Children with retinoblastoma (RB) typically survive their cancer due to advances in early diagnosis and treatment. Extraocular invasion and metastasis, and secondary malignant tumor carry a very high mortality rate. Prerequisites for metastasis include tumor initiating capacity, altered cellular adhesion and cell motility, resistance to extracellular death signals and disruption of the basement membrane and extracellular matrix. All those changes can be determined by the cell of origin and the genetic instability of the tumor, responding to the multiple layers of pressure such as hypoxia, from the tumor microenvironment or niche. The interaction between tumor cells and the tumor stroma is regulated by several metastasissuppressor proteins and microRNA. This knowledge has important implications for our understanding and the treatment of extraocular spreading of RB.
Objective To observe the choroidal thickness of patients with chronic central serous chorioretinopathy (CSC) in affected eyes and unaffected fellow eyes.Methods Forty-five chronic CSC patients diagnosed by fundus pre-set lens, fundus fluorescein angiography (FFA) and indocyanine green angiography were enrolled in this study. The patients included 36 males and nine females, with a mean age of (46.18plusmn;8.20) years, with a mean duration of (16.34plusmn;7.23) months. Thirty-six patients were affected unilaterally and nine patients affected bilaterally. The patients were divided into affected eyes group (group A, 51 eyes) and unaffected fellow eyes group (group B,39 eyes). Fifty age-, sex- and diopter- matched normal subjects (50 eyes) were enrolled in this study as control group (group C). Enhanced depth imaging (EDI) choroidal scans were obtained in all eyes by using spectral-domain optical coherence tomography. Subfoveal choroidal thickness (SFCT) and choroidal thickness at 3 mm nasal (NCT3 mm), temporal (TCT3 mm), superior (SCT3 mm), inferior (ICT3 mm) to the fovea were measured.Results The mean SFCT of group A, B and C were (436.76plusmn;87.01), (394.71plusmn;61.63), (294.86plusmn;75.30) mu;m respectively. The mean SFCT of group A and B were thicker than group C, the difference was significant among three groups (F=44.791,P＜0.001). There were difference between group A, B, C in NCT3 mm, TCT3 mm, SCT3 mm and ICT3 mm (F=15.816, 22.823, 15.147, 11.527;P＜0.001). The mean SFCT in affected eyes of unilateral patients was (416.34plusmn;79.44) mu;m, which was thicker than that in unaffected fellow eyes (t=2.897, P=0.007). Conclusion Choroidal thickness increased significantly in affected eyes and unaffected fellow eyes in patients with chronic CSC.
ObjectiveTo observe the effect of phase Ⅱenzyme inducer 5, 6-dihydrocyclopenta 1, 2-dithiole-3-thione (CPDT) on nuclear factor erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) signal pathway and oxidative stress in the retina of type 2 diabetic rats.
MethodsThirty-five male Wistar rats were randomly divided into two group, normal group and model group. Model group were further randomly divided into two group, diabetic group and CPDT intervention group. There were 8 rats in the normal group and 27 rats in the model group. Diabetic group and CPDT intervention group were given high fat and high sugar diet for 2 months. After 12 hours of fasting, type 2 diabetic rat model was induced by intraperitoneal injection of low dose of streptozotocin. CPDT was added into the high fat and high sugar diets at 1 week after the diabetic model was established in the CPDT intervention group. Eight weeks after CPDT treatment, blood glucose, serum malondialdehyde (MDA), blood lipid, Nrf2 and hemeoxygenase-1 (HO-1) expression were evaluated.
