Objective To observe the expression of programed death-1 (PD-1) and its ligands including PD-L1 and PD-L in peripheral blood mono-nuclear cells (PBMCs) of patients with diabetic retinopathy (DR) patients. Methods Forty patients with DR (DR group) and 20 healthy controls (control group) were included in this study. There were 20 patients with non-proliferative DR (NPDR) and 20 patients with proliferative DR (PDR). Peripheral Blood samples were obtained from two groups. Real time polymerase chain reaction (RT-PCR) was used to analyze PD-1, PD-L1, and PD-L2 mRNA expression in PBMCs. The clinical data was analyzed in DR group and controls, also in PDR group and NPDR group. Results The results of RT-PCR showed that the expression of PD-1 and PD-L1 mRNA in DR group were significantly lower than those in the control group (t=-2.060, -2.562; P=0.043, 0.013). There was no significant difference in PD-L2 mRNA expression between DR and control group (t=-0.857，P=0.395). Compared with the NPDR group, the lower expression level of PD-1 mRNA and higher expression level of PD-L1 and PD-L2 mRNA in PDR group were observed, but the differences were not statistically significant (t=-1.335, 0.987, 0.131; P=0.190, 0.334, 0.897). Conclusion PD-1 and PD-L1 mRNA expression in PBMCs of DR patients is decreased compared with controls, but there are no differences in PD-L2 mRNA expression in them.
Objective To observe the histological changes and apoptosis of retinal cells in pigmented rabbits treated by transpupillary thermotherapy (TTT) with different laser power. Methods Fourteen pigmented rabbits (28 eyes) were divided averagely into seven groups（control group, 50, 70, 90, 110, 130, and 150 mW group）according to different laser power of TTT. Light microscopy was performed to observe the histological changes, and TDT-mediated biotin-dUTP nick-end labeling (TUNEL) technique and flow cytometry (FCM) examination were used to detect the apoptotic cells 24 and 48 hours after photocoagulation, respectively. Results The color of retinal burn speckles changed from offwhite to white and super white with the diameter enlarged gradually as the laser power of TTT increased. The results of light microscopy revealed that compared with the control group, the retinal tissue did not change much in 50-70 mW group; in 90-130 mW group, the retinal structure was integrated, but the cone and rod cells became swollen and condensed nuclei and cytoplasmic vacuolization were seen in the inner nuclear layer. The difference of retinal structure in 50-130 mW group 24 and 48 hours after photocoagulation and control group was not significant. In 150 mW group, tumefaction and degeneration were observed in each layer of retina and the inner and outer segments of photoreceptor cells lost 24 hours after photocoagulation, and obvious necrosis and cell loss of retinal tissues were detected hours after photocoagulation. The results of TUNEL examination indicated that positive cells were found in outer nuclear layer in each photocoagulation group which increased as the laser power of TTT was enhanced; the apoptosis gradually involved the inner nuclear layer and ganglion cell layer. The results of flow cytometry (FCM) examination showed the peak of apoptotic cells in each photocoagulation group 24 hours after photocoagulation. Conclusion Under certain subthreshold photocoagulation (50-70 mW), retinal tissue of rabbits does not change much but apoptosis of photoreceptor cells increase significantly. As the laser power of TTT increases, the retinal tissues become swollen, degenerated and even necrotic; cellular apoptosis gradually involves the inner nuclear layer and ganglion cell layer. (Chin J Ocul Fundus Dis, 2006, 22:249-252)
Objective To detect the apoptosis of vascular endothelial cells and retinal pigment epithelial (RPE) cells in vitro induced by verteporfin-photodynamic therapy. Methods Cultured vascular endothelial cells and human RPE cells were incubated with verteporfin at a concentration of 1.0 mu;g/ml which was equivalent to the initial plasma level of verteporfin in clinical therapy. Each kind of cells were divided into 6 groups according to different time of incubation: 0, 5, 15, 30, 60, and 120 minutes group. After incubated, the cells were illuminated by the laser light with the maximum wavelength of absorption of verteporfin (wavelength: 689 nm, power density: 600 mW/cm2) with the power of 2.4 J/cm 2for 83 seconds. The percentage of cellular apoptosis was measured by flow cytometry 3 hours after PDT, and the measurement was repeated thrice. Results The proportion of cellular apoptosis 3 hours after PDT were 0.01plusmn;0.01, 0.25plusmn;0.02, 0.32plusmn;0.02, 0.41plusmn;0.04, 0.49plusmn;0.03 and 0.61plusmn;0.02, respectively in 0-120 minutes group of vascular endothelial cells; and 0.02plusmn;0.01, 0.22plusmn;0.01, 0.31plusmn;0.02, 0.38plusmn;0.03, 0.47plusmn;0.05 and 0.58plusmn;0.03 respectively in 0-120 minutes group of RPE cells. The proportion of cellular apoptosis of both kinds of the cells increased as the incubation time was prolonged. There was no significant difference of the percentage of cellular apoptosis between the accordant time groups in the two kinds of cells (P＞0.05). Conclusions Cellular apoptosis can be quickly induced by verteporfin-PDT both in human vascular endothelial cells and RPE cells; under the same condition in vitro, PDT has no obvious selection for the apoptosis of the two kinds of cells. (Chin J Ocul Fundus Dis, 2006, 22: 253-255)
