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@#Objective: To investigate the expression of Artemin in chondrosarcoma and its effect on proliferation and migration of endothelial cells, and to explore the mechanism. Methods: A total of 40 chondrosarcoma tissue samples (low degree (Ⅰ), 20 cases; high degree (Ⅱ,Ⅲ), 20 cases) surgically resected from patients, who were treated in Lianyungang HospitalAffiliated to Nanjing University of Chinese Medicine from May, 2015 to April, 2019, were collected for this study. Another 20 cases of normal cartilage tissue specimen from patients with amputations due to car accidents were served as control. The expressions of Artemin, vascular endothelial growth factor (VEGF), Ki-67 and CD31+ vascular density in tumor tissues were detected by immunohistochemistry.After being treated with 10 ng/ml Artemin, the changes of VEGF, stromal cell derived factor-1 (SDF-1), matric metalloproteinase 2 (MMP2) and MMP9 in the supernatant of SW1353 cell culture were detected by enzyme-linked immunosorbent assay (ELISA), and the effects of Artemin-treated chondrosarcoma cells on the migration and proliferation of ECV304 cells were detected by Transwell migration assay and MTT cell proliferation assay, respectively. Results: The expressions of Artemin and Ki-67 in the tissues of low-level group were significantly higher than those in the control group (all P<0.01); the expressions ofArtemin and Ki-67 in the tissues of high-level group were significantly higher than those in the low-level group (all P<0.01). The expression of Artemin was positively correlated with VEGF level and vascular density in chondrosarcoma tissues (all P<0.01);Artemin promoted the secretion of VEGF by chondrosarcoma cells, but had no significant effect on the secretion of SDF-1, MMP2 and MMP9. Artemin induced the proliferation and migration of ECV304 cells by promoting the secretion of VEGF by chondrosarcoma cells (all P<0.01). Conclusion: Artemin is highly expressed in chondrosarcoma tissues and has a positive correlation with the expression of VEGF and vascular density. Artemin can enhance the angiogenesis induced by chondrosarcoma.
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Background Choroidal neovascularization (CNV) is one of the causes of blindness in multiple eye diseases.Researches showed that complement system participates in the pathogenesis of CNV.Objective This study was to construct the recombinant of complement factor B-small interference RNA (CFB-siRNA) expression vector and to observe its inhibitory effect on human umbilical vein endothelial cells (ECV-304).Methods CFB gene primers were designed based on human CFB gene,and an expression vector of CFB-siRNA was constructed by inserting CFB-siRNA into pRNAT-U6.1/Neo plasmid.Recombinant plasmids were confirmed by the digestion analysis of restriction endonuclease,and all inserted sequences were verified by DNA sequencing.The recombinant pRNAT-U6.1/CFB-siRNA plasmid and the blank plasmid were transfected into ECV-304 cells in the CFB-siRNA group and blank plasmid group by electroblot,respectively,and non-transfected cells served as the normal control group.The cells were observed under the fluorescence microscope 48 hours after transfection,and the transfective efficiency was calculated.The relative expression of CFB mRNA in the cells of different groups was detected by semi-quantitative reverse transcription PCR (RT-PCR).MTT was employed to calculated the growth inhibitory rates of the cells 24,48 and 72 hours after transfection.The percentages of the cells in different cell cycles were detected by flow cytometry.Results The sequence of the target vector was identical to the designed sequence.The green fluorescence protein (GFP) was seen in both the CFB-siRNA group and the blank plasmid group.The relative expression levels of CFB mRNA were 0.07 ±0.04,0.14 ±0.02 and 0.14 ±0.03 in the CFB-siRNA group,the blank plasmid group and the normal control group,respectively,a significant difference was obtained among the three groups (F=233.05,P =0.00);the expression level of CFB mRNA in the CFB-siRNA group was significantly declined in comparison with the blank plasmid group and the normal control group (both at P＜0.05).The growth inhibitory rates of the cells were (23.45 ±0.01) ％,(33.48 ±0.02) ％ and (45.49±0.01) ％ at 24,48 and 72 hours after transfection,respectively,a significant difference was obtained among the three groups (Fgroup =212.99,P =0.00);the growth inhibitory rates in CFB-siRNA group were significantly higher than that in the blank plasmid group and normal control group (all at P＜ 0.05).The percentages of G1 phase cells were (44.4 ±0.5) ％,(25.8 ±0.4) ％ and (27.9 ± 0.6) ％ in the CFB-siRNA group,the blank plasmid group and the normal control group respectively,a significant difference was obtained among the three groups (F=58.98,P=0.00).The percentages of G1 phase and G2 phase cells in the CFB-siRNA group were significantly higher than those in the blank plasmid group and the normal control group (all at P＜0.05).Conclusions Recombinant pRNAT-U6.1/CFB siRNA inhibits the proliferation of ECV-304 cells effectively by arresting the cells in G1 intermediate phase of the growth cycle.