ResultsType 2 diabetic model was successfully established in 25 rats, the success rate was 92.6%.The level of blood lipid of diabetic group was higher than those of the normal group (FTC=65.866, FTG=25.441, FLDL-C=38.889; P=0.000). Blood glucose was significant different between all groups (χ2=25.812, P=0.000), and was significantly higher in diabetic group than that in normal group and CPDT intervention group. The serum MDA content was significant different between all groups (F=59.545, P=0.000), and was significantly higher in diabetic group than that in normal group (t=10.523, P=0.000) and CPDT intervention group (t=7.766, P=0.000). The mRNA level of retinal Nrf2 and HO-1 was significant different between all groups (FNrf2=19.503, PNrf2=0.000;FHO-1=9.737, PHO-1=0.001), and was higher in CPDT intervention group than the diabetic group (tNrf2=3.399, PNrf2=0.002;tHO-1=2.167, PHO-1=0.039). The protein level of retinal Nrf2 and HO-1 was significant different between all groups (FNrf2=112.823, FHO-1=119.361; P=0.000), and was higher in CPDT intervention group than the diabetic group (tNrf2=6.203, tHO-1=6.388; P=0.000). Immuno-staining showed that Nrf2 and HO-1 were mainly expressed in retinal ganglion cell layer, inner plexiform layer and inner nuclear layer, and were significant different between all groups (FNrf2=16.206, FHO-1=46.790; P=0.000). They also were higher in CPDT intervention group than the diabetic group (tNrf2=3.172, PNrf2=0.003;tHO-1=6.321, PHO-1=0.000), was higher in diabetic group than that in normal group (tNrf2=2.679, PNrf2=0.011;tHO-1=3.482, PHO-1=0.001).
ConclusionCPDT may activate Nrf2/ARE pathway, induce Nrf2 and HO-1 expression, decrease serum MDA and blood glucose, and thus reduce oxidative stress injury in the retina of type 2 diabetic rats.
Objective To observe the expression of vascular endothelial growth factor receptor-1 (VEGFR-1) and VEGFR-2 in hypoxic chorioretinal endothelial cells of monkeys (RF/6A), and to evaluate the effect of minocycline. Methods RF/6A was cultured and divided into four groups: control group, hypoxia group, hypoxia and low dose of minocycline group (0.5 mu;mol/L), hypoxia and medium dose of minocycline group (5 mu;mol/L), and hypoxia and high dose of minocycline group (50 mu;mol/L). Real-time reverse transcriptionpolymerase chain reaction (RT-PCR) and immunohistopathological staining were used to measure the mRNA and protein expression of VEGFR-1 and VEGFR-2, respectively. Results RT-PCR showed that the expression of VEGFR-1 mRNA did not vary significantly between groups (F 24 h=0.17,F 48 h=1.53,F72 h=2.04;P＞0.05). Compared with hypoxia group, the expression of VEGFR-2 mRNA in all minocycline treated groups were significantly downregulated (low minocycline, medium minocycline, high minocycline: t=4.69, 20.16, 17.12; P＜0.001). The immunohistopathological study showed the cells with positive staining of VEGFR-1 can be observed in all groups, and the staining was relatively weak and mainly located in cell membrane and cytoplasm. The optical density value analysis showed that the protein expression of VEGFR-1 did not vary significantly between groups at all time points（F 24 h=0.251,F 48 h=0.340,F72 h=0.589;P＞0.05）. The VEGFR-2 positive staining cells were also observed in all groups, and the staining was relatively high. Brown staining particles of VEGFR-2 were observed in the cell membrane with minor staining particles in cytoplasm. The staining density of VEGFR-2 was significantly higher in hypoxia group than control group. Compared with the hypoxia group, the protein expression of VEGFR-2 in minocycline treated groups was significantly lower（F 24 h=19.147,F 48 h=14.893,F72 h==11.984; P＜0.05). Conclusion The expression of VEGFR-2 is upregulated in RF/6A, and minocycline somewhat shows an inhibition effect.
Objective To investigate the cellular viability and mitochondrial reactive oxygen species (ROS) production of the Müller cells under high glucose condition, and explore the protection role of the 5,6-dihydrocyclopenta-1, 2-dithiole-3-thione (CPDT) on Müller cells. Methods Müller cells from Sprague Dawley rats were divided into 5 groups randomly, including 25 mmol/L normal glucose group (group A) and 65 mmol/L high glucose group (group B). High glucose group with 45, 60, 70 μmol/L CPDT and cultured them 72 hour was set as group C, D and E. Water soluble tetrazolium salt (WST)-8 was used to measure the cellular viability. Flow cytometry was used to measure the active oxygen and apoptosis index. The expression of nuclear factor erythroid 2-related factor 2 (Nrf2), hemeoxygenase-1 (HO-1), Bcl-2 and Bax protein were measured by Western blot. Results Compared with group A, the WST-8 showed that the viability of Müller cells apparently decreased in group B (t=39.59,P＜0.05). Compared with the group B, the viability of Müller cells had changes in group C (t=0.97,P＞0.05), but recovered in group D and E (t=?4.17, ?7.52;P＜0.05). Compared with group A, the FCM showed that the mitochondrial ROS levels was higher in group B (t=?30.99,P＜0.05). Compared with group B, the mitochondrial ROS levels were decreased in group D (t=27.68,P＜0.05). Compared with group A, Bax, Nrf2 and HO-1 increased (t=–11.03, –63.17, –11.44;P＜0.05), while the bcl-2 decreased in group B (t=7.861,P＜0.05). Compared with the group B, Nrf2, HO-1 and Bax decreased (t=15.11, 26.59, 6.27;P＜0.05), while the bcl-2 increased in group D (t=?6.53,P＜0.05). Conclusions Under the high glucose, CPDT may reduce the mitochondrial ROS levels and the expression of Nrf2, HO-1 and Bax protein of Müller cells. It may inhibit apoptosis through activating the Nrf2/HO-1 pathway and balancing of level of Bcl-2 protein and mitochondrial ROS.
