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)
Purpose To study changes of cell cycle of vascular endothelial cell in non-proliferative diabetic retinopathy. Methods Alloxan induced Wistar-rats were employed and immunohistochemistry,Western blotting methods were used. Results The vascular endothelial cells of retinas of 8～20 weeks diabetic rats were observe to be cyclinD1,cyclinD3,cyclinB1,p21 and p27 positive stained with light and electronmicroscopies.CyclinE immuno-stained vascular endothelial cells was observed occasionally.Meanwhile,the evidences of morphologic changes of the vascular en dothelial cells were proved:less plasma,thinner cell,more bubble organelles than those of controls.But,the ultra-structures of pericytes and other type of retinal cells did not change and they also immunostain negative.Komas blue and Western blotting methods also proved that the vascular endothelial cells of retina of 20th week diabetic rats expressed cyclinD1,cyclinB1,p21 and p27 protein. Conclusion Glucose induced retinal vascular endothelial cells of 8～20th weeks diabetic rats enter cell cycle and were arrested at G1/S restriction point.This study also suggested that retinal vascular endothelial cells may possess the ability to resist glucose damage and mechanism of selfstability during very early stage of diabetes. (Chin J Ocul Fundus Dis,2000,16:173-176)
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 explore the mechanism of microvascular spasm after limb ischemia-reperfusion. METHODS: The rabbit hindlimb normothermic tourniquet ischemia model was employed. The tendon on the dorsum of the foot was exposed for observation of microvessels. The responses of arterioles on tendon surface to topical application of 10(-6) mol/L noradrenaline (NE) (a vasoconstrictor), 10(-6) mol/l acetylcholine(Ach) (an endothelium-dependent vasodilator) and 10(-4) mol/L nitroglycerin(NTG) (an endothelium-independent vasodilator) were observed at the period of ischemia and following 30 minutes of reperfusion after 2 hours and 5 hours of ischemia by use of intravital microscopy. RESULTS: No significant changes in the responses of arterioles to NE, Ach and NTG were noted following 30 minutes of reperfusion after 2 hours of ischemia compared with pre-ischemia. The constrictor responses of arterioles to NE were still not significantly altered following 30 minutes of reperfusion after 5 hours of ischemia, however, the dilation responses to Ach and NTG were significantly decreased (to Ach P lt; 0.01; to NTG, P lt; 0.05). CONCLUSION: Reperfusion after 5 hours of ischemia significantly impairs both the endothelium-dependent and endothelium-independent vasodilation, meanwhile preserves constrictor responses to NE, these may contribute to the genesis of the vasospasm in ischemia reperfusion.
Objective To detect the levels of vascular endoth elial growth factor (VEGF) in aqueous humor and vitreous of patients with neovascular glaucoma (NVG) and infer their possible effect on the development of neovascularization of iris. Methods The concentration of VEGF in 22 samples of ocular fluid of aqueous humor and vitreous respectively obtained from 11 patients with NVG undergone intraocular surgery were measured by using enzyme linked immunosobent assay (ELISA) for quantitative analysis. As control, 12 samples of ocular fluid of 6 patients with macular hole were detected by the same methods. Results The mean [AKx-]plusmn;s VEGF concentrations in aqueous humor and vitreous from patients with NVG were [(1.451plusmn;0.247)、(1.610plusmn;0.125) ng/ml] higher than those in the cotrol group [(0.189plusmn;0.038)、(0.201plusmn;0.055) ng/ml], there was a significant difference between the two groups statistically (t=12.007，Plt;0001；t=26.0 57，Plt;0.001). Conclusion The patients with NVG have significantly increased level of VEGF in ocular fluid, and VEGF might fill the role in mediating active iris neovascularization. (Chin J Ocul Fundus Dis, 2001,17:305-306)
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 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 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)
PURPOSE:To investigate the content of vascular endothelial growth factor (VEGF)in vitreous of patients with retinal vascula'r proliferative diseases. METHODS:The concentration of VEGF in undiluted vitreous samples from patients with retina vein occlusion (RVO) (n=7),Eales disease (n=7)and controls (n=7) was measured by enzyme linked immunosorbent assay (EL1SA). RESULTS:The levels of vitreous VEGF were significantly higher 13 folds in patients with RVO(4.67plusmn;3.38)ng/ml and 5 folds in patients with Eales disease(1.79plusmn;0.44)ng/ml than in controls (0.35plusmn;0.15)ng/ml separately(P＜0. 01). CONCLUSIONS:VEGF might play a part in mediating the neovascularization of retinal vascular diseases. （Chin J Ocul Fundus Dis,1997,13:171-173）
Objective To probe a selective cultural method for bovine retinal endothelial cells (BREC) and pericytes (BRP) in vitro.Methods With the isolation of active retinal blood vessels, BREC were cultured in a fibronectin coated substrate and Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% human serum and 100μg/ml heparin, while homogeneous cultures of retinal pericytes were obtained when isolated microvessels were seeded to uncoated dishes and grown in DMEM supplemented with 20% fetal bovine serum. BREC were identified by acetylated-low density lipoprotein (Dil-Ac-LDL) incorporation and immunohistochemical method of Von Willebrand factor, while BRP were identified by the immunohist ochemical method of α-isoform of smooth-muscle actin. Results The purity of selectively cultured BREC and BRP was more than 98%, being reproducible. BREC got together around the microvessel fragments with the small-cyprinoid-like configuration at first，and could phagocytize Dil-Ac-LDL with the expression of fluorescence in cytoplasm. The expressions of Von Wllebrand factor and α-isoform of smooth-muscle actin were positive and negative in BREC respectively, while were negative and positive in BRP respectively.Conclusion BREC and BRP with high purity can be obtained by using selective culture and coated-dishes respectively which are simple and repeatable methods. (Chin J Ocul Fundus Dis，2004，20：23-26)