PURPOSE: To produce monoclonal antibodies directed against tumor-associated antigens expressed of retinoblastoma-derived tissue culture cell line SO-RBS0. METHODS:Hybridization was performed and the specificity of the antibody was tested by immunofluorescent and immunohistochemical methods. RESULTS:Two hybridomas secreted specific monoclonal antibody against retinoblastoma cells were produced ,and the isotype of the monoclonal antibody was IgG2a CONCLUION:The monoclonal antibodies were specific and highly active against retinoblastoma cells and might be used as immunoconjugate.
Objective To observe the morphological changes of dendrite and soma in retinal ganglion cells (RGCs) which subsisted in early diabetic rats. Methods The RGCs of 3-months-course diabetic rats and coeval normal rats were marked by gene gun techniques. To collect RGCs photographs by Leica microscope with Z axis and CCD camera；to observe the changes of diameter, variance of structural features in dendritic field and somata after classification which according to the size and morphology. Thy-1 antibody marks on the retinal RGCs, taking a photograph under fluorescent microscope, counting the changes of retinal RGCs density in early diabetic rat. Results In three-month diabetic rats,the density of retinal RGCs was decreased obviously. Morphological changes of RGCs in the dendritic fields were observed with gene gun technique. There was no severe variation in all kinds of the bole of cell dendrite, in which some only showed crispation partially and sparseness also twisting in the dendritic ramus. The mean diameter of dendritic field and soma in class A of diabetic rats was (401plusmn;86) mu;m, the mean diameter of dendritic field in control group was (315plusmn;72) mu;m,compared with each other, there is statistically significant differences (t=21.249，Plt;0.001); the mean diameter of soma in class A of diabetic rats was (24plusmn;6) mu;m, the mean diameter of soma in control group was (22plusmn;5) mu;m, compared with each other, there is no statistically significant differences (t=0.927，Pgt;0.05); the mean diameter of dendritic field and soma in class B of diabetic rats were (170plusmn;36)、(14plusmn;2) mu;m respectively, in control group were (165plusmn;36)、(16plusmn;2) mu;m, the mean diameter of dendritic field and soma in class C of diabetic group were(265plusmn;78)、(17plusmn;5) mu;m respectively, in control group were (251plusmn;57)、(17plusmn;4) mu;m , compared with each other, there are on statistically significant differences(t=1.357，0.798，0.835，1.104，Pgt;0.05). Conclusions In short-term diabetes, the survived RGCs show good plasticity in adult diabetic rats, especially in class A. The changes of dendrites were more sensitive than the soma, which could be the leading index of the morphologic changes of RGCs in the early stage. The good plasticity showed by the RGCs and the time window from changing in dendrite to cell death provide us many evidences not only for the research but also for the nerve protection in clinic. (Chin J Ocul Fundus Dis,2008,24:249-254)
Objective To observe the effect of pigment epithelium-derived factor (PEDF）on glutamate metabolism in diabetic rat retina. Methods 78 Sprague-Dawley rats were randomly divided into the model group, model control group, PEDF intervention group and intervention control group. There were some dead and euglycemia rats at the end of experiment, so only 12 rats in each group were included in the statistical analysis. The diabetic retinopathy rat model of the model, PEDF intervention and intervention control group were induced with streptozotocin injection. The rats in the model group were not intervened. The monthly-age matched normal rats of model group were in the model control group. The left eyes of rats were received intravitreal injection with 5 mu;l (0.1 mu;g/mu;l) PEDF (PEDF intervention group) or 5 mu;l phosphate buffer solution (intervention control group). The expressions of L-glutamate/L-aspartate transporter in retina were analyzed by western blot and real time RT-PCR techniques and glutamate content in retina was analyzed by high-pressure liquid chromatography (HPLC). Cultured rat Muuml;ller cells were divided into the control,experimental, PEDF intervention and intervention control group, GLAST expressions were detected by fluorescence immunofluorescence and real-time RT-PCR techniques. The glutamate up-take activity of Muuml;ller cells was determined by intracellular [3H] labeled D, L-glutamate concentration with scintillation counting. Results Western blot and real-time RT-PCR showed that GLAST expression decreased (real-time RT-PCR：t=8.86,Plt;0.01；Western blot：t=3.42,P＜0.05), glutamate content increased（t=4.01,P＜0.05）in model group compared with the model control group; GLAST expression increased (real-time RT-PCR：t=3.56,P＜0.05；Western blot：t=3.52,P＜0.05), glutamate content decreased（t=4.36,P＜0.05）in the PEDF intervention group compared with the intervention control group. Real-time RT-PCR and fluorescence immunofluorescence showed that high glucose down-regulate GLAST expressions in Muuml;ller cells (rea-time RT-PCR：t=3.48,P＜0.05；fluorescence immunofluorescence：t=4.72,P＜0.05 ) and impair glutamate uptake activity of Muuml;ller cells (t=3.81, Plt;0.05). Under high glucose conditions, PEDF up-regulated GLAST expression significantly (real-time RT-PCR：t=6.82,P＜0.01；fluorescence immunofluorescence：t=3.72,P＜0.05) and ameliorated the glutamate up-take activity of Muuml;ller cells（t=4.14, Plt;0.05). Conclusions In diabetic rats, PEDF may improve the activity of GLAST in Muuml;ller cells, thus ameliorate retinal glutamate metabolism and inhibit death of retinal ganglion cells.
