Objective To investigate the effect of surface propertyof different polyether-ester block copolymers［poly(ethylene glycol-terephthalate)/poly(butylene terephthalate), PEGT/PBT］ on the growth of smooth muscle cells (SMCs) and endothelial cells(ECs). Methods Three kinds of copolymers were synthesized, which were 1000-T20 (group A), 1000PEGT70/PBT30 (group B) and 600PEGT70/PBT30 (group C). The water-uptake and contact angle of three polyether-ester membranes were determined. The canine aorta smooth muscle cells and external jugular vein endothelial cells were primarily harvested, subcultured, and then identified. The proliferation of SMCs and ECs on the different polyether-ester membranes were investigated. Results The water-uptake of three copolymers arranged as the sequence of group C＜group A＜group B, and contact angle as the sequence of group C＞group A＞group B, indicating group B being more hydrophilic. However, smooth musclecells andendothelial cells grew poorly on the membrane of group B after low density seeding, but proliferated well on the membranes of group A and group C. Conclusion In contrast with more hydrophilic 1000PEGT70/PBT30, moderately hydrophilic 1000-T20 and 600PEGT70/PBT30 has better compatibility with vascular cells. The above results indicate that the vascular cells can grow well on moderately hydrophilic PEGT/PBT and that PEGT/PBT can be used in vascular tissue engineering.
Objective To investigate the effect of arginase (Arg) inhibitor N-ω-Hydroxy-L nor-Arginine (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.
Objective To establish a rapid in vitro culture method of human choroidal endothelial cells (HCEC) and the cellular Characteristics to provide an in vitro model for researches of choroiretinal diseases which involved the HCEC. Methods The human choroidal tissues were digested in two steps by trypsin and collagenase, and the HCEC were obtained and cultured after the digested cell suspension was sorted and purified with magnetic beads of CD31 Dynabeads. The characteristics of HCMEC were observed by the morphologic observation method, transmission electron microscopy, and immunohistochemical staining with FⅧ factor, CD31, and CD34. Results The cultured HCEC were polygonal and oval, and after amalgamation, the cells had slabstone-like appearance. After the subculture, the configuration of HCEC remained the same, and represented cobblestone appearance with less magnetic beads attached on the cellular surface after HCEC converged into a single layer. The Weibel-Palade body which is the characteristic marker of endothelial cells was found. The staining of FⅧ fatcor, CD31, CD34 were positive. Conclusion HCEC can be cultured in vitro successfully with our method, which is easy to get sufficient number of highly purified HCEC. (Chin J Ocul Fundus Dis, 2007, 23: 126-129)
Intravitreal injection of anti-VEGF drugs has gradually become the first-line treatment for diabetic retinopathy (DR). However, diabetic macular edema (DME) caused by DR blood-retinal barrier damage is less sensitive to anti-VEGF drugs.Therefore, it is necessary to find supplementary drugs or alternative drugs that can effectively protect the structure of the blood vessel wall. Melatonin is a hormone mainly secreted by the pineal gland, which can play a number of functions in the human body such as regulating biological rhythms, scavenging free radicals, and anti-inflammatory. In recent years, studies have shown that melatonin can improve neuronal degeneration and protect blood vessel structure through multiple mechanisms in retinopathy. In terms of its protective effect on the retinal capillary structure, melatonin can improve the damage of early DR endothelial cells and pericytes through anti-oxidative stress, anti-inflammatory, and inhibiting cell apoptosis so as to protect the integrity of the blood-retinal barrier structure. It suggests that melatonin may provide new ideas for the prevention and treatment of DR, especially with DME.
ObjectiveTo explore the effects of transthyretin (TTR) on biological behavior of retinal microvascular epithelial cell (RMVEC). MethodsRMVEC was cultured in medium with 0 μmol/L and 4 μmol/L TTR. The proliferation, migration and healing abilities (0, 24, 48 hours) of RMVEC with different concentrations of TTR were measured by methyl thiazol tetrazolium (MTT) assay, transwell assay and scarification test. ResultsMTT assay shows that RMVEC with the concentrations of 4 μmol/L TTR [absorbance (A) value=0.17±0.02] glows faster than with the concentrations of 0 μmol/L TTR (A value=0.40±0.03), the difference was statistically significant (t=15.47, P=0.000 1). The transwell assay shows RMVEC with the concentration of 4 μmol/L TTR [(140±7) cells] migrants faster than RMVEC with the concentration of 0 μmol/L TTR [(227±14) cells], the difference was statistically significant (t=5.44, P=0.000 6). The scarification test shows that the RMVEC with the concentration of 4 μmol/L TTR [(134.4±45.4) μm] heals faster than the RMVEC with the concentration of 0 μmol/L TTR [(330.0±23.1) μm], the difference was statistically significant (t=8.25, P<0.01). The cells in 48 hours and 4 μmol/L group were healed completely, but not healed in 0 μmol/L group. ConclusionTTR can promote the proliferation, migration and healing abilities of RMVEC.
ObjectiveTo observe RNA-Seq analysis of gene expression profiling in retinal vascular endothelial cells after anti-vascular endothecial growth factor (VEGF) treatment.MethodsRetinal 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.ResultsThe 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, ITGB1BP2, NCF1 and UNC5C were related to production of inflammation; AKR1C4, ATP1A3, CHST5, LCTL were related to energy metabolism of cells; DAB1 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.ConclusionsHigh 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.
