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.
ObjectiveTo investigate the effects of thrombospondin-1 active fragment (TSP-1) synthetical peptide VR-10 on proliferation and migration of rhesus choroidal-retinal endothelial (RF/6A) cell and the expressions of apoptosis relative genes in RF/6A cell. MethodsThe survival rate of RF/6A cell were detected by methyl thiazolyl tetrazolium, and migration ability was measured by transwell chamber after exposure to 1.0 μg/ml TSP-1 and synthetic peptide VR-10 (0.1, 1.0, 10.0 μg/ml) for different times (6, 12, 24, 48 hours). Caspase-3 and factor associated suicide (FAS) protein levels were measured by Western blot. The mRNA level of bcl-2 and FAS ligand (FASL) were measured by reverse transcription-polymerase chain reaction (RT-PCR). ResultsThe survival rate of RF/6A cells was determined by the treatment time and concentration of TSP-1(1.0 μg/ml) and the synthetic peptide VR-10 (0.1, 1.0, 10.0 μg/ml). The lowest survival ratio of RF/6A was 78% (P < 0.001) when cells were treated by 10 μg/ml synthetic peptide VR-10 after 48 hours. TSP-1 and synthetic peptide VR-10 could inhibit migration of RF/6A cells in transwell chamber (P < 0.001). 10.0 μg/ml synthetic peptide VR-10 had the strongest effect, 1.0 μg/ml TSP-1 was the next. Migration inhibition rate was increase with the increase of the concentration of VR-10 (P < 0.001). There was no significant differences between 0.1 μg/ml and 1.0 μg/ml VR-10 (P=0.114). Western bolt showed that RF/6A cell in control group mainly expressed the 32×103 procaspase-3 forms. To 10.0 μg/ml VR-10 treated group, it showed decreased expression of procaspase-3 (32×103) and concomitant increased expression of its shorter proapoptotic forms (20×103). Compared with control group, expression of FAS peptides were significantly increased in 10.0 μg/ml VR-10 treated group. Compared with control group, expression of FasL mRNA was significantly increased in 10.0 μg/ml VR-10 treated group(t=39.365, P=0.001), but the expression of bcl-2 mRNA was decreased(t=-67.419, P=0.000). ConclusionTSP-1 and synthetic peptide VR-10 had the ability to inhibit proliferation and migration of endothelial cell, and also induce apoptosis by increasing FAS/FASL expression and repressing bcl-2 expression.
ObjectiveTo explore repressive effects of transthyretitin (TTR) on the growth of human retinal endothelial cells (hREC) under high glucose and hypoxia environment.MethodshRECs 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.ResultsHypoxia 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).ConclusionUnder high glucose and hypoxia, transthyretitin suppress the growth of hREC through Akt/Bcl-2/Bax, but not Akt/eNOS signaling pathway.
ObjectiveTo observe the immunological regulation effects of human umbilical cord mesenchymal stem cells (hUCMSC) on glucose-damaged rhesus retinal vascular endothelial cells (RF/6A). MethodshUCMSC and RF/6A were co-culture according to 1:1 ratio in the co-culture system (Transwell plates), hUCMSC cells were added to upper chamber, while the lower chamber containing 25mmol/L glucose and RF/6A. There were three groups including RF/6A blank control group, high glucose treated RF/6A group, and high glucose treated RF/6A with hUCMSC co-culture group. MTT was used to measure the RF/6A cell viability. Western blot was used to to detect protein level of Foxp3. Enzyme-linked immunosorbent assay (ELISA) was used to detect the concentration of interleukin (IL)-17. ResultsMTT assay revealed that at the first day, the survival rate of the three groups had no significant difference (F=0.030, P > 0.05). On day 3 and day 7, the cell viability of the high glucose group was significantly lower than that of the control group (t=36.072, 27.890; P < 0.05), the cell viability of the high glucose treated RF/6A with hUCMSC co-culture group was higher than that of high glucose group (t=36.072, 19.650; P < 0.05).Western blot analysis showed that Foxp3 in high glucose RF/6A group was significantly lower than that in the control group at day 7 after culture (t=7.826, P < 0.05) and high glucose RF/6A with hUCMSC group (t=19.936, P < 0.05). ELISA showed that IL-17 in the high glucose group, high glucose with hUCMSC co-culture group was significantly higher than that of the control group (F=1 267.503, P < 0.05), while IL-17 in the hUCMSC co-culture group was significantly lower than that in high glucose group (t=17.386, P < 0.05). ConclusionhUCMSC can regulate the expression of Foxp3 and IL-17 to increase the proliferative ability of RF/6A, which was suppressed by high glucose.
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 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 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.
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)
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 ...