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Aim To investigate the effect of MTX(included(±)MTX,(+)MTX and(-)MTX)on the proliferation of ECV304 cells and to explore its mechanisms.Methods ECV304 cells were cultured.The cell proliferation was determined by MTT.The morphological changes were inspected by inverted microscope.Cell cycle phases were assayed by propidium iodide staining flow cytometry.Results ECV304 cells were treated with(+)MTX,(-)MTX and(±)MTX at 1～150 μmol·L~(-1) for 24,48,72 h.The results showed that the proliferation of ECV304 cells was significantly inhibited under different conditions.The order of the inhibited efficacy was(+)MTX>(±)MTX>(-)MTX.The morphology of ECV304 cells were changed by(+)MTX,(-)MTX and(±)MTX treatment,which included the cell shrinkage,chromatin condensation.After administration of 10 μmol·L~(-1) of(+)MTX,(-)MTX and(±)MTX for 48 h,the cell cycle phases were assayed by propidium iodide staining flow cytometry.The result showed DNA replication was interfered by(+)MTX,(-)MTX and(±)MTX treatment.Conclusions The proliferation of ECV304 cells has the chiral selective effects by(+)MTX and(-)MTX treatment,and the inhibition on ECV304 cells proliferation of(+)MTX is significantly stronger than that of (-)MTX.
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Objective To investigate the cytolytic activity of extracellular cytolytic toxin rVVC of Vibrio vulnificus on the apoptosis of human ECV304 cells, and to analyze the activities of Caspase-3,-8 and -9. Methods The cytotoxic effect of refolded rVVC on the growth and apoptosis of ECV304 cells was identified by MTT, Hochest33342/PI fluorescent staining, flow cytometry and DNA agarose electrophoresis analysis, respectively. The activities of Caspase-3, -8 and -9 was measured using a colorimetric method. Results The viability of human ECV304 cells exposed to rVVC was inhibited by rVVC after 24 h. 2.0 HU/ml rVVC groups had a better cytotoxic effect to human ECV304 than that of 0.5 HU /ml rVVC groups. The apoptosis of human ECV304 cells in 2.0 HU/ml rVVC+40 μmol/L Z-VAD-FMK groups was relative reduced than that of 2.0 HU/ml of rVVC groups. After 0.5 h treatment with 2.0 HU/ml of rVVC, the Caspase-3 activity in human ECV304 cells increased gradually and reached the peak at 3 h (versus control groups, P<0.01). The activity of Caspase-8 and -9 remained unchanging. Conclusion The rVVC has cytotoxic effect on human ECV304 and the cytolysin is probably correlated with Caspase-3.
