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 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 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 effect of the damage and functional change of vascular endothelial cells (VEC) on diabetic retinopathy(DR). Methods Circulating endothelial cell (CEC) number and plasma endothelin(ET) level were measured in 18 normal control subjects and 55 patients with diabetes mellitus (DM) consisting of 20 cases of DM with out retinopathy,20 cases of DM with-background diabetic retinopathy and 15 cases of DM with proliferative diabetic retinopathy. Results CEC number and plasma ET level in DM were significantly higher than those in normal subjects(Plt;0.001)respectively.With the progression of DR,CEC number was significantly elevated and plasma ET level was gradually elevated.There was significant positive correlation between CEC number and plasma ET level (r=0.738,Plt;0.001,n=55). Conclusion VEC damage and elevated plasma ET level induced by VEC damage may play an important role in the development and progression of DR. (Chin J Ocul Fundus Dis,2000,16:166-168)
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 evaluate glaucoma and anterior chamber angle changes after intravitreal silicone oil injection(SOI). Methods The intraocular pressure(IOP) and anterior chamber angles of 34 e yes(33 patients)undergone pars plana vitrectomy and SOI were observed. Results Glaucoma occurred in 9 eyes(26%),in which silicone oil glaucoma(SOG) accounted for 77%(7/9).The changes of the superior part of anterior chamber angle were peripheral anterior synechiae in 11 eye(32%) and SO emulsification droplets in 22 eyes(64%) in 1～4.5 months after surgeries.Glaucoma occurred in 6 eyes of 7 eyes undergone peripheral iridectomy at the inferio r part of the iris after the closure of iridectomy holes (plt;0.05). Conclusion High incidence of glaucoma was found in this series of patients after intravitreal silicone oil injection,and the main causes of SOG were closure of the inferior iridecomy hole and silicone emulsification. (Chin J Ocul Fundus Dis, 2001,17:105-107)
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 evaluate the relative factors of effect of vitrectomy on corneal endothelial cells. Methods Retrospective analysis of the results of corneal endothelium microscopy performed on 213 eyes of 213 patients undergone vetrectomy operations including single vitrectomy (78 eyes), vitrectomy combined with cataract extraction (135 eyes), silicone oil injection (34 eyes), and C3F8 injection (53 eyes) before and after 1 week, 1 and 3 moths of these surgical procedures. Results There was no significant difference between pre- and postoperative corneal endothelium density in single vitrectomy group and vitrectomy combined with cataract extraction with posterior capsule integrity group (Pgt;0.05). The corneal endothelium density significantly decreased postoperatively in C3F8or silicone oil injection group with broken posterior capsule (Plt;0.05). Conclusion C3F8 and silicone oil may damnify corneal endothelium in patients undergo vitrectom y combined with cataract extraction with broken posterior capsule. (Chin J Ocul Fundus Dis，2004，20：101-103)