Objective To study the effect of splenectomy on the anti-tumor immunity in rats with induced hepatocellular carcinoma (HCC). Methods At the second and fourth month of the induced HCC, the NK cell activity, TNF-α level and total lymphcyte in blood were measured in the group of splenectomy and the control group. Results There were no different in the total lymphcyte and TNF-α in the blood in two groups, but there were significant difference in the NK cell activity between the group of splenectomy and the control group (Ｐ＜0.05). Conclusion There are some change in the anti-tumor immunity after splenectomy in rats, in which NK cell activity is at low level continuously. TNF-α isn′t affected after the second month after splenectomy.
Objective To establish a rat model of blood ocular barrier breakdown induced by anterior segment intraocular analogic surgery. MethodsOne hundred and fifty healthy adult male rats were randomly divided into control group and model group, 75 rats in each group. The rats were anesthetized with 1 ml/kg ketamine hydrochloride/xylazine hydrochloride solution. Three way pipes were attached to a phosphate buffer infusion bag and two intravenous catheters. One catheter was inserted 30° obliquely through the transparent cornea anterior to the limbus into the rat's anterior chamber. Then the needle was withdrawn and the sheath was indwelling. Another catheter was connected with a manometer. Intraocular pressure was varied from 0 to 12 mm Hg (1 mm Hg=0.133 kPa) 60 times, 30 times per min. The catheter was removed. The eyes were treated with ofloxacin ophthalmic solution after surgery. The 1st, 2nd, 3rd, 5th and 7th day after surgery, the integrity of the blood ocular barrier was assessed by immunohistochemical staining for albumin and quantitative measurement using Evan′s blue as a tracer. ResultsAlbumin immunohistochemical staining of the control group was confined to the iris and retinal blood vessels. The choroid was stained at each time point after surgery. Albumin immunohistochemical staining of the model group was abundant around the iris and the retinal vasculature on the 1st day after surgery. The albumin diffused throughout the iris and the retina on the 2nd and the 3rd day after surgery. The albumin reached the retinal vessels on the 5th and 7th day after surgery. The aqueous humor Evans blue leakages of the model group were higher than those of the control group on the 1st, 2nd, 3rd and 5th day after surgery. The differences were statistically significant (t=25.781, 37.433, 25.150, 19.171； P<0.01). The Evans blue leakage of the model group was close to that of the control group on the 7th day after surgery. The difference was no statistical significant（t=1.303, P=0.209）. The retinal Evans blue leakages of the model group were higher than those of the control group on the 1st, the 2nd and the 3rd day after surgery. The differences were statistically significant (t=11.997, 14.622, 23.014; P<0.01). The Evans blue leakage of the model group was close to those of the control group on the 5th and 7th day after surgery. The differences were not statistically significant（t=2.027, 0.756; P=0.058, 0.459）. Conclusion This study establishes a rat model of blood ocular barrier breakdown induced by imitating the injury to the anterior segment during intraocular surgery.
ObjectiveTo explore the inhibition effect of Cysteine-rich 61(CCN1;Cyr61) specific siRNA expression vector on RNV in a mouse model of oxygen-induced retinopathy (OIR). MethodsOne hundred and twenty healthy C57BL/6J mice were chosen and randomly divided into the experimental group and control group, with 60 mice in each group. The experimental group was intravitreously injected with CCN1siRNA recombinant plasmids. The control group was injected with vector plasmids. Adenosine diphosphate-ase stained retina flat-mounts was performed to assess the retinal vascular profiles, retinal section with HE staining was applied to count the number of new vascular cell nuclei and the protein and mRNA expression of CCN1 and vascular endothelial growth factor (VEGF) were detected by immunohistochemistry, Western blot and Real-time RT-PCR. ResultsCompared with control group, regular distributions, good branches and reduced density of retinal neovascularization were observed in the experimental group. The number of nucleus of vascular endothelial cells breaking through the inner limiting membrane was obviously less in the experimental group than that in the control group (t=8.756, P < 0.05). The expression of CCN1 and VEGF were obviously decreased in the experimental group compared with the control group (all P < 0.05). ConclusionThe development of RNV of ROP can be markedly inhibited by RNA interference targeting CCN1, and CCN1siRNA may provide an effective method for preventing vascular proliferative retinopathy.
