Objective The observe the effects of interferon-inducible protein-10 (IP-10) on proliferation, migration and capillary tube formation of human retinal vascular endothelial cells (HREC) and human umbilical vein endothelial cells (HUVEC). Methods The chemokine receptor (CXCR3) mRNA of HREC and HUVEC were quantified by reverse transcriptase polymerase chain reaction (RT-PCR). In the presence of the different concentrations of IP-10, the difference in proliferation capacity of HREC and HUVEC were analyzed by cell counting kit-8 (CCK-8) methods. Wound scratch assay and threedimensional in vitro matrigel assay were used for measuring migration and capillary tube formation of HREC and HUVEC, respectively. Results RT-PCR revealed both HREC and HUVEC expressed CXCR3. The proliferation of HREC in the presence of IP-10 was inhibited in a dosagedependent manner (F=6.202,P＜0.05), while IP-10 showed no effect on the inhibitory rate of proliferation of HUVEC (F=1.183,P＞0.05). Wound scratch assay showed a significant reduction in the migrated distance of HREC and HUVEC under 10 ng/ml or 100 ng/ml IP-10 stimulation (F=25.373, 23.858; P＜0.05). There was no effect on the number of intact tubules formed by HREC in the presence of 10 ng/ml or 100 ng/ml IP-10. The number of intact tubules formed by HREC in the presence of 1000 ng/ml IP-10 was remarkably smaller. The difference of number of intact tubules formed by HREC among 10, 100, 1000 ng/ml IP-10 and nonintervention group was statistically significant (F=5.359,P＜0.05). Conclusion IP-10 can inhibit the proliferation, migration and capillary tube formation ability of HREC and the migration of HUVEC.
Objective To evaluate which is better method zymogen or low temperature frozen in removing vascular endothelial cell so as to lay a foundation for creating a kind of brace which is not to be rejected and the same as own blood vessel. Methods Fresh and not damaged umbilical blood vessel was collected from natural labour women, human umbilical blood vessel was remove carefully from normal foetus, then was put into disinfectant at 37℃ for 24 hours. They were divided into 3 groups:normal group(NG),zymogen group(ZG) and low temperature frozen group(LG). ZG: 0.1% collagenⅡ enzyme was addedin umbilical blood vessel and closed the both sides and the vascular endothelialcell was removed in 37℃ water. LG:Umbilical blood vessel was put into liquidnitrogen for 24 hours after frozened step by step, and then it was put into 37℃ water for 30-60 s and the vascular endothelial cells were washed away by normal saline. NG:Umbilical blood vessel was kept into 4℃ Kerb’s liquid. The bacteria were culturedin each group. The samples were stained by HE,elastic fiber and collagen fiberwere observed by light and scanning electron microscope. The difference of compliance was compared. Human leukocyte antigen ABC(HLA-ABC) and HLA-DR were observed by immunohistochemical method and the expression of antigen of umbilical blood vessel was analysed. Results In LG, umbilical vascular endothelial cells were removed completely; artery showed vertical smooth muscle and vein showed elastic membrane. InZG, umbilical vascular endothelial cells were removed completely after 20 minutes;artery showed vertical smooth muscle cells and vein showed lower endothelial layer. The vascular compliance in LG was higher than that in NG, and the latter was also higher than that in ZG,but showing no significant differences (Pgt;0.05). The compliance of umbilical vein was 2-3 times as much asthat of umbilical artery.The expression of HLA-ABC and HLA-DR in LG andZG were lower than that in NG, showing significant differences (Plt;0.01). Conclusion Low temperature frozen methodand zymogen method(0.1% collagen Ⅱ enzyme for 20 min) can remove vascular endothelial cells of human umbilical blood vessel completely.Low temperature frozenmethod was better than zymogen method.
Objective\ To promote the differentiation of cultured endothelial cells and enhance their resistance to fluid shear stress.\ Methods\ Using the mended parallel plate flow apparatus and peristalsis pump providing fluid shear stress, endothelial culture models were established in vitro with the same environment factors as steady culture. According to the increasing degree of shear stress, the experiment included:(1) Group A, exposing to the gradual increasing fluid shear stress, (2) Group B, exposing to step ...
Objective To discuss the endothelial cell which was modified by exogenous anticoagulant genes contribute to the increase of antithrombosis activity of lined vascular prosthesis and the influence to other physiological functions of endothelial cells. Methods This summarized paper was made on literature review of recent years. Results The transfection of genes, including plasminogen activator (tPA, uPA, Urokinase), thrombomoduline (TM) and hirudin, etc, to endothelial cells resulted in not only the increase of antithrombosis activity of local vascular, but also the decrease of endothelial cell function in adherence and proliferation. Conclusion The increase of antithrombosis activity of lined vascular prosthesis has been done by exogenous genes. However, this technique ought to be studied, intensively.
The autograft and non-autograft cannot meet the needs of clinical vascular surgery. Since there are possibilities of thrombus formation in artificial vascular grafts, the methods for deposing the graft using physical and chemical ways or simply seeding with endothelial cells cannot produce satisfactory grafts for vascular operations until now. In order to increase the anticoagulative capacity of artificial vascular graft, it is rational to use genetic engineering methods modifying the endothelial cells to make it express anticoagulative factors stably. Although seeding artificial graft with the genetically engineered endothelial cells can possibly produce a satisfactory graft for vascular surgery, some problems still need to be solved.
