ABSTRACT
Objective To construct the connective tissue growth factor (CTGF) recombinant interference vector (shRNA) and observe its inhibitory effect on the expression of endogenous CTGF in retinal vascular endothelial cells.Methods The human CTGF shRNA was constructed and the high-titer CTGF shRNA lentivirus particles was acquired via three-plasmid lentivirus packaging system to infect retinal vascular endothelial cells.The optimal multiplicity and onset time of lentivirus infection were identified by tracing down the red florescent protein in interference vector.The cells were classified into three groups:blank control group,infection control group and CTGF knockdown group.The differences in cells migrating ability was observed through Transwell allay.The mRNA and protein expression of CTGF,fibronectin,a-smooth muscle actin (α-SMA) and collagen Ⅰ (Col Ⅰ) were quantified through real-time PCR testing and Western blot system.Data between the three groups were examined via one-way analysis of variance.Results The result showed that an optimal multiplicity of 20 and onset time of 72 hours were the requirements to optimize lentivirus infection.Transwell allay result showed a contrast in the number of migrated cells in the CTGF knockdown group and that in the blank control group and infection control group (F=20.64,P=0.002).Real-time PCR testing showed a contrast in related gene expression (CTGF,fibronectin,α-SMA and Col Ⅰ) in the CTGF knocked-down group and that in the blank control group and infection control group (F=128.83,124.44,144.76,1 374.44;P=0.000,0.000,0.000,0.000).Western blot system showed the statistical significance of the contrasted number of related protein expression (CTGF,fibronectin,α-SMA and Col Ⅰ) in the knockdown group and that in the blank control group (F=22.55,41.60,25.73,161.68;P=0.002,0.000,0.001,0.000).Conclusion The success in producing CTGF shRNA lentivirus particle suggests that CTGF shRNA lentivirus can effectively knock down CTGF expression.
ABSTRACT
Objective To observe RNA-Seq analysis ofgene expression profiling in retinal vascular endothelial cells after anti-vascular endothecial growth factor (VEGF) treatment.Methods Retinal vascular endothelial cells were cultured in vitro,and the logarithmic growth phase cells were used for experiments.The cells were divided into the control group and high glucose group.The cells of two groups were cultured for 5 hours with 5,25 mmol/L glucose,respectively.And then,whole transcriptome sequencing approach was applied to the above two groups of cells through RNA-Seq.Now with biological big data obtained as a basis,to analyze the differentially expressed genes (DEGs).And through enrichment analysis to explain the differential functions of DEGs and their signal pathways.Results The gene expression profiles of the two groups of cells were obtained.Through analysis,449 DEGs were found,including 297 upregulated and 152 downregulated ones.The functions of DEGs were influenced by regulations over molecular biological process,cellular energy metabolism and protein synthesis,etc.Among these genes,ITGB 1BP2,NCF 1 and UNC5C were related to production of inflammation;AKR1C4,ATP 1A3,CHST5,LCTL were related to energy metabolism of cells;DAB 1 and PRSS55 were related to protein synthesis;SMAD9 and BMP4 were related to the metabolism of extracellular matrix.GO enrichment analysis showed that DEGs mainly act in three ways:regulating biological behavior,organizing cellular component and performing molecular function,which were mainly concentrated in the system generation of biological process part and regulation of multicellular organisms.Pathway enrichment analysis showed that gene expressions of the two cell groups were differentiated in transforming growth factor-β (TGF-β) signaling pathway,complement pathway and amino acid metabolism-related pathways have also been affected,such as tryptophan,serine and cyanide.Among them,leukocyte inhibitory factor 9 and bone morphogenetic protein 4 play a role through the TGF-β signaling pathway.Conclusions High glucose affects the function of retinal vascular endothelial cells by destroying transmembrane conduction of retinal vascular endothelial cells,metabolism of extracellular matrix,and transcription and translation of proteins.