ObjectiveTo observe the expression of Caspase-3, Caspase-8, Bcl-2, Bax in the rat retina of ocular ischemic syndrome (OIS). Methods30 Brown Norway rats were randomly divided into experimental group and control group, 15 rats in each group. The rats in experimental group were established a model through ligating the bilateral common carotid artery. At 3 months after modeling, the retinal thickness and ganglion cell (RGC) density were measured by hematoxylin eosin staining; the expression of Caspase 3, Caspase 8, Bax and Bcl-2 in the retina was measured by quantitative real-time reverse transcription polymerase chain reaction. ResultsThe RGC density in control group and experimental group was 61.97±9.07 and 38.1±5.98, respectively. Compared to the control group, the RGC density was diminished in the experimental group (t=3.059, P < 0.01). A significant decrease was found in the total retina (t=3.036), inner plexiform (t=3.715), inner nuclear (t=3.339) and outer plexiform (t=3.341) thickness (P < 0.05). However, no change of the thickness was evident in the outer nuclear layers (t=2.000, P > 0.05). The levels of protein and RNA expression of Caspase 3, Caspase 8 and Bax in the retina were increased in experimental group (F=17.036, 7.787, 11.431, 11.162, 17.763, 12.183; P < 0.05), while the Bcl-2 expression were decreased (F=10.298, 12.047; P < 0.05). ConclusionsThere is obvious expression of apoptosis-associated proteins in the rat retina of OIS. Caspase 3, Caspase 8 and Bax expression are increased, while Bcl-2 expression are decreased.
ObjectiveTo study the inhibitory effects of pigment epithelium derived factor (PEDF) on oxygen-induced retinal neovascularization in mice, and to investigate the possible involvement of interleukin-1β (IL-1β) in the neovascular-inhibitory function of PEDF. Methods A total of 140 postnatal day (P)7 C57BL/6 mice were randomly divided into normal control group, oxygen-induced retinopathy (OIR) model group, PEDF treatment group and PBS treatment control group. All mice except normal control group with their mothers were exposed to (75±2)% oxygen environment for 5 days and then kept in room air for another 5 days to establish the OIR model. Mice in normal control group were kept in room air only. At P12 and P14, respectively, mice in PEDF treatment group received intravitreous injections of 1 μl PEDF (2 μg/μl), while PBS treatment control group received the same volume of PBS (10 mmol/L, pH7.4).All mice were euthanized at P17 and eyes were isolated. The changes of retinal vessels were observed on retinal flat mounts and cryosections by fluorescence microscopy. Retinal specimens were prepared for IL-1β protein and mRNA analysis by Western blot and real time fluorescence quantitative reverse transcription-polymerase chain reaction (Real-time RT-PCR). ResultsChanges of retinal vessels had been viewed by fluorescence microscopy on flat-mounted retina, the relative retinal neovascularization areas were significantly increased in OIR model group compared with normal control group (t=15.02, P < 0.01), and the relative retinal neovascularization areas were obviously smaller in PEDF treatment group than those in PBS treatment control group (t=5.96, P < 0.01). Fluorescence staining revealed that retinal vascular tufts were extending from outer plexiform layer (OPL) to ganglion cell layer (GCL) of the retina along with multiple interconnections; Neovascular tufts in OIR model group and PBS treatment control group were presenting distinctly more than those of normal control group and PEDF treatment group. The specific expression levels of IL-1β protein in retinas of OIR mice by Western-blot analysis were higher than those of normal control group(t=3.35, P < 0.05), While these of PEDF treatment group showed a considerable decline in comparison with PBS treatment control group (P < 0.01), and there were no difference in normal control group and PEDF-treated group (F=11.764, P > 0.05). Similarly, expression levels of IL-1β mRNA tested by Real-time RT-PCR were obviously increased in the OIR model group when compared to normal control group(t=4.43, P < 0.01). After treated with PEDF, expression levels of IL-1β mRNA showed a considerable decrease when compared to PBS treatment control group (P < 0.01), and there were no difference in normal control group and PEDF-treated group (F=11.15, P > 0.05). ConclusionsPEDF can inhibit oxygen-induced retinal neovascularization. The mechanism may be related to that PEDF can downregulate the expression of IL-1β in retina.