Objective To observe the expression of cyclin dependent kinase 5 (Cdk5) and p25 in the pathogenesis of retinitis pigmentosa (RP) in Royal College of Surgeon (RCS) rats and its relationships with apoptosis. To explore the mechanism of Cdk5 and p25 induced photoreceptor apoptosis in the pathogenesis of RP. Methods Retinas of RCS and RCS-rdy+ rats were obtained at the ages of postnatal day 17, 25, 35, 60. The retinal structure and thickness of outer nuclear layer were measured by optical microscopy. The expression of Cdk5, p25, cleave-caspase 3 in the retina was evaluated by immunohistochemistry. The protein expression of cleave-caspase 3 in the retina was determined by Western blot. The apoptosis of retinal cells was examined by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL). The mean absorbance value of apoptotic cells was analyzed by SPSS 17.0 software. Results The retinal thickness of the RCS rats was significantly reduced in comparison to the RCS-rdy+ rats as the postnatal days progressed, particularly in the layer of rods and cones and the outer nuclear layer. The expression level of Cdk5, p25, cleave-caspase 3 of RCS rats increased from postnatal 17 days to postnatal 35 days, while decreased on postnatal 60 days; but there was no obvious change of above indexes in RCS-rdy+ rats. The protein expression of cleave-caspase 3 in the RCS rats was significantly increased with progression of postnatal days to postnatal 35; but there was no obvious similar change in RCS-rdy+ rats. The results of TUNEL showed that the apoptotic cells significantly increased in the outer nuclear layer of RCS rats from postnatal 17 days to postnatal 35 days, while decreased on postnatal 60 days; but there was no obvious change of above index in RCS-rdy+ rats. This study showed that there were significant correlations between the following variables: Cdk5 expression and p25 expression, Cdk5 expression and cleave-caspase 3 expression, Cdk5 expression and apoptotic cells, p25 expression and cleave-caspase 3 expression, p25 expression and apoptotic cells, cleave-caspase 3 expression and apoptotic cells. The partial correlation coefficients were 0.949, 0.808, 0.959, 0.887, 0.979, 0.852, respectively and the P value was 0.000. Conclusions The apoptotic cells significantly increases and the expression level of Cdk5, p25, cleave-caspase 3 of RCS rats increases from postnatal 17 days to postnatal 35 days. The tendency of apoptotic cells to increase is consistent with the change of Cdk5, p25, cleave-caspase 3 expression. The apoptosis of photoreceptor cells is related to increasing expression of Cdk5 and p25 in RCS rats. Cdk5 may be involved in the development of RP in RCS rats.
ObjectiveTo observe the expression of Toll-like receptor 4 (TLR4) and inflammatory cytokines, leucocytic density and permeability in retina of diabetic rat.
MethodsA total of 106 Brown Norway rats were randomly divided into experimental group and control group with 53 rats in each group. Diabetic model was established in experimental group by intraperitoneal injection of streptozotocin, and control rats received intraperitoneal injection of an equal volume of citric acid-sodium citrate buffer. Four weeks later, the retinas were collected for further analysis. TLR4 RNA and protein expression were measured by quantitative polymerase chain reaction and Western blot. Inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), monocyte chemo-attractant protein-1 (MCP-1), were measured by enzyme-linked immunosorbent assay in rat retina homogenate. Leukocyte density in the retina was measured by acridine orange fundus angiography. The retinal permeability was evaluated by Evans blue (EB) staining.
