Objective To study the feasibility of transplanting human saphanous vein endothelial cells to luminal surface of blood vessel prosthesis and to play a theoretical foundation for the clinical application of autologous endothelial cell transplantation. Methods Human saphanous vein endothelial cells were harvested with 0.1% collagenase and cultivated in vitro for 13.08±1.24 days. The cultures were confirmed as endothelial cells with the fourescent linked anti-Ⅷ antigen antibodies. The content of both 6-keto-PGF1α and Von Willebrand factor (vWF) in the supernatant were detected with ELISA and radioimmunoassay. The multiplied cells were lined in vitro onto the luminal surface of expanded polytetraflouroethylene (ePTFE) grafts precoated with fibrin glue and fibronectin, then cultivated again for 9 days. Results 11.46±2.69×１０６ of available endothelial cells could be regularly obtained, the number of endothelial cells increased 147.93±88.68 times when culture were terminated. All the cells diploid cells with a purity of 99%. The content of both 6-keto-PGF1α and vWF in the media showed no significant difference between the primary and subculture passages. The luminal surface of grafts was covered completely by a spindlelike endothelial monolayer and an even fibrin glue matrix could be seen underneath. Conclusion Endothelial cells derived from human saphanous veins might be feasible to be transplanted onto the luminal surface of ePTFE and present a potential clinical application.
Objective To determine the effect of thiazolidinediones (TZDs) on early retinopathy in rats with experimental diabetes. Methods In 40 rats, diabetic models were set up in 36 by one-off intraperitoneal injection with streptozotocin (STZ), and other 4 were in the normal control group. Twenty-four diabetic rats with the disease-duration of more than 6 months underwent intravitreous injection (with rosiglitazone or pioglitazone in 10 rats, respectively), and the rest 4 rats werenprime;t injected with drugs as the diabetic positive control group. Immunohistochemical treptomycin-avidin-biotin-complex (SABC) method, in situ hybridization of retinal vascular endothelial growth factor (VEGF) mRNA, and TdT-dUTP terminal nick-end labelling (TUNEL) were performed on the ocular paraffin section to detect the cellular apoptosis. The difference of VEGF expression and cellular apoptosis between TZDs and control group was observed and analyzed. Results The results of immunohistochemical staining and hybridization in situ were negative in the normal control group. The positive expression rate of VEGF was lower in rosiglitazone and pioglitazone group than which in the diabetic positive control group, and there was no obvious differences of positive expression of VEGF mRNA and cellular apoptosis between the 2 groups. Conclusion TZDs (rosiglitazone and pioglitazone) may inhibit the positive expression of VEGF protein in retina of STZ-induced diabetic rats to some extent, but not affect the growth of VEGF in retina. (Chin J Ocul Fundus Dis, 2006, 22: 7-10)
Objective To investigate the effect on proliferation and apoptosis of vascular endothelial cells exposed to high glucose. Methods The cultured human umbilical vein endothelial cells (HUVEC) were treated with high glucose at concentrations of 10,20,30,40,50 mmol/L for 2,4,6,8,10,12,14 days,cpm value was studied by using tritium-labelled thymidine deoxyribose(3H-TdR)incorporation assay.Flow cytometry was used to detect apoptosis index,proliferation index and cell cycle. Results Treated with high glucose,the proliferation index and cpm were significantly reduced in a dose and time dependent manner than the control groups(Plt;0.05).The apoptosis index of HUVEC were higher compared with the control groups(Plt;0.05).The percent of the cultured cells in G1 phase in all high glucose groups were increased,the percent in S phase were largely decreased(Plt;0.05). Conclusion Exposed to high glucose,the apoptosis of HUVEC was accelerated and the suppressive effect of high glucose on the proliferation of HUVEC was observed.Endothelial cells were possibly arrested in G1/S checkpoint. (Chin J Ocul Fundus Dis,2000,16:177-180)