Objective To investigate the effects of heparanase and vascular endothelial growth factor (VEGF) and their correlation in CoCl2 induced human retinal microvascular endothelial cells (HRECs) in an hypoxia model. Methods Human eyes were selected to establish CoCl2induced HRECs hypoxia model in this study. Four experimental groups were studied: normal control group, hypoxia group (CoCl2 100 μmol/L, 48 hours),PI-88 group (specific competitive inhibitor of heparanase: phosphomannopentaose sulfate, PI-88,5 μg/ml, combined with CoCl2 100 μmol/L, 48 hours) and PBS control group. Heparanase, VEGF and Pol Ⅱ expression in HRECs of normal and hypoxia group were analyzed with immunofluorescence. Western blot was used to evaluate the expression of heparanase and VEGF in HRECs of normal, hypoxia, PI88 and PBS control groups. ResultsImmunofluorescence studies showed that the expression of heparanase and VEGF in cytoplasm was intense in hypoxia HRECs, but faint in normal group. Heparanase was also observed in the nucleus of hypoxia HRECs. Western blot results showed that the expression of Hpa and VEGF protein was increased significantly in hypoxia group compared with normal group (Hpa：F=-4。005, P＜0.05；VEGF：F=-4.063, P＜0.05), and VEGF was decreased in HRECs treated with PI-88(F=5。963, P＜0.05). ConclusionsHeparanase is upregulated that resulted in increase of VEGF expression, therefore enhanced angiogenesis in CoCl2 induced hypoxia HRECs.
ObjectiveTo observe the expression in vitro and the influence of adenovirus-mediated recombinant Tum5 gene to the proliferation, migration and tubing of Rhesus RF/6A cell under high glucose. MethodsTo construct the adenovirus vector of recombinant Tum5 gene (rAd-Tum5), and then infected RF/6A cell with it. The Flow Cytometry was used to detect the infection efficiency. RF/6A cells were divided into normal group, high glucose (HG)-control group (HG group), empty expression vector group (HG+rAd-GFP), and HG+rAd-Tum5 group. Western blot was used to detect the expression of Tum5. The CCK-8 test was applied to detect the proliferation of RF/6A cell, the Transwell test was applied to detect the migration and the Matrigel test was applied to detect the tubing of RF/6A cell under high glucose. The proliferation, migration and tubing of RF/6A were tested respectively by CCK-8 test, Transwell test and Matrigel test. ResultsThe adenovirus vector of recombinant Tum5 gene was successfully constructed. The infection efficiency of rAd-Tum5 in RF/6A cell was 50.31% and rAd-GFP was 55.13% by the Flow Cytometry. The results of Western blot indicated that Tum5 was successfully expressed in RF/6A cell. The result of CCK-8 test, Transwell test and Matrigel test indicated that there were statistical differences between all groups in proliferation, migration and tubing of the RF/6A cell (F=44.484, 772.666, 137.696;P < 0.05). The comparison of each group indicated that the HG group was higher than normal group (P < 0.05). There were no statistical differences between HG group and HG+rAd-GFP group (P > 0.05). However, the HG+rAd-Tum5 group was less than HG group (P < 0.05), and the same to HG+rAd-GFP (P < 0.05). ConclusionThe adenovirus vector of recombinant Tum5 gene can inhibit the proliferation, migration and tubing of RF/6A cell under high glucose.
Organoids are three-dimensional structures formed by self-organizing growth of cells in vitro, which own many structures and functions similar with those of corresponding in vivo organs. Although the organoid culture technologies are rapidly developed and the original cells are abundant, the organoid cultured by current technologies are rather different with the real organs, which limits their application. The major challenges of organoid cultures are the immature tissue structure and restricted growth, both of which are caused by poor functional vasculature. Therefore, how to develop the vascularization of organoids has become an urgent problem. We presently reviewed the progresses on the original cells of organoids and the current methods to develop organoids vascularization, which provide clues to solve the above-mentioned problems.