Objective To observe the inhibitory effect of recombined adenovirus of p21 (rAd-p21) on the proliferation of Rhesus retinal vascular endothelial cell line (RF/6A). Methods RF/6A cells were cultured in vitro which divided into phosphate buffered solution (PBS) group, rAd-p21 transfection group and negative control (NC) group. Plasmid vectors were transfected into RF/6A cells. The expression of p21 mRNA and protein in RF/6A cells were measured by RT-PCR and Western blot respectively. The cell cycle distribution was analyzed by flow cytometry. Endothelial-cell tube formation assay was performed in Matrigel.Results The expression of p21 mRNA and protein in rAd-p21 transfection group were higher than that in PBS and negative control group. The cell cycle distribution showed that the proportion of G0/G1 cells in rAd-p21 transfection group ［(67.45plusmn;11.61) %］ was apparently higher than that in the negative control group ［(41.55plusmn;8.99)%］ and PBS group ［(40.76plusmn;6.66) %］ (F=21.284, P=0.000). The number of endothelialcell tubes in the rAd-p21 transfection group (3.86plusmn;1.21) was apparently less than that in the negative control group (7.62plusmn;2.69) and PBS group (8.25plusmn;3.19) (F=7.138，P=0.004). Conclusions The p21 mRNA and protein can stably express in RF/6A cells after rAd-p21 transfection. RAd-p21 can inhibit the proliferation of RF/6A cells.
Objective To evaluate the inhibited effects of small interfering RNA targeting Rac1 (Rac1-siRNA) on rat retinal neovascularization in retinae. Methods Retinal vein occlusion was induced by retinal photodynamic medthod in 25 Sprague-Dawley rats. Rac1-siRNA vector DNA was injected into the vitrous of one eye of those rats (gene intervention group), and empty vector DNA was injected into the fellow eye (blank control group). Rac1-siRNA vector was injected in other 25 SD rats without retinal vein occlusion (blank intervention group). Two weeks after injection, fluorescein isothiocyanate (FITC)-dextran was perfused into the hearts of all the rats, and the retinal wholemount was made to observe the neovascularization. The numbers of endothelial cells which break through the internal limiting membrane were counted after hematoxylin-eosin staining. Results A massive of neovascularization and FITC leakage were found in blank control group. Small part of neovascularization and a little FITC leakage were observed in the gene intervention group. Retinal vessels were normal in blank intervention group. Compared with blank contrast group and blank intervention group, the difference of the mean numbers of endothelial cells which broke through the internal limiting membrane in the gene intervention group was significant（t=? P=0.000??lt;0.05）. Conclusion Rac1-siRNA can inhibit retinal neovascularization induced by retinal vein occlusion in rats.