Objective To investigate the effect of hypoxia on the exp ression and function of integrin receptor αvβ3 of bovine retinal vascular endotheliocytes. Methods Bovine retinal vascular endotheliocy tes in the culture dishes coated by vitronectin was put into the normal and hypoxemic condition, respectively. Enzyme linked immunosorbent assay and cell adhesion analysis were used to detect the expression and function of integrin receptor αvβ3 in bovine retinal vascular endotheliocytes, respectively. Results Under the condition of hypoxia, the expression of αvβ3 increased gradually, and reached the peak at the 48th hour. The expression of αvβ3 at the 60th and 72nd hour in hypoxia group was higher than that in the normal group. Bovine retinal vascular endotheliocytes absorbed more Vn of extra-cellular matrixes (ECM) after cultured under hypoxemic condition for 24 hours.Conclusion Hypoxia may up-regulate the expression of αvβ3, which promote the adsorbability of endotheliocytes.(Chin J Ocul Fundus Dis，2004，20：360-363)
Objective To observe the expression of p53, bcl-2 genes, vascular endothelial cell growth factor（VEGF）, basic fibroblast growth factor（bFGF）, insulin-like growth factor-I （IGF-I）, and the receptors of these factors of retinal vascular endothelial cells (VECs) of 1- to 20-week diabetic rats, and the relationship between the expressions and cell cycle arrest.Methods Retinal sections of diabetic rats induced by alloxan were immunohistochemically stained and observed by light microscopy (LM) and electron microscopy (EM). Dot blotting and Western blotting were used to determine the expression of mRNA, proteins of p53 and bcl-2. Results Under LM, immunohistochemical positive expression of p53 and bcl-2 were found on the vessels of ganglion cell layer and inner nuclear layer of retinae of 8- to 20-week diabetic rats; under EM, these substances were observed depositing in VECs. The retinal VECs also expressed VEGF, bFGF, IGF-I and their receptors. There was no positive expression of other cell types in these retinae, all cell types of retinae in control group, or all cells of retinae of diabetic rats with the course of disease of 1 to 6 weeks. The result of dot blotting revealed that retinal tissue of 20-week diabetic rat expressed p53 and bcl-2 mRNA, and the result of Western blotting revealed that they also expressed p53 and bcl-2 proteins. But retinal tissues of control group did not. Positive expression of bax was not found in the retinae in control group or 1- to 20-week diabetic rats. Conclusion p53, bcl-2 may introduce cell cycle arrest of VECs of retinae in 8- to 20-week diabetic rats. High glucose might stimulate the expression of VEGF, bFGF, IGF-I and their receptors, and the growth factors may keep VECs surviving by self-secretion. (Chin J Ocul Fundus Dis，2003，19：29-33)
Objective To observe apoptotic and proliferative characteristics of the retinal vascular end othelial cells (RVECs) of the 1～16 weeks diabetic rats and p53 and bcl-2 expressions of the rats,in order to probe the pathogenic mechanism of diabetic retinopathy(DR). Methods Models of diabetic Wistar rats were made by alloxan venous injection.The retinal blood vessels were filled by ink,the wholemounts and paraffin-embedded sections of the retinas were made,TUNEL staining and Immunohistochemical ABC staining were used,and light microscopy was taken,in succession. Results Apoptosis of the RVECs was not found.Compared with control group,the morphologic features of the RVECs and the structure of the retinal blood vessels remained unchanged.In the period from the 10th to the 16th week,the immunohistochemical stain of PCNA,BrdU,p53,and bcl-2 for RVECs revealed positive results,but there was no any sign of the RVECs stacking and proliferating or new blood vessels forming in the retinas.In control group,the reaction of immunological stain of the aforementioned parameters was negative. Conclusions No accelerated apoptosis and proliferation of the RVECs in the 1～16 week diabetic rats happen after alloxan injection.Almost all of the RVECs were stimulated to enter the cell cycle in the 10th week.Expression of p53 and bcl-2 might play an important role in stabilizing the RVECs in early stage of diabetes. (Chin J Ocul Fundus Dis, 1999, 15: 157-159)
Objective To investigate the relationship between the expression of vascular endothelial growth factor(VEGF)and the retinoblastome(RB)differentiation degree and the infitration capability. Method The VEGF expression in RB tissues of 40 cases was analysed by using LSAB immunohistochemical method. Results The VEGF expression in differentiated RB tissues of 13 cases was markedly lower than that in non-differentiaed RB tissues of 27 cases(P＜0.05);The VEGF expression in RB tissues of the optic nerve infiltrated group(14 cases) was significantly higher than of the optic nerve noninfiltrated group(26 cases)(P＜0.05)． Conclusion The results indicate that the VEGF expression is signficantly related with the differentiation degree and infiltration capability of RB. (Chin J Ocul Fundus Dis, 1999, 15: 238-240)