ABSTRACT
Objective To observe RNA-Seq analysis of gene expression profiling in human retinal vascular endothelial cells after anti-vascular endothecial growth factor (VEGF) treatment.Methods Cultured the retinal vascular endothelial cells in vitro and logarithmic growth phase cells were used for experiments.The cells were divided into VEGF group and VEGF combined with anti-VEGF drugs group.The VEGF group cells were treated with 50 ng/ml VEGF for 72 h to simulate the high VEGF survival conditions of vascular endothelial cells in diabetic retinopathy.VEGF combined with anti-VEGF drug group cells was treated with 50 ng/ml VEGF and 2.5 μg/ml anti-VEGF drugs for 72 h to imitate the microenvironment of cells following the anti-VEGF drugs treatment,and whole transcriptome sequencing approach was applied to the above two groups of cells through RNA-Seq.Now with biological big data obtained as a basis,to analyze the differentially expressed genes (DEGs).And through enrichment analysis to explain the differential functions of DEGs and their signal pathways.Results The gene expression profiles of the two groups of cells were obtained.Through analysis,328 DEGs were found,including 194 upregulated and 133 downregulated ones.The functions of DEGs were influenced by regulations over molecular biological process,cellular energy metabolism and protein synthesis,etc.Among these genes,SI,PRX and HPGD were related to protein synthesis,BIRCT to cellular apoptosis,and ABLIM 1 and CRB2 to retinal development,and ABCG 1,ABCA9 and ABCA12 were associated with the cholesterol of macrophage and the transfer of phospholipid.GO enrichment analysis showed that DEGs mainly act in three ways:regulating biological behavior,organizing cellular component and performing molecular function.Pathway enrichment analysis showed that gene expressions of the two cell groups were differentiated in ECM receptor pathway,and Notch,mitogen-activated protein kinase,transforming growth factor (TGF)-β and Wnt signal pathways.Among them,the gene expression in TGF-β signal pathway attracts most attention,where the DEGs,such as CAMK2B,COL3A1,CYGB,PTGER2 and HS6ST2,among others,were closely related to fibrosis process.Conclusion The anti-VEGF drugs may enhance the expression ofCAMK2B,COL3A1,CYGB,PTGER2 and others genes related to TGF-β signal pathway and aggravate retinal fibrosis disease.
ABSTRACT
Objective To explore repressive effects of transthyretitin (TTR) on the growth of human retinal endothelial cells (hREC) under high glucose and hypoxia environment. Methods hRECs were divided into 8 groups, including normal glucose group (5.5 mmol/L glucose), hypoxia group, high glucose group (25.0 mmol/L glucose), high glucose and hypoxia group, normal glucose group+TTR, normal glucose and hypoxia group+TTR, high glucose group+TTR, high glucose and hypoxia group+TTR. Flow cytometry was used to analyze cellular apoptosis. The expression level of Akt, p-Akt, eNOS, Bcl-2 and Bax protein were measured by Western blot. Results Hypoxia could induce apoptosis as the apoptosis rate of normal and hypoxia group was higher than normal group (χ2=25.360, P<0.05), high glucose and hypoxia group was higher that high glucose group (χ2=17.400, P<0.05). The cell apoptosis rate of high glucose and hypoxia group+TTR were increased significantly as compared with high glucose and hypoxia group (χ2=9.900, P<0.05). There was no statistically significant difference on the cell apoptosis rate between normal group and high glucose group, normal group+TTR and normal group, high glucose group+TTR and high glucose group, normal and hypoxia group+TTR and normal and hypoxia group (P>0.05). Western blot showed that the expression of Akt did not change significantly in all eight groups(F=2.450, P>0.05). Compared to normal group, the expression of p-Akt, eNOS, Bcl-2 in normal and hypoxia group were decreased (t=9.406, 5.306, 4.819), and the expression of Bax (t=-4.503) was increased (P<0.05). Compared to high glucose group, same trend was found in high glucose and hypoxia group (t=8.877, 7.723, 6.500, -14.646; P<0.05). The expression of p-Akt in normal and hypoxia group+TTR was higher than normal and hypoxia group (t=-5.024, P<0.05) ,but there was no difference on the expression of eNOS, Bcl-2, Bax between these two groups (t=-2.235, -2.656, -0.272;P>0.05). Compared to high glucose and hypoxia group, the expression of p-Akt and Bcl-2 in high glucose and hypoxia group+TTR were decreased (t=4.355, 4.308; P<0.05), the expression of Bax was increased (t=-4.311, P<0.05), and there was no difference on the expression of eNOS between these two groups (t=-1.590, P>0.05). There was no statistically significant difference in the expression of p-Akt, eNOS, Bcl-2, Bax between high glucose group and normal group (t=-3.407, -4.228, -4.302, -2.076; P>0.05), normal group+TTR and normal group (t=-4.245, -4.298, -2.816, -1.326; P>0.05), high glucose group+TTR and high glucose group (t=4.016, -0.784, 0.707, -0.328; P>0.05). Conclusion Under high glucose and hypoxia, transthyretitin suppress the growth of hREC through Akt/Bcl-2/Bax, but not Akt/eNOS signaling pathway.
