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 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 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)
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)
Objective To observe the expression of vascular endothelial growth factor (VEGF) in aqueous humor and vitreous body in eyes with proliferative vitreo-retinal diseases, and to investigate the role of VEGF plays in the pathoge nesis of proliferative vitreo-retinal diseases. Methods The concentration of VEGF in aqueous humor and vitreous body in eyes with proliferative vitreoretinopathy (PVR), retinal vein occlusion (RVO), proliferative diabetic retinopathy (PDR), and neovascular glaucoma (NVG) were measured by double antibodies sandwich enzyme-linked immunosorbent assay (ELISA). Results The concentration of VEGF in aqueous humor and vitreous body in eyes with PVR, RVO, PDR and NVG were obviously higher than that in the control group (Plt;0.05), respectively. Among all of the diseases, the concentration of VEGF in aqueous humor and vitreous body decreased orderly in NVG, PDR, RVO and PVR (Plt;0.05). The concentration of VEGF in vitreous body in eyes with PVR, RVO, PDR and in the control group were much higher than that in aqueous humor in corresponding groups (Plt;0.05). There was a negative correlation between the disease history and content of VEGF in aqueous humor and vitreous body in patients with PVR (r=－0.819, －0.823；Plt;0.05). The disease history positi vely correlated with the concentration of VEGF in aqueous humor and vitreous body in patients with RVO (r=0.913, 0.929；Plt;0.05), and the time of vitreous hemorrhage positively correlated with the concentration of VEGF in aqueous humor and vitreous body in patients with PDR (r=0.905, 0.920；Plt;0.05). Conclusion The concentration of VEGF in aqueous humor and vitreous body in patients with proliferative vitreo-retinal diseases significantly increases, and VEGF may play an important role in the pathoge nesis of proliferative vitreo-retinal diseases. (Chin J Ocul Fundus Dis, 2006, 22: 313-316)
Objective To construct specifically expressed vascular endothelial growth factor (VEGF)165 gene in retina. Methods Rho promoter, specifically expressed in retina, was amplified by polymerase chain reaction (PCR) from the genomic DNA of a BLAB/C rat, then it was cut with restriction enzymes and cloned into the plasmid pcDNA3.1+-VEGF165 to form recombinant plasmid pcDNA3.1+-rho-VEGF165. The correct recombinant plasmid pcDNA3.1+-rho-VEGF165 was identified by restriction enzymes and PCR, and was transferred by jetPEI into cultured human navel vein endothelial cells and human retinal pigment epithelial (RPE) cells. The expression of VEGF protein in human navel vein endothelial and RPE cells was detected by immunocytochemical staining and protraction of the growth curve of the cells. Results In human RPE cells, the expression of VEGF protein was more in recombinant plasmidpcDNA3.1+-rho-VEGF165 than that in plasmidpcDNA3.1+-rho-VEGF165 ; in human navel vein endothelial cells, no obvious difference of the expression of VEGF protein between recombinant plasmid pcDNA3.1+-rho-VEGF165 and plasmid pcDNA3.1+-rho-VEGF165 was found. Conclusions The construction of pcDNA3.1+-rho-VEGF165 carrier may provide the basic material for the study of the nosogenesis of VEGF in retinal neovascularization, and establish the foundation to set up the model of transgenic mice with VEGF specific expressing in retina. (Chin J Ocul Fundus Dis, 2005,21:106-108)
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)