Objective To investigate the interference effect of nerve growth factor (NGF) on apoptosis of retinal cells in experimental retinal detac hment (RD). Methods Twenty seven Sprague-Dawely rats were selected, and the left and right eyes were in the experimental control group and NGF group, respectively. After the RD model was set up by subretinal injection with sodium hyaluronate, 5mu;l NGF（1mu;g/mu;l）was injected into the vitreous body of the right eyes which were in the NGF group; 5mu;l PBS was injected into vitreous body of left eyes which were in the experimental control group. The injection was performed once every 4 days till the end of the observation period. The eye balls of the 27 rats were extrafted 1.5, 3, 6, 12 hours, 1 day, 2, 4, 8 , 16, and 32 days after the RD model was established. Another 2 rats were selected as the normal control, which underwent none of the injections but eyeball extraction at the end of the observation period. TUNEL and transmission electron microscopy were used to detect the apoptosis of the retinal cells. Cell counts and statis tical analysis were used to assess results. Results Typical apoptosis cells were observed in the early time of RD. Apoptosis was found in each retinal layers, especially in inner and outer nuclear layers. The number of apoptosis cells increased as the time of RD was prolonged（Plt;0.01）. It was also found that apoptosis cells in NGF group were less than that in the experimenta l control group（Plt;0.01）. Conclusion Intravitreous injection exogenous NGF may inhibit the apoptosis of retinal cells in experimental RD. (Chin J Ocul Fundus Dis, 2006, 22: 333-335)
Objective To observe the effect of blue light on apoptosis of cultured human retinal pigment epithelial (RPE) cells in vitro. Methods Human RPE cells were exposed to blue light, and the cells were divided into 3 groups: group A, with various intensity of illumination; group B: with same intensity but different time of illumination; group C: with same intensity and time of illumination but different finish time of the culture. The apoptosis of RPE cells was observed by TdT-dUTP terminal nick-end labeling (TUNEL) and annexin V-fluoresein isothiocyanate (FITC)/propidium iodide (PI) flow cytometry, and transmission electron microscopy. Results The positive cells stained by TUNEL shrinked and turned round, whose nuclei concentrated and congregated like the crescent or hat. Cracked nuclei and membrane bleb were found. Swollen mitochondrial, disappeared inner limiting membrane of mitochondria, and dilation of the rough endoplasmic reticulum with metabolite were observed by transmission electronmicroscopy. In group A, mild damage of RPE cells was found when the threshold value of the intensity of illumination was less than（500±100）lx, and the apoptosis and necrosis of RPE cells aggravated as the intensity of illumination increased; in group B, as the time of illumination extended, the number of apoptotic RPE cells didn′t increase while the necrosis increased; in group C, 6 and 12 hours after illumination, apoptosis of cells was the main injury, while apoptosis with necrosis was found and necrotic cells increased as the time of illumination was prolonged. Conclusions Illumination with blue light may cause damages of human RPE cells in vitro, with the modalities of apoptosis, apoptotic necrosis and necrosis. The extent of injury is dependent on intensity and duration of the illumination. (Chin J Ocul Fundus Dis, 2005, 21: 384-387)
ObjectiveTo evaluate the protective effect of estrogen on survival of retinal ganglion cells (RGCs) after transient retinal ischemia-reperfusion (RIR) in rats.MethodsRIR was induced in 60 ovariectomized adult rats (OVX) by increasing intraocular pressure via an intracameral catheter. All of the rats were divided into two groups randomly: in experimental group, the rats underwent a subcutaneous injection with 17β-estrodiol（100 μg/kg） 2 hours before retinal ischemia; and in the control group, saline water was injected correspondingly. The number of RGCs and the thickness of the inner retinal layers were mesured by HE staining method before and 12, 24, 48, and 72 hours after reperfusion. TdT-mediated biotin-dUTP nick end labelling (TUNEL) staining technique was used to examine the apoptosis of RGCs.ResultsTwenty-four and 48 hours after reperfusion, the number of apoptotic cells in experimental group was obvious lower than that in the control group(Plt;0.05), and the number of RGCs in experimental group was higher than that in the control group(Plt;0.05).ConclusionEstrogen can protect retinal neurons from transient RIR in ovariectomized rats.(Chin J Ocul Fundus Dis, 2005,21:177-179)