Objective To construct small hairpin RNA (shRNA) expression plasmid targeting rat opticin gene.Methods Four pairs of opticin oligonucleotides were synthesized and inserted into the plasmid vector, resulting into four plasmids: shRNA-1, shRNA-2, shRNA-3 and shRNA-4. Then the four constructed shRNA expression vectors and empty vector were transfected into rat ciliary non-pigment epithelium (NPE) cells by lipofectmaine 2000. Nontransfected NPE cells were set as control group.The expression of opticin mRNA and protein were measured by Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot respectively.Results The opticin mRNA expression of the shRNA-1,shRNA-2,shRNA-3,shRNA-4 group were decreased compared with the control group (F=10.239，P＝0.000)；the inhibitory rate were 85.7%，62.87%，54.87% and 48.77% respectively.The opticin protein expression of the shRNA-1,shRNA-2,shRNA-3,shRNA-4 group were also decreased compared with the control group (F=17.870，P=0.000)；the inhibitory rate were 78.7%，34.6%，31.1% and 16.8% respectively.Conclusions The shRNA-1 expression plasmid has most potent inhibitory effect on opticin expression in rat ciliary NPE cells.
ObjectiveTo investigate the influence of Ataxia-telangiectasia mutated (ATM) activation on cellular oxidative stress induced by high glucose in bovine retinal capillary endothelial cells(BRECs). Methods The BRECs were treated by different culture medium with various glucose concentrations (5 mmol/L glucose, 30 mmol/L glucose, 30 mmol/L glucose+10 μmol/L KU55933) as normal glucose group, high glucose group and treatment group respectively.After the cells incubated for 48 hours, the protein expression of ATM, P-ATM, Mitogen-Activated Protein Kinase P38(P38), P-P38, Extracellular signal-regulated kinases(ERKs), P-ERKs was detected by Western blot; cellular ROS level was detected by Reactive Oxygen Species Assay Kit; propidium iodide/Hoechst staining was used for analysis of apoptosis; the expression of vascular endothelial growth factor (VEGF) in the supernatant was determined by Enzyme-Linked Immunosorbent Assay (ELISA); the paracellular permeability between endothelium cells was detected by FITC-dextran. ResultsCompared with the protein level of P-ATM, P-P38 and P-ERKs in high glucose group increased. Especially, P-P38, P-ERKs expressed much more than in high glucose group. The secretion of VEGF in high glucose group was higher than that in the normal glucose group but less than that in treatment group. The same tendency existed in ROS assay, apoptosis assay and paracellular permeability measuring. ConclusionsHigh glucose induced altered activation of ATM which might play a protective role in cellular oxidative stress. Deficiency of ATM might lead to ROS explosion, cell apoptosis and dysfunction of endothelial barrier. The mechanism might be associated with P38, ERKs and VEGF.
Objective:To observe the inhibited effect and its mechani sm of estro gen on lightinduced apoptosis of retinal photoreceptor cells. Methods:Twenty female Sprague-Dawley rats were randomly divided into two groups: ovariectomized (OV) group and OV and estrogen (E2) replacement (OV+E2) group, with 10 rats in each group. All of the rats were exposed to the cyclic illumination under 12 hou r light and 12 hour dark condition with the light intensity of (600plusmn;35.4) lx ( a total of 14 times). All of the right eyes were extracted and the thickness of r etinal outer nuclear layer (ONL) was measured. Terminal deoxynucleotidyl Transfe rasemediated dUTP nick end labeling (TUNEL) method was used to evaluate positi v e apoptosis cells in ONL. The expression of nitric oxide synthase (NOS) in retin al cells was detected by immunohistochemistry with image analysis method. Results:The thickness of ONL in OV group was obviously thinner than that in the OV+ E2 group. The number of positive apoptosis of the cells was (0.0275plusmn;0.0069) c el ls/mu;m2 in OV group and (0.0162plusmn;0.0054) cells/mu;m2 in OV+E2group; the di fferen ce between the two groups was significant (t=4.1370，P=0.0012). The values o f in tegral optical density of NOS positively stained cells in retinal inner nuclear layer was (0.3675plusmn;0.0662) in the OV group and (0.2941plusmn;0.0350) in OV+E2 group ; the difference between the two groups was significant (t=3.4885, P=0.0031). Conclusion:Estrogen can protect retina from light injuries by regu lating NOS synthesis and inhibiting apoptosis of photoreceptor cells.