ABSTRACT
Objective To explore repressive effects of transthyretitin (TTR) on the growth of human retinal endothelial cells (hREC) under high glucose and hypoxia environment. Methods hRECs were divided into 8 groups, including normal glucose group (5.5 mmol/L glucose), hypoxia group, high glucose group (25.0 mmol/L glucose), high glucose and hypoxia group, normal glucose group+TTR, normal glucose and hypoxia group+TTR, high glucose group+TTR, high glucose and hypoxia group+TTR. Flow cytometry was used to analyze cellular apoptosis. The expression level of Akt, p-Akt, eNOS, Bcl-2 and Bax protein were measured by Western blot. Results Hypoxia could induce apoptosis as the apoptosis rate of normal and hypoxia group was higher than normal group (χ2=25.360, P<0.05), high glucose and hypoxia group was higher that high glucose group (χ2=17.400, P<0.05). The cell apoptosis rate of high glucose and hypoxia group+TTR were increased significantly as compared with high glucose and hypoxia group (χ2=9.900, P<0.05). There was no statistically significant difference on the cell apoptosis rate between normal group and high glucose group, normal group+TTR and normal group, high glucose group+TTR and high glucose group, normal and hypoxia group+TTR and normal and hypoxia group (P>0.05). Western blot showed that the expression of Akt did not change significantly in all eight groups(F=2.450, P>0.05). Compared to normal group, the expression of p-Akt, eNOS, Bcl-2 in normal and hypoxia group were decreased (t=9.406, 5.306, 4.819), and the expression of Bax (t=-4.503) was increased (P<0.05). Compared to high glucose group, same trend was found in high glucose and hypoxia group (t=8.877, 7.723, 6.500, -14.646; P<0.05). The expression of p-Akt in normal and hypoxia group+TTR was higher than normal and hypoxia group (t=-5.024, P<0.05) ,but there was no difference on the expression of eNOS, Bcl-2, Bax between these two groups (t=-2.235, -2.656, -0.272;P>0.05). Compared to high glucose and hypoxia group, the expression of p-Akt and Bcl-2 in high glucose and hypoxia group+TTR were decreased (t=4.355, 4.308; P<0.05), the expression of Bax was increased (t=-4.311, P<0.05), and there was no difference on the expression of eNOS between these two groups (t=-1.590, P>0.05). There was no statistically significant difference in the expression of p-Akt, eNOS, Bcl-2, Bax between high glucose group and normal group (t=-3.407, -4.228, -4.302, -2.076; P>0.05), normal group+TTR and normal group (t=-4.245, -4.298, -2.816, -1.326; P>0.05), high glucose group+TTR and high glucose group (t=4.016, -0.784, 0.707, -0.328; P>0.05). Conclusion Under high glucose and hypoxia, transthyretitin suppress the growth of hREC through Akt/Bcl-2/Bax, but not Akt/eNOS signaling pathway.
ABSTRACT
Objective To investigate the effect of arginase (Arg) inhibitor N-ω-Hydroxy-L norArginine (nor-NOHA) on high glucose cultured rhesus macaque retinal vascular endothelial cell line (RF/6A) in vitro.Methods The RF/6A cells were divided into the following 4 groups:normal control group (5.0 mmol/L of glucose,group A),high glucose group (25.0 mmol/L,group B),high glucose with 125 mg/L nor-NOHA group (group C),and high glucose with 1％ DMSO group (group D).The proliferation,migration ability and angiogenic ability of RF/6A cells were measured by Methyl thiazolyl tetrazolium (MTT),transwell chamber and tube assay respectively.The express of Arg Ⅰ,eNOS,iNOS mRNA of RF/6A cells were measured by real-time polymerase chain reaction (RT-PCR),Enzyme-linked immuno sorbent assay (ELISA) was used to detect the expression of NO and interleukine (IL)-1b of RF/6A cells.Results The proliferation,migration,and tube formation ability of group A (t=2.367,5.633,7.045;P＜0.05) and group C (t=5.260,6.952,8.875;P＜0.05)were significantly higher than group B.RT-PCR results showed the Arg Ⅰ and iNOS expression in group B was higher than that in group A (t=6.836,3.342;P＜0.05) and group C (t=4.904,7.192;P＜0.05).The eNOS expression in group B was lower than that in group A and group C (t=4.165,6.594;P＜0.05).ELISA results showed NO expression in group B was lower than that in group A and group C (t=4.925,5.368;P＜0.05).IL-1b expression in group B was higher than that in group A and group C (t=5.032,7.792;P＜0.05).Conclusions Nor-NOHA has a protective effect on cultured RF/6A cells in vitro and can enhance its proliferation,migration and tube formation.The mechanism may be inhibiting the oxidative stress by balancing the expression of Arg/NOS.