Objective To observe the influence of rAAV-mediated antisense vascular endothelial growth factor (rAAV-aVEGF165) on the expression of retinal VEGF in diabetic rats. Methods 40 Sprague-Dawley rats induced diabetic rat model by intraperitoneal injection with streptozotocin (STZ). 32 rats were involved in study besides death and blood sugar recovery in experimental process, 16 spragud-Dawleg (SD) rats were received intravitreal injection with rAAV-aVEGF165 (1010 pfu) as experimental group, another group of Sprague-Dawleg (SD) rats were injected with phosphate buffered saline (PBS) as control group. One and five month after model establishment, the expression of retinal VEGF was evaluate by immunhistochemistry and Western blot; the retinal vasular was examined by transmission electron microscopy. Results On 1 month,the expression of retinal VEGF was lowest in each group. On 5 month, the expression of retinal VEGF was decreased in experimental group which compared to control, the difference are statistically significant （t=23.87,Plt;0.01）. The transmission electron microscopy results showed that retina has no obvious chages in experimental group, however,contral group showed fragmental thickening and splitting of basement membrane, swelling and deformation of endothelia cells,fingerlike prcess into the capillary cavity,and uneven distibution of heterochromatin in pericytes. Conclusion rAAV-aVEGF165 can reduce the expression of retinal VEGF thereby preventing occurrence and development of diabetic retinopathy. rAAV is an effective vectors of eye antisense gene. （Chin J Ocul Fundus Dis，2008，24：255-258）
PURPOSE:To measure the concentration changes of tumor necrosis factor a (TNF-alpha;)in vitreous during the development of experimental PVR induced by macrophages and explore the initial and mediated factors which stimulate the cellular proliferation. METHODS:Rabbit PVR model was induced by macrophages and the vitreous was taken at the 7th,14th,21st and 28th day and 4 eyes in each group. The TNF-alpha; levels in vivreous of the above examinated and control eyes were measured with an ELISA kit. RESULTS:The TNF-alpha; level in the vitreous reached its peak 434mu;g/ml at 21st day in the mod-el, then rappidly decreased to 122mu;g/ml at 28th day. CONCLUSION:The changes of TNF-a in the vitreous of the PVR model were parallel to the natrual phases of the development of PVR,indicating TNF-alpha; may play an important role in initiating and mediating the inflammation and cellular proliferation in the vitreous. （Chin J Ocul Fundus Dis,1997,13: 231-233）
Objective To observe the relationship between retinal microglial activations and ganglion cell (RGC) damages in early-stage diabetic rats. Methods A total of 20 SpragueDawley（SD）rats were randomly divided into 4 groups (each with 5 rats): 1 month control group, 1 month diabetes group, 3 month control group, 3 month diabetes group. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ). The RGCs of all rats were retrograde labeled by carbocyanine dye DiI injected at the superior colliculi.Microglial cells and RGCs in retinal flat-mounts and sections were stained immunohistochemically and recorded under confocal microscope.Results The diabetic microglial cells were amoeboid and ovoid with fewer processes on retinal flat mounts. The density of microglial cells which phagocytosed DiI particles in the RGC layer significantly increased in the 3month diabetes group（P＜0.01）. The density of microglial cells in the RGC layer significantly increased in the 1- and 3- month diabetes group（P＜0.05）. However there were more microglial cells in the RGC layer in the 3- month diabetes group than the 1-month diabetes group（P＜0.0001）. Significant correlation was found between the amount of microglial cells and that of RGCs in the early-stage of diabetes. Conclusions Microglial cell activation has close relationship with the RGC damages in early-stage diabetic rats.