ObjectiveTo determine the signal pathway of specifically expressed oncostatin M(OSM) in lens inducing retinal degeneration in transgenic mice.MethodsA sequence-truncated OSM cDNA (661 bp) of mice was linked to αA-crytallin promoter, and was micro-injected into unicellular embryo to set up the model of transgenic mice. Reversal transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA expression of gp130/OSMRβ receptor in the retinae of OSM transgenic and non-transgenic mice. Rabbit anti-phosphorylated STAT-3 antibody was used to detect the protein expression of phosphorylated STAT-3，and mouse anti-cytochrome C antibody was used to detect the distributing of cytochrome C in retinae. ResultsExpression of gp130/OSMRβmRNA was found in retina of non-transgenic mice. At the 17.5th day in the embryonic stage, significant accumulation of the phosphorylated STAT-3 was detected in the retinal nucleolus in OSM transgenic retina. At the first day after birth, intensive staining of cytochrome C in OSM transgenic retina was found. Conclusionsspecifically expressed OSM in lens may act on gp130/OSMRβ receptor in retinae, activate STAT-3, and cause the release of cytochrome C from mitochondria, which eventually induces widespread retinal degeneration.(Chin J Ocul Fundus Dis, 2005,21:167-169)
Objective To investigate the effects of genistein with different concentration on proliferation and apoptosis of cultured human retinal pigment epithelial (RPE) cells in vitro. Methods The effect of genistein with the concentration of 5，10，25，50，75，and 100 mg·L-1on the proliferation of cultured RPE was examined by tetrazolium salt (MTT) assay and AgNORs staining. The cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) methods, in the mean time, the morphologic changes of cell apoptosis were observed by light microscopy and transmission electronic microscopy, the results of which were compared with the normal RPE cells. Results Genistein with the concentration of 25, 50, 75, and 100 mg·L-1had a dose-dependent and time-dependent antiproliferative effects on RPE cells with the inhibitory rate of 12.0%-64.6% (P＜0.05). The results of AgNORs staining showed that the number of AgNORs in the nucleolus decreased when treated by genistein. In TUNEL staining, the median of percent of apoptotic RPE cells was 7.6%, 9.8%, 13.7% when treated with 50 mg·L-1genistein, 10.3%, 16.4%, 23.4% when treated with 75 mg·L-1genistein, and 15.4%, 21.2%, 35.8% when treated with 100 m g·L-1genistein respectively for 24, 48, and 72 hours. After the treatme nt with 50 mg·L-1genistein for 48 hours, the apoptosis in the nucleolus of RPE cells was found. Conclusions Genistein with different concentrations has a dose-dependent and time-dependent antiproliferative effect on RPE cells. Genistein can induce the apoptosis of RPE cells when it reaches a certain extent of concentration. (Chin J Ocul Fundus Dis，2004，20：241-244)
Objective To investigate the mechanism of the toxic effect of N methyl N-nitrosourea (MNU) on photoreceptor cell apoptosis of rat’s retina. Methods Thirty 50-day-old female Sprague-Dawley ( SD ) rats were intraperitoneally injected with MNU (60 mg/kg) and were put to death by dislocation of cervical vertebra 12, 24, 48, and 72 hours and 7 days after the injection, respectively. The photoreceptor cell apoptosis was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and transmission electron microscope. The expression of proliferating cell nuclear antigen (PCNA), vimentin and glial fibrillary acidic protein (GFAP) was detected at different time after injection by immunohistochemical methods. Results The apoptotic index of the retina in the posterior pole was (33. 6±2. 3), (46. 5±5. 7), (20. 1±5. 3), (8. 2±3. 6) and (2. 5±1. 3~//oo at the 12th,24th, 48 th, and 72nd hour and on the 7th day, respectively, after injection. Karyopyknosis was found in most photoreeeptor cells 24 hours after injection. The expression of PCNA was found in internal granularlayer and between internal granular layer and choroid 24 hours after injection, reached the peak after 72 hours, and reduced obviously after 7 days. The positive expression of GFAP and vimentin was found in internal and external granular layer 24 hours after injection, reached the peak after 72 hours, and reducedobviously after 7 days.Conclusion MNU may selectively lead the photoreceptor cell apoptosis and proliferation of Mvller cells. (Chin J Ocul Fundus Dis，2004，20：33-36)
Objective To investigate the effects of epidermal growth factor (EGF)，fibroblast growth factor(FGF), and bovine serum on proliferation and apoptosis of the cultured fetal human retinal cells.Methods EGF and FGF were added or not to the medium of fetal human retinal cells cultured by bovine serum in vitro. The number of cells, bromodeoxyuridine(BrdU) incorporation and Tdt-mediated dUTP nick end labelling(TUNEL) were detected to determine the proliferation and apoptosis. Immunohistochemical staining of neuron specific enolase(NSE), Thy1.1, glial fibrillary acidic protein(GFAP) and scan electromicroscopy were performed to identify cell components. The expression of transcription factor c-fos, c-jun and apoptosis regulation factor bcl-2 and Bax were examined by immunohistochemical staining to explore the underlying mechanism.Results The increased number of NSE and Thy1.1 positive cells and BrdU incorporation, and decreased apoptotic cells were found in the groups treated with EGF and FGF. Meanwhile, the up-regulation of c-fos, c-jun and bcl-2 were also found. Conclusion EGF and FGF can promote the survival and proliferation of cultured retinal cells by up-regulating the expression of c-fos, c-jun and bcl-2. (Chin J Ocul Fundus Dis，2003，19：113-116)