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AIM:To explore the effect of β-asarone on vascular endotheliam and adhesion molecule expression of endothelium induced by β-amyloid peptide from Alzheimer's disease and to estimate the injury repair.METHODS:Cultured ECV304 cells were incubated with freshly solublizeal Aβ1-42 and the mixture of Aβ1-42 and β-asarone,the expression of three central adhesion molecules,CD106,CD62P,CE62E and Ca2+ concentration were examined and apoptosis was recorded by Flow eytometry.Test viability of cells by MTT methods.RESULTS:The results showed that in model group and treated group,ligation of endothelial CD106,CD62P,CE62E,markers for endothelial cell activation and Ca2+ concentration,leads to a lot of release.The livability decreased and the apoptosis increased.Further more,simultaneous treatment of ECV304 cells with β-asarone resulted in the decrease significandy in these three adhesion molecules described above and Ca2+ concentration as well as the livability upper and apoptosis lower.CONCLUSION:CD106,CD62P,CE62E,important inflammational factor of Aβ-induced endothelial injury,may be promotion of the inflammatory scade in vascular endothelial.β-asarone may protect ECV304 cell apoptosis by regulate Ca2+ and expression of cell surface markers.
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Objective: To investigate the effect of silencing Smad4 gene expression by using small interfering RNA (siRNA) on proliferation and migration of ECV304 cell line and explore the role of Smad4 in angiogenesis. Methods: Two siRNA sequences targeting Smad4 gene including siRNA1 and siRNA2 were designed and then synthetized. The two sequences of siRNA were transfected into ECV304 cells via lipofectamine mediation. The alteration of Smad4 mRNA and protein expression was examined by real-time RT-PCR and Western blot, respectively. The proliferation capacity of ECV304 cells was measured by cell cycle analysis and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) flow cytometry. The monostratal wound healing test was used to determine the migration capacity of ECV304 cells. Results: siRNA2 was successfully screened out of the two sequences. Transfection of siRNA2 significantly knocked down the Smad4 mRNA and protein expressions. The proliferation and migration capacity of ECV304 cells are significantly enhanced after transfection with siRNA2. Conclusions: Highly effective and specific siRNA targeting Smad4 gene can be successfully screened by RNA interference technique. Selective silencing of Smad 4 gene greatly promotes the proliferation and migration of ECV304 cells.
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Objective To explore the role of oxygen free radicals in the proliferation of ECV304 induced by AngⅡ.Methods The lines of human umbilical vein endothelial cell(ECV304)cultured in vivo were divided into three groups which were treated by AngⅡ,AngⅡ+N-acetyl-L-cysteine(NAC),and normal culture medium.First we observed the proliferous effect of ECV304 induced by AngⅡat different concentration with improved MTT and microscope.Then the contents of oxygen free radicals(?OH)in three groups were detected by spectrophotometer.Results ECV304 incubated with AngⅡ(0.03125～1?mol/L)for 12 hours increased the proliferation rate(P 0.05 vs.control group).Conclusions ECV304 induced by AngⅡcan produce oxygen free radicals(?OH),and the contents of oxygen free radicals(?OH) increase with the prolongation of time and the enlargement of dose;Antioxidant NAC can inhibit the proliferation of ECV304 induced by AngⅡ,this effect may be related with reducing the content of oxygen free radicals(?OH);oxygen free radicals(?OH)may be one of the major mocleculars which play an important role in the signal transduction of ECV304 proliferation.
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Objective To study the effect of the ultra-filtration extract from Angelica sinensis and Hedysarum polybotrys’s mixture on the VEGF mRNA expression of ECV-304 cell. Method Angelica sinensis and Hedysarum polybotrys’s mixture were refined by ultra-filtration technique. ECV-304 cells were cultured as model, and its proliferation was detected by MTT colorimetry. VEGF mRNA expression was observed by semi-quantitative RT-PCR. Results The ultra-filtration extract from Angelica sinensis and Hedysarum polybotrys’s mixture could markly promote the growth of ECV-304 cells, there was significant difference between experimental and control group (P
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Objective To discover the effective component of Rhizoma Tupistrae chinensis with inhibiting angiogenesis. Methods Several parts of Rhizoma Tupistrae chinensis were obtained by using different extraction solvents. They were screened by the model of ECV304 cell membrane chromatography with anti-VEGF antibody as a control drug. The inhibition of effective component (ZGQ-C) on ECV304 proliferation in vitro was examined by MTT assay. Results The inhibition rate (40.51%,51.11%,63.54%,74.32%,81.26%) was correlated with the ZGQ-C concentration. The ECV304 cell had significantly changed in morphology. Conclusion The ZGQ-C is an effective component for inhibiting angiogenesis. The screening results on the model have a good correlation with that of MTT assay.