Objective To investigate the effects of Hep-A and Hep-B on vascular endothelial growth factor (VEGF)-induced breakdown of blood-retinal barrier. Methods The mice were subcutaneously injected vehicle, Hep-A or Hep-B 10 mg/kg twice a day for 5 days. Then, 1 μl of 10-6mol/L VEGF were intravitreous injected. After 6 hours, 13.7×104Bq/g3H-mannital were injected intraperitoneally. The mice were sacrificed and the retinas, lungs, kidneys were removed and examined for radioactivity. The result were analyzed using SPSS software to calculate and compare retina/lung and etina/kidney leakage ratio among groups of different treatment. Result The retina/lung and retina/kidney leakage ratio were 0.38±0.04 and 0.21±0.03 respectively in normal mice; increased significantly to 1.05±0.11 and 0.46±0.04 respectively in model mice, both Plt;0.01 compared to those in normal mice; decreased to 0.59±0.06 and 0.32±0.03 respectively in mice treated with Hep-A, both Plt;0.01 compared to those in model mice; decreased 0.54±0.04 and 0.35±0.03 in mice treated with Hep-B,both Plt;0.01 compared to those in model mice. Conclusion Hep-A and Hep-B can significantly reduce VEGF-induced breakdown of blood-retinal barrier in mice. Chin J Ocul Fundus Dis，2004，20：352-354)
ObjectiveTo observe the inhibitory effect of berberine (BBR) on the apoptosis of human retinal vascular endothelial cells (hREC) under high glucose environment.MethodshREC was divided into blank control group (NC group), high glucose group (HG group), BBR treatment group (BN group), and BBR+high glucose treatment group (BH group). The cells of each group were cultured in Dulbecco's modified eagle medium; 5.5 and 30.0 mmol/L glucose were added to the medium of the NC group and HG group, respectively; 5.0 mmol/L glucose and 5.0 mmol/L BBR was added to the BN group; 30.0 mmol/L glucose and 5.0 mmol/L BBR was added to the medium of the BH group. Flow cytometry was used to observe the apoptosis rate of each group. Western blotting was used to detect the relative expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2 related X protein (Bax), and Cytochrome C (Cyt-C) and cysteine aspastic acid-specific protease 3 (Caspase-3) proteins in each group of cells. The difference between the two groups was tested by t test, and the difference among multiple groups was analyzed by one-way analysis of variance. ResultsThe results of flow cytometry showed that compared with the NC group, the apoptosis rate of the HG group significantly increased, and the difference was statistically significant (P＜0.01); compared with the HG group, the apoptosis rate of the BH group significantly reduced, the difference was statistical significance (P＜0.05). Western blot test results showed that, compared with the NC group, the relative expression of Bax and Caspase-3 protein in the HG group increased, and the relative expression of Bcl-2 protein decreased. The difference was statistically significant (P＜0.01). Compared with the HG group, the relative expression of Bax, Cyt-C, and Caspase-3 protein in BH group cells decreased, and the relative expression of Bcl-2 protein increased, and the difference was statistically significant (P＜0.01). ConclusionBBR can inhibit hREC apoptosis by affecting the expression of apoptotic protein under high glucose environment.
Objective To evaluate the possible role of vascular endothelial growth factor (VEGF) in the pathogenesis of retinal neovascularization resulting from retinal ischemia． Methods Retinal vein occlusion of rabbits model was established with argon laser photo coagulation to retinal vein．The VEGF mRNA expression was observed in ischemic retina and retinal neovascular tissue with in situ hybridization technique． Results VEGF mRNA expression was detected in inner ischemic retina and retinal neovascular tissue that were corespondent to the distribution of retinal ischemia．The best VEGF mRNA expression was detected in retinal neovascular tissue． Conclusion VEGF might play an important role in the pathogenesis of vascular proliferative retinopathy． (Chin J Ocul Fundus Dis, 2001,17:5-7)