and its receptors and the formation of lymphatic vessels and lymphatic metastasis in gastric cancer. VEGF-C high expression might be an early event in lymphatic metastasis and could be considered as an independent predictive factor of lymphaticmicrometastasis. By inhibition of gastric cancer cell from secrete VEGF-C or blockage of the interaction of VEGF-C with VEGFR3, it was possible to inhibit tumor angiogenesis and the invasion and distant spread of cancer cells, thereby decreased mortality and improve survival. ConclusionVEGF-C and its receptors may promote the formation of lymphatic vessels and lymphatic metastasis in gastric cancer. It may be an effective way to gastric cancer for the treatments against VEGF-C and its receptors.
Objective To observe the proliferation and migration of endothelial cells after 30% total burn surface area (TBSA) of deep partial thickness scald, and the effect of basic fibroblast growth factor (bFGF) on angiogenesis during wound healing.Methods A total of 133 male Wistar ratswere divided randomly into normal control (n=7), injured control group (n=42), bFGF group (n=42) andanti-c-fos group (n=42). The apoptosis expression of fibroblasts was determinedwith in situ hybridization and the changes of proliferation cell nuclear antigen(PCNA), focal adhesion rinase(FAK), c-fos and extracellular signalregulated kinase(ERK) proteins expression were detected with immunohistochemistry staining technique after 3 hours, 6 hours, 1 day, 3 days, 7 days, 14 days and 21 days of scald.Results In injured control group and bFGF group, theproliferation rate of the vascular endothelial had evident changes 7 days and14 days after scald； the expression of FAK was increased 14 days after scald. ERK proteins expression was different between injury control group and bFGF group at initial stage after scald. Stimulation of ERKs by bFGF led to up-regulation of c-fos and b expression of FAK. Conclusion Exogenous bFGF extended the influence on wound healing process by ERK signaling pathway, affecting migration cascade of vascular endothelial cell. The oncogene proteins play an important role on accelerating angiogenesis duringwound healing.
Objective To observe the influences of estradiol (E2), basic fibroblast growth factor (bFGF), and tamoxifen (TAM) on the proliferation of hemangioma vascular endothelial cell (HVEC). Methods Two strawberry hemangioma from 2 infants (case 1 and case 2) were prepared for HVEC culture. The HVEC on passage 3 were cultured in estrogenfree improved minimum essential medium (IMEM) and subjected to various treatments with 100 pg/ml 17-β-E2, 10 ng/ml bFGF, and 1×10-6 mol/L 4-OH-tamoxifen(4-OH-TAM). The experiment was divided into 5 groups: group 1(IMEM, control group), group 2(17-β-E2), group 3(bFGF), group 4(17-β-E2／bGFG) and group 5(17-β-E2／bGFG／4-OH-TAM). The cell count(CC) and DNA proliferation index (PI) were determined. Results Two cases of HVEC were successfully cultured in vitro. The HVEC showed cobblestoneslike under microscopy and factor Ⅷrelated antigen(also named as von Willebrand factor，vWF) was positive by immunochemical staining. At 9 days in case 1: CC and PI remained unchanged in the control group; CC and PI were slightly increased in group 2, being 1.4 and 1.6 times as much as those in the control group respectively (P＜0.05); CC and PI significantly increased in group 3, being2.6 and 2.3 times as much as those in the control group respectively (P＜0.01); CC and PI increased remarkably in group 4, being 3.7 and 2.9 times as much as those in thecontrol group respectively (P＜0.01); CC and PI were down to the levels of controls in group 5(P＞0.05). The results in case 2 were similar to those in case 1. Conclusion In vitro, the promoting effect of bFGF on HVEC proliferation is much ber than that of estrogen. Estrogen and bFGF enhance this proliferation in a synergistic manner, which can be inhibited by tamoxifen.
Objective
To explore the effects of Zhaoke defibrase and anti alpha;vbeta;3mAb (23C6) on the adhesion and immigration of bovine retinal vascular endothelial cells.
Methods
The culture dishes coated with vitronectin (Vn) and collagen，assays of adhesion and immigration were performed 60 minutes after different concentration of Zhaoke defibrase and anti-alpha;vbeta;3 mAb was added to the bovine retinal vascular endothelial cells. The apoptosis of bovine retinal vascular endothelial cells induced by Zhaoke defibrase and anti-alpha;vbeta;3 mAb was detected by electron microscopy.
Results
Both Zhaoke defibrase and anti-alpha;vbeta;3 mAb inhibited the adhesion and immigration of bovine retinal vascular endothelial cells in a dose-dependent manner. The inhibited concentration (IC50) of Zhaoke defibrase was less than 0.05 mu;mol/L, while (IC50) of anti-alpha;vbeta;3 mAb was more than 2.5 mu;mol/L. 81.8% endothelial cells adhering to Vn were inhibited by 0.1 mu;mol/L Zhaoke defibrase, while 76.3% by endothelial cells adhering to Vn were inhibited by 10 mu;mol/L anti-alpha;vbeta;3 mAb. Typical apoptosis cells were found in bovine retinal vascular endothelial cells after affected by Zhaoke defibrase and anti-alpha;vbeta;3 mAb.
Conclusion
Both Zhaoke defibrase and anti- alpha;vbeta;3mAb can significantly inhibit the adhesion and immigration of bovine retinal vascular endothelial cells to extracellular matrix， and the mechanism may lie in inducing the apoptosis of endothelial cells.
(Chin J Ocul Fundus Dis, 2005,21:118-121)
Abstract: Objective To study the expression of E-selectin on vascular endothelial cells of nude mice liver induced by esophageal carcinoma cells, in order to find out the function of E-selectin in the metastasis of esophageal carcinoma into the liver. Methods Twelve Balb/c nude mice aged from 6 to 8 weeks with their weight ranged between 20 and 25 grams were selected in our research. The mice were equally distributed into the experimental group and the control group(n=6). EC9706 cell solution (5×10.6/0.02 ml) were injected beneath the splenic capsule of the mice in the experimental group. One hour later, spleen was removed. For the mice in the control group, after laparotomy, phosphate buffer without EC 9706 was injected beneath the splenic capsule and spleen was also removed one hour after the injection. Eight hour later, we resected the liver of the nude mice, and expression of E-selectin on vascular endothelial cells of the liver was detected with reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC). Results In the experimental group, 8 hours after injection of EC9706 cells (5×10.6), the results of RT-PCR showed expression of E-selectin mRNA in the liver, and IHC showed a positive protein expression of E-selectin in the cytosol and membrane of hepatic sinus vessels.However, no E-selectin mRNA expression was found in the control group and IHC showed a negative protein expression of E-selectin. Conclusion Human esophageal carcinoma cell line EC9706 can induce balb/c mice liver vascular endothelial cell E-selectin expression, which shows that EC9706 may stay in the liver and form etastatic focus.