ObjectiveTo observe the effect of pyrimidine bundle-binding protein-associated splicing factors (PSF) on the function of hypoxia-induced human retinal microvascular endothelial cells (hRMECs).MethodsA three-plasmid system was used to construct lentivirus (LV)-PSF. After LV-PSF infected hRMECs in vitro, the infection efficiency was measured by flow cytometry. Real-time quantitative PCR (RT-PCR) was used to detect the expression of PSF mRNA in hRMECs infected with LV-PSF. The experiment was divided into two parts, in vivo and in vitro. In vivo experiments: 20 healthy C57B/L6 mice at the age of postnatal 7 were randomly divided into normal group, oxygen-induced retinopathy (OIR) group, OIR+LV-Vec group, and OIR+LV-PSF group, each group has five mice. Mice in 3 groups were constructed with OIR models except the normal group and the mice in OIR group were not treated. The mice in the OIR + LV-Vec group and the OIR+LV-PSF group were injected with an empty vector (LV-Vec) or LV-PSF in the vitreous cavity, respectively. The effect of LV-PSF on the formation of retinal neovascularization (RNV) was observed then. In vitro experiments: hRMECs were divided into normal group, hypoxia group, vector group, and PSF high expression group. HRMECs in the normal group were cultured in vitro; hRMECs in the hypoxic group were restored to normal culture conditions for 3 h after 3 h of hypoxia stimulation; hRMECs in the vector group and PSF high expression group were infected with LV-Vec and LV-PSF for 48 h, and hRMECs were returned to normal culture conditions for 24 h with hypoxia stimulation for 3 h. The effect of PSF on cell proliferation was observed by MTT colorimetry. Cell scratch test and Transwell migration experiment were used to observe the effect of PSF on cell migration ability under hypoxia stimulation. RT-PCR was used to observe the mRNA expression of HIF-1α, VEGF and PSF in each group of cells.ResultsThe LV-PSF of stably expressing PSF was successfully constructed. The infection efficiency was 97% determined by flow cytometry. The level of PSF mRNA in hRMECs infected with LV-PSF was significantly increased and detected by RT-PCR. In vivo experiments: The RNV area of the mice in the OIR group and the OIR + LV-Vec group was significantly increased compared to the normal group (t=18.31, 43.71), and the RNV area of the mice in the OIR + LV-PSF group was smaller than that in the OIR group (t=11.30) and OIR + The LV-Vec group (t=15.47), and the differences were statistically significant (P＜0.05). In vitro experiments: MTT colorimetry results showed that the proliferative capacity of hRMECs in the hypoxic group was significantly enhanced compared with the normal group (t=2.57), and the proliferative capacity of hRMECs in the PSF high expression group was significantly lower than that of the normal, hypoxic, and vector groups (t=5.26, 5.46, 3.73), the differences were statistically significant (P＜0.05). The results of cell scratch test showed that the hRMECs could be stimulated by the hypoxia stimulation for 3 hours to restore the normal condition for 24 hours or 48 hours (t=8.35, 13.84; P＜0.05). Compared with the vector group, cell migration rate in the PSF-high expression group was not significant (t=10.99, 18.27, 9.75, 8.93, 26.94, 7.01; P＜0.05). Transwell experiments showed that the number of cells stained on the microporous membrane was higher in the normal group and the vector groups, while the number of cells stained in the PSF high expression group was significantly reduced (t=9.33, 6.15; P＜0.05). The results of RT-PCR showed that the mRNA expression of HIF-1α and VEGF in hRMECs in the hypoxic and vector groups increased significantly compared with the normal group (t=15.23, 21.09; P＜0.05), but no change in the mRNA expression of PSF (t=0.12, 2.15; P>0.05); compared with the hypoxia group and the vector group, the HIF-1α and VEGF mRNA expression in hRMECs in the PSF high expression group were significantly decreased (t=10.18, 13.10; P＜0.05), but the PSF mRNA expression increased (t=65.00, 85.79; P<0.05).ConclusionPSF can reduce the RNV area in OIR model mice. PSF may inhibit hypoxia-induced proliferation and migration of hRMECs through the HIF-1α/VEGF signaling pathway.
Objective To explore an optional condition to induce mouse embryonic stem cell(ESC) to differentiate into endothelial cells so as to provide seedcells for tissue engineered vascular. Methods The embryos from one pregnant 12.5days mouse was harvested to culture the mouse embryonic fibroblasts(MEF). The ESC was reanimated by common method, and used to cultured into embryoid body(EB) in vitro. The EB which was used to induce into endothelial cells was divided into two groups. The EB was cultured in the EB medium with 3ng/ml transforming growth factor β1, 50 ng/ml vascular endothelial cell growth factor and 1 μmol/L potent and selective inhibitor of activin receptorlike kinase receptors in experimental group. The EB was cultured in the EB medium in the control group. After 14 days, RTPCR and immunohistochemistry were used to detect vWF and CD34, to analyze the morphology and type of the differentiated cells fromESC. Results The primary MEF had a high proliferation activity. At the 3rdday, the fusion rate of MEF was about 90% with a fusiform shape. The cells was fusiform shape and arranged compactly with fullness of nucleus and 2-3 entoblasts. The 3rd5th generations EB was polygonal with fullness of cytoplasm and 3-4 entoblasts. ESC could maintain undifferentiated state, and the cells unit lookedlike bird nest with smooth margin; the cells was small at size and b refractivity with high rate of nuclein and rapid proliferation. At 3 days of dropculture, EB can seen grossly and at 3 days of suspension, large and transparent EBformed. EB was spread radiately with an intensive adhesion at the 2nd day. In experimental group, many round cells was differentiated around EB from the 4thday to the 7th day, and form tubular structures from the 10th day to the 14th day. The vWF and CD34 were expressed. In control group, EB could not form tubularstructures, and the vWF and CD34 were not expressed. Conclusion ESC can differentiate into endothelial cells under some conditions, and form vessellike structure under condition culture, which can provide sources of seed cells for tissue engineered vessel.