ABSTRACT
Objective To explore transthyretin (TTR) effect on retinal vascular endothelial cells (hREC) under high glucose and hypoxia environment.Methods hREC and human retinal pigment epithelial cell (hRPEC) were cultured at low-glucose (LG),high glucose (HG) and hypoxia.The glucose concentration was increased from 5.5 mmol/L up to 25 mmol/L,and hypoxia was induced by 200 μmol/L CoCl2.The cells were divided into LG group,LG-hypoxia group,HG group,HG-hypoxia group according to the different cell culture environment.The growth index was detected at 0,4,8,16,24,36,48,60,72 hours after cultured.Furthermore,hREC and hRPEC were also cultured with additional TTR (4 μmol/L),respectively.Then transwell co-culture system was employed to reveal the effects of hRPEC on the growth of hREC.Results At 72 hours after cultured,the growth index of hREC and hRPEC in LG group were increased as compared with LG-hypoxia group and HG group (hREC:F=17.098,22.970;P＜0.05.hRPEC:F=45.442,9.011;P＜0.05);the growth index of hREC and hRPEC were decreased in HG group and HG-hypoxia group (hREC:F=146.184,P＜0.05;hRPEC:F=27.907,P＜0.05).Additionally,hREC could be significantly repressed by added TTR during culture with high concentration of glucose (F=161.430,24.106;P＜0.05).hREC could be significantly increased by added TTR during culture with low concentration of glucose (F =200.486,48.662;P ＜ 0.05).In co-culture process,hRPEC revealed inhibition activity against hREC under both natural and abnormal environment (LG group:F=15.711,P＜ 0.05;LG-hypoxia group:F =45.659,P＜0.05;HG group:F =7.857,P ＜0.05;HG-hypoxia group:F=6.348,P＜0.05).Conclusion Under high glucose and hypoxia environment,the growth of hREC from neovascular could be inhibited by TTR.
ABSTRACT
Background Previous retinal vascular mounting only obtained part of retinal vessels for the study of retinal diseases,and thus it is difficult to comprehensively assess these diseases.So optimizing the trypsin digestion method to show the complete retinal capillary network is very important for the study of retinal diseases.Objective This study was to modify the preparing way of trypsin digested whole retinal capillary network and offer a basis for a quantitative analysis of cells and capillaries.Methods Thirty Wistar rats were divided into model group (20 rats) and control group (10 rats).Streptozotocinum(STZ) of 1.25％ dissolved in 0.05 mmol/L sodium citratehydrochloric acid buffer was intraperitoneally injected to establish diabetes models in the model group,and the equal volume of solvent was used in the same way in the control group.Eight months after injection,100 ml PBS was injected via ventriculus sinister and released via cut right atrium,and then 100 ml 4％ paraformaldehyde was injected into the ventriculus sinister.The rat retinas containing part of the optic nerve were entirely isolated,and digested by trypsin,and vitreous,inner limiting membrane and neural retinal tissue were removed.The whole retinal capillaries network was mounted on the slide.The ghost pericytes and acellular capillaries in the middle retinal area were identified and counted under the optical microscope.Results The retinal mount exhibited that the retinal vessels were stained by hematoxylin and periodic acid schiff.The vessels network presented with the entire type in shape with the radical central retinal arteries and veins and their branches.The capillary showed the shallow-and deep-layer networks between the small arteries and veins.Pericytes distributed and protruded vessel wall and formed the ghost cells without nuclei.The diameter of acellular capillaries was 20％ or more than that of near capillaries,and no cellular nuclei or ghost cell was found through the vessel.Conclusions The experimental technique for setting-up of cleaned vasculature and mounting vessels on glass microscopic slide provides intact vessels,which is helpful for the evaluation of retinal vascular morphology and quantitative analysis.