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DHA, one of w-3 fatty acids, modulates cell growth or death though the changes of apoptotic signaling in human endothelial ECV304 cells. We investigated the effects of DHA on the changes of apoptotic signaling in human vascular endothelial ECV304 cells using lipid peroxidation (LPO) metabolites. LPO could be originated by dietary polyunsaturated fatty acids such as linoleic acid (LA), arachidonic acid (AA) and docosahexaenoic acid (DHA). DHA caused cell death of ECV304 cells compared to LA, AA or control as evidenced by changes in cell morphology and MTT assay. LPO levels was significantly elevated by 10 fold in DHA-treated ECV 304 cells and caspase-3 activity was increased by DHA corresponding to increasing incubation times compared to control. One of reasons of the cell death in DHA-treated ECV304 cells could be expected that caspase activity, marker for mitochondrial damages, might be triggered by the increasing LPO levels. Our results strongly indicated that DHA induced LPO production has an important role on apoptotic signaling pathway in ECV304 cells. LPO production in endothelial cells which was metabolized by oxidation of dietary PUFA, might be one of risk factors in the initial progression of atherosclerosis.
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Humans , Apoptosis , Arachidonic Acid , Atherosclerosis , Caspase 3 , Cell Death , Endothelial Cells , Fatty Acids , Fatty Acids, Unsaturated , Linoleic Acid , Lipid Peroxidation , Risk FactorsABSTRACT
Objective: To construct an expression system containing the N-terminal segment of Thrombospondin-1 (TSP-1) in E. coli. To investigate the activity of TSF. Methods: thbs1 gene fragment was amplified from human fetal cord vein endothelial cells and inserted into plasmid pET32c ( + ) which was then transfected and expressed in E. coli. Investigate the effect of recombinant TSF to the proliferation of ECV304 in vitro with MTT. The expression level of CD36 was assayed by FACS. Results: Recombinant TSF was purified. TSF could restrain the proliferation of ECV304 with CD36 low-expression. Conclusions:Low dose of rTSF was a latent assistant treatment of anti-cancer.
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Aim To illustrate the role of Tanshinone IIA(TanIIA)extracted from dan-shen root in atherosclerosis and its different targets by investigating the effect on the expression of NF-?B、I?B-? and the mRNA of ICAM-1 and VCAM-1 in human umbilical vascular endothelial cell 304(ECV304).Mehods The TNF-?-induced cultured ECV-304 cell injury model was established to analyze the expression of NF-?B、I?B-? by cell ELISA method and the mRNA of ICAM-1 and VCAM-1 by RT-PCR method of the cells cultured with different denses of TanIIA.Results The results of ELISA method showed that the expression of the NF-?B was reduced significantly and the expression of the I?B-? was enhanced significantly in ECV304 which were cultured with higher denses of TanIIA.The results of RT-PCR method showed that the mRNA of ICAM-1 and VCAM-1 induced by TNF-? was significantly refrained.Conclusions TanIIA has a significant effect on inhibiting the mRNA of ICAM-1 and VCAM-1 induced by the NF-?B activation,so it can play an impotant role in resisting atherosclerosis.