Objective To quantitatively assess the damage of blood-retinal barrier (BRB) in rats with diabetic retinopathy using dynamic contrast enhanced-magnetic resonance imaging (DCE-MRI). Methods Forty 3-week-old Sprague-Dawley (SD) rats were randomly divided into experiment and control group. The rats in experiment group underwent intraperitoneal injection of streptozotocin. The rats with blood glucose over 16.65 mmol/L and ldquo;+++rdquo; of urine glucose were considered as diabetes and were further divided into four subgroups according to the course of diabetes mellitus (2, 4, 6, and 8 months).The rats in control group underwent intraperitoneal injection with the same volume of buffer and were divided into four subgroups (with 5 rats in each subgroup) according to the coordinate age of rats in experimental group.All of the eyeballs were scanned by DCE MRI and enucleation was performed after intraperitoneal injection with pentobarbitone.The data were analyzed by SPSS 12.0 statistical software.Results All the rats in experiment group became diabetic models. There was no obvious BRB permeability in control group and in 2- and 4-months experiment group.The average BRB permeability rate in 6 and 8 month experiment groups were (0.1399plusmn;0.0065) and (0.1816plusmn;0.2756) mm3/min respectively (Z=-2.121, Plt;0.05). Retinal edema and cellular disorganization appeared at 4 months and became more severe when diabetes course extended.Conclusions DCE MRI can measure the BRB permeability rate accurately and assess the extent of BRB damage quantitatively in rats with diabetic retinopathy.
Objective To observe the inhibition effect of the hypoxia inducible factor-1alpha; (HIF-1alpha;) specific siRNA on the expression of vascular endothelial growth factor (VEGF) mRNA in retinal tissues in diabetic rat. Methods This is a randomized controlled study. HIF-1alpha; specific siRNA recombinant plasmid was built in pSilencer2.1-U6neo vector. Fifty-four healthy Sprague Dawley (SD) rats were divided into control group (15 rats) and experimental group (39 rats). The experimental rats were induced with streptozotocin injection for diabetic retinopathy model, and then randomly divided into diabetic retinopathy (DR) group (15 rats), vector group (12 rats) and gene therapy group (12 rats). LipofectamineTM2000 mixed with pSilencer2.1-U6neo plasmid or HiF-1alpha; siRNA plasmid were injected into the vitreous in the vector group and gene therapy group respectively. Nothing was transfected into DR and control group. The expression of VEGF mRNA in retinas was measured by real-time RT-PCR. The inhibition efficiency of VEGFmRNA was calculated at 24, 48, 72 hours and 1 week after injection respectively. Significant differences between groups were evaluated by oneway analysis and LSD-t analysis. Results HIF-1alpha; siRNA recombinant plasmid was confirmed by enzyme digestion and sequence analysis. Real-time RT-PCR revealed that the expression of VEGFmRNA was faint in the control group, increased obviously in the DR and vector group, decreased in the gene therapy group. There was no statistically significant between DR and vector group (t=0.669，0.142,0.151，0.025；P=0.514，0.889，0.882，0.980). The expression of VEGFmRNA in the gene therapy group were obviously decreased compared with DR and vector group (t=8.768, 13.695, 11.285, 8.253;P=0.000). The inhibition efficiency of VEGFmRNA was 32.76%, 43.60%, 47.70%, 50.86% at 24, 48, 72 hours and 1 week after injection. Conclusions The expression of VEGFmRNA can be efficiently inhibited by HIF-1alpha; siRNA recombinant plasmid.