Objective To explore morphological recellularization level of bioprosthetic valve scaffold (BVS) and to provide researching means for fabricating tissue engineered heart valve in vitro.Methods The homograft bioprosthetic aortic tube valve was selected as BVS, which was conserved by liquid nitrogen, and its endothelial cells (ECs) were removed by 0.1% sodium dodecylsulphate (SDS). As implantation cells, the endothelial cells (ECs) differentiating from human bone marrow mesenchymal stem cells (MSCs) in vitro were implanted with high-density seeding (gt;10 5 cells/cm2) on the BVS, which was covered by fibronectin (80 μg/ml) in advance. The complex structure was statically cultured in DMEM (high glucose) with 20% FBS and VEGF (10 ng/ml) for about 20 days in vitro and stained by 0.5% AgNO3. The morphological structure was observed and photographed by stereomicroscope to detect the recellularization level. Results The ECs of the bioprosthetic valve were notonly removed completely, but also the collagen fiber and elastic fibers were reserved. The ECs differentiating from MSCs were successfully implanted on the HBS, whose recellularization levels on 7th, 14th and 20th day were 73%, 85%, and 92% respectively. Conclusion AgNO3 staining technique is effective, convenient, and economic in evaluating the recellularization level of BVS. It is an effective method in morphological observation for fabricating tissueengineered heart valve in vitro.
Objective To observe the expression of vascular endothelial growth factor receptor-1 (VEGFR-1) and VEGFR-2 in hypoxic chorioretinal endothelial cells of monkeys (RF/6A), and to evaluate the effect of minocycline. Methods RF/6A was cultured and divided into four groups: control group, hypoxia group, hypoxia and low dose of minocycline group (0.5 mu;mol/L), hypoxia and medium dose of minocycline group (5 mu;mol/L), and hypoxia and high dose of minocycline group (50 mu;mol/L). Real-time reverse transcriptionpolymerase chain reaction (RT-PCR) and immunohistopathological staining were used to measure the mRNA and protein expression of VEGFR-1 and VEGFR-2, respectively. Results RT-PCR showed that the expression of VEGFR-1 mRNA did not vary significantly between groups (F 24 h=0.17,F 48 h=1.53,F72 h=2.04;P＞0.05). Compared with hypoxia group, the expression of VEGFR-2 mRNA in all minocycline treated groups were significantly downregulated (low minocycline, medium minocycline, high minocycline: t=4.69, 20.16, 17.12; P＜0.001). The immunohistopathological study showed the cells with positive staining of VEGFR-1 can be observed in all groups, and the staining was relatively weak and mainly located in cell membrane and cytoplasm. The optical density value analysis showed that the protein expression of VEGFR-1 did not vary significantly between groups at all time points（F 24 h=0.251,F 48 h=0.340,F72 h=0.589;P＞0.05）. The VEGFR-2 positive staining cells were also observed in all groups, and the staining was relatively high. Brown staining particles of VEGFR-2 were observed in the cell membrane with minor staining particles in cytoplasm. The staining density of VEGFR-2 was significantly higher in hypoxia group than control group. Compared with the hypoxia group, the protein expression of VEGFR-2 in minocycline treated groups was significantly lower（F 24 h=19.147,F 48 h=14.893,F72 h==11.984; P＜0.05). Conclusion The expression of VEGFR-2 is upregulated in RF/6A, and minocycline somewhat shows an inhibition effect.
ObjectiveTo observe RNA-Seq analysis of gene expression profiling in human retinal vascular endothelial cells after anti-vascular endothecial growth factor (VEGF) treatment.MethodsCultured 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.ResultsThe 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 ABLIM1 and CRB2 to retinal development, and ABCG1, 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.ConclusionThe anti-VEGF drugs may enhance the expression of CAMK2B, COL3A1, CYGB, PTGER2 and others genes related to TGF-β signal pathway and aggravate retinal fibrosis disease.
Objective To observe the influences of uncoupling protein 2 (UCP-2) rs660339 variants transfection on cell proliferation and apoptosis of human umbilical vein endothelial cell (HUVEC). Methods Two UCP-2 green fluorescent protein (GFP) lentivirus constructs were created with the rs660339 locus carried C or T (UCP-2C or UCP-2T), respectively. HUVEC were cultured after lentiviral infection of UCP-2C or UCP-2T. The expression of UCP-2C or UCP-2T was detected with real time polymerase chain reaction. Cell proliferation and cell apoptosis were compared among negative control (NC) group, UCP-2T group and UCP-2C group using CCK-8 cell viability and flow cytometry. Western blot and immunostaining were employed to examine the expression of Bcl-2 gene. Results The lentivirus constructs were successfully created. ＞80% of the transfected cells were found to express GFP under fluorescent microscope. The mRNA levels of UCP-2 gene were significantly increased (F=29.183,P=0.001) in the UCP-2T group and UCP-2C group. The CCK-8 assay revealed that on day two (F=15.970,P=0.004), day three (F=16.738,P=0.004), day four (F=5.414,P=0.045) post-infection, UCP-2T and UCP-2C group showed significantly greater proliferation than the NC cells. The apoptotic rate in the UCP-2T and UCP-2C group was significantly lower than NC group (F=277.138,P=0.000), and the apoptotic rate of UCP-2T was significantly lower than that of UCP-2C (P=0.003). The protein levels of Bcl-2 in the UCP-2T and UCP-2C group were significantly greater than that in the NC group (F=425.679,P=0.000), and the Bcl-2 expression of UCP-2T was greater than that of UCP-2C (P=0.002). The Bcl-2 density in the UCP-2T and UCP-2C group were greater than that in the NC group (F=11.827,P=0.008), while there was no difference between UCP-2T and UCP-2C group (P=0.404). Conclusion The variants of UCP-2 rs660339 may influence HUVEC proliferation and apoptosis, and UCP-2T showed a stronger effect of inhibiting apoptosis than UCP-2C.