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Aim To study the protective effects of Ganoderma lucidum polysaccharides peptide(GLPP)on ECV304 from oxidative injury.Methods Cultured ECV304 were injured by oxygen free radicals derived from tBOOH.Various concentrations of GLPP(12.5,25,50,100 mg?L-1)were added into culture medium.The survival rate of cells was measured by MTT assay.The morphological change of cells and injury of mitochondria were examined under the light and electron microscopes.The percentage of apoptosis of ECV304,labeled with AnnexinV/PI,was measured by flow cytometry.Results GLPP(12.5,25,50,100 mg?L-1)could reduce oxidative injury induced by tBOOH in ECV304 cells.The survival rate of cells treated with GLPP increased.The light microscopic examination showed that the injured cells decreased in GLPP-treated groups.Under the electron microscope it was found that GLPP(50 mg?L-1,incubated for 24 h)could protect the organelle such as mitochondria from oxidative injury and cells from apoptosis by tBOOH.The result of flow cytometry showed that the total percentage of apoptosis in control,GLPP and injury treated group was 2.24%?0.43%,24?6.4%(P
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AIM To investigate the pathways of Ca 2+ entry into ECV304 endothelial cell and the effect of angiotensin Ⅱ(AⅡ) on calcium activated non setective cation channel(CAN). METHODS The cell attachment and whole cell configurations of patch clamp technique were used to record channel activity. RESULTS (1) The single channel conductance is ? o=(12 90?2 11) pS( n =4) for Ca 2+ passing through CAN of ECV304 cell in condition of pipette solution without K + and Na + but composed 120 mmol?L -1 CaCl 2. The channel current amplitude and open time can be enhanced by 1?10 -7 mol?L -1 AⅡ. The enhanced conductance in CAN is ? 1=(22 18?2 29) pS( n =4). The results of whole cell recording are identified with single channel recording. (2) The whole cell configuration was carried out for recording voltage dependent Ca 2+ channel in ECV304 cell. The peak current amplitude was (29 32?3 56) pA( n =4). This current was inhibited to (6 00?3 94) pA( n =4) by nifedipine and activated by BayK8644. CONCLUSIONS (1)Ca 2+ enters ECV304 cell via Ca 2+ activated non selective cation channel and voltage dependent L type calcium channel. (2) AⅡ can significantly enhance the calcium entry via CAN in ECV304 cell.
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Objective To investigate the effect of strong ion-irradiation and active macrophage on vascular endothelial cells. Methods The vascular endothelial cells(ECV-304) were divided into 3 groups: ECV304(A), ECV-304 with radiation (B)and ECV-304 with active macrophage U937 and radiation(C). The survival rates, cell cycle and apoptosis of ECV-304 were determined 48 hours after irradiation. The concentrations of TGF-?1 and VEGF in the cultured supernatant were determined by ELISA method.The protein expression of the endothelial receptor KDR in ECV-304 cells was detected by immunofluorescence and flow-cytometry while its mRNA expression was detected by RT-PCR. Results After high dose of ion-irrdiation, the cellular proliferation activity decreased, apopototic cells increased and the cell ratio in G2/M phase increased.In addition,The secretion of VEGF and TGF-?1 was promoted with the increased KDR expression. Active macrophages could alleviate the decrease of cellular proliferation and the increase of apoptotic cells and cell ratio in G2/M phase induced by ion-irriation.Furthermore,the VEGF secretion and the KDR expression were enhanced and the secretion of TGF-?1 was inhibited by the active macrophages. Conclusions Ion irradiation could induce the decrease of cellular proloferation, the increase of apoptotic cells and the cells in G2/M phase.This might partly be related to the inhibiting roles of TGF-?1 secreted by the ECV-304 cells.Active macrophages might protect the endothelial cells from radiation injury.
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Objective:To develop an experimental three-dimensional model by ECV304 cells(human umbilical vein endothelial cell line) for investigating the mechanisms of angiogenesis in vitro.Methods:ECV304 cells were seeded onto three-dimensional collagen gels made of rat-tail collagen.When the endothelial cells were cultured and grown to near confluence,treated with bFGF for 3 to 12 days,and then assessed with inverted phase contrast microscope.Results:The endothelial cells migrated into the gels,formed complex networks by cell cords at different levels through the bottom view,and sprouted capillary-like structures through the side view.Conclusion:ECV304 cells are capable of expressing some early events of angiogenesis in the three-dimensional collagen gels:proliferating,migrating and sprouting and so on.It should be useful for studying angiogenesis in vitro.
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AIM: To research the effect of serum from patients with blood stasis syndrome(BSS) associated with hypertension disease on vWF、TM、EPCR excreted by ECV-304 and Tanshinone Ⅱ_A's intervention. METHODS: Cultivated human umbilical vein endothelial cells(CRL-1730) were divided into four groups,according to different serum of patients with BSS associated with hypertension disease,BSS(model) group,non-blood stasis group,health group and control group.The incubation time was 24 h.Tanshinone Ⅱ_A was of different concentrations(40、20、10、5 ?g/mL),all Tanshinone Ⅱ_A groups' cells were incubated by patients' serum and Tanshinone Ⅱ_A for 24 h.vWF,TM and EPCR were assayed by enzyme-linked immunosorbent assay(ELISA). RESULTS: vWF,TM and EPCR secreted were higher in the model group,the non-blood stasis group and the health group than those in the control group;vWF,TM and EPCR secreted were higher in the model group,the non-blood stasis group than those in the health group;Thereinto,the difference was distinct between the model group and the health group(P
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AIM:To explore the effect of ?-asarone on vascular endothelium and adhesion molecule expression of endothelium induced by ?-amyloid peptide from Alzheimer's disease and to estimate the injury repair.METHODS:Cultured ECV304 cells were incubated with freshly solublized A?_ 1-42 and the mixture of A?_ 1-42 and ?-asarone,the expression of three central adhesion molecules,CD106,CD62P,CE62E and Ca 2+ concentration were examined and apoptosis was recorded by Flow cytometry.Test viability of cells by MTT methods.RESULTS:The results showed that in model group and treated group,ligation of endothelial CD106,CD62P,CE62E,markers for endothelial cell activation and Ca 2+ concentration,leads to a lot of release.The livability decreased and the apoptosis increased.Further more,simultaneous treatment of ECV304 cells with ?-asarone resulted in the decrease significantly in these three adhesion molecules described above and Ca 2+ concentration as well as the livability upper and apoptosis lower.CONCLUSION:CD106,CD62P,CE62E,important inflammational factor of A?-induced endothelial injury,may be promotion of the inflammatory scade in vascular endothelial.?-asarone may protect ECV304 cell apoptosis by regulate Ca 2+ and expression of cell surface markers.
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AIM: To establish a quantitative measurement method of ECV-304 cell migration in a scratch wound model in vitro. METHODS: The method of ECV-304 cell scratch wound model was improved. The ECV-304 cell migration was measured quantitatively using a computer-assisted video microscopic method with image-analysis system. The effects of heparin and thrombospondin-1 on ECV-304 cell migration were observed in the scratch wound model in vitro. RESULTS: The average width and length of cell scratch wound was (0.95?0.08) mm and (51.20?3.40) mm respectively in 120 experiments. The peak of cell migration reached about 24 h after culture. The stimulated effect of heparin and inhibited effect of thrombospondin-1(TSP-1) on cell migration were observed. CONCLUSION: A quantitative measurement method of ECV-304 cell migration in vitro is established. The stimulated and inhibited effects on ECV-304 cell migration were proven respectively using this method.
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ECV304 was reported first in 1990 as a spont aneously-transformed and immortalized cell line derived from a Japanese HUVEC. S ubsequently, many studies validated that the ECV304 is a permanent endothelial cell line. It has been used widely as an endothelial cell model and an useful re search tool in biomedicine and pharmacology. However, several distinct differenc es exist between ECV304 and HUVEC. Some studies even pointed out that ECV304 is not of HUVEC origin. According to the research data including ours, this reporte dly endothelial-derived permanent human cell line ECV304 may be dedifferentiated towards an epithelial phenotype. It is therefore not an appropriate cell line t o study endothelial cell biology. But cultured ECV304 cells can still be used as a model, tool or target in the pathophysiological and pharmacological studies, depending on whether or not their functional expression or markers are suitable for the research work.