ResultsTLR4 expression was significantly increased in diabetic rats of experimental group compared with non-diabetic rats of control group (F=1.606, 0.789; P < 0.05). Inflammatory cytokines (TNF-α, IL-1β and MCP-1) were significantly increased in retina of diabetic rats of experimental group versus non-diabetic rat of control group (F=24.622, 5.758, 4.829; P < 0.05). The retinal leukocyte density was (6.2±0.5)×10-5, (2.2±0.3)×10-5 cells/pixel2 in experimental and control group respectively, the difference was statistically significant (F=2.025, P < 0.05). The amount of retinal EB leakage was (23.41±4.47), (13.22±3.59) ng/mg in experimental and control group respectively, the difference was statistically significant (F=21.08, P < 0.05).
ConclusionTLR4 and inflammatory cytokines expression, leucocytic density and permeability increased significantly in retina of diabetic rat.
Retinal neovascularization is a complicated pathophysiological process as a result of imbalance between angiogenic and antiangiogenic factors. Correct understanding of the signaling pathways, exploring the critical factors involved in retinal angiogenesis, looking for new strategies by reconstructing the new vessels are helpful for knowing the mechanism of the occurrence and development of reitnal neovascularization, which would be good for preventing and treating retinal neovascularization diseases.
Objective To investigate the relationship of the expression between heat shock protein (HSP) 70 and 90, and Survivin and its effects on the proliferative activity in retinoblastoma (RB) cells. Methods Expression of Survivin, HSP70 and 90, and Ki-67 in conventional paraffin samples from 43 patients with RB and 6 healthy people was detected by streptavidin-biotin peroxidase (SP) immunohistochemical method. Ki67 labeling index was used to evaluate the proliferative activity in RB. Results In 43 cases of RB, positive expression of HSP70 and 90 and Survivin was found in 28 (65.12%), 37 (86.05%) and 27 (62.79%) cases, respectively. None of the 6 normal retinal tissue expressed HSP70, HSP90 or Survivin. Positive expression of Survivin was more frequent in positive expressions of HSP90 than that in negative expressions of HSP90 (P＜0.05). Ki67 labeling index was higher in positive expressions of HSP90 and positive expressions of Survivin than that in their negative expressions respectively (P＜0.05). Meanwhile, higher Ki67 labeling index was found in positive HSP90Survivin expressions than that in negative HSP90Survivin expressions and those cases where only HSP90 or Survivin was found (P＜0.05). Expression of HSP70 did not correlate with that of Survivin, nor had any significant effect on Ki67 labeling index (P＞0.05). Expression of HSPs and Survivin and Ki67 labeling index did not correlate with histological types (P＞0.05). Conclusion Expression of HSP90 correlates with that of Survivin in RB. Co-existence of Survivin and HSP90 probably plays an important role in the genesis of RB.
Objective To observe the influences of uncoupling protein 2 (UCP-2) rs660339 variants transfection on cell proliferation and apoptosis of human umbilical vein endothelial cell (HUVEC). Methods Two UCP-2 green fluorescent protein (GFP) lentivirus constructs were created with the rs660339 locus carried C or T (UCP-2C or UCP-2T), respectively. HUVEC were cultured after lentiviral infection of UCP-2C or UCP-2T. The expression of UCP-2C or UCP-2T was detected with real time polymerase chain reaction. Cell proliferation and cell apoptosis were compared among negative control (NC) group, UCP-2T group and UCP-2C group using CCK-8 cell viability and flow cytometry. Western blot and immunostaining were employed to examine the expression of Bcl-2 gene. Results The lentivirus constructs were successfully created. ＞80% of the transfected cells were found to express GFP under fluorescent microscope. The mRNA levels of UCP-2 gene were significantly increased (F=29.183,P=0.001) in the UCP-2T group and UCP-2C group. The CCK-8 assay revealed that on day two (F=15.970,P=0.004), day three (F=16.738,P=0.004), day four (F=5.414,P=0.045) post-infection, UCP-2T and UCP-2C group showed significantly greater proliferation than the NC cells. The apoptotic rate in the UCP-2T and UCP-2C group was significantly lower than NC group (F=277.138,P=0.000), and the apoptotic rate of UCP-2T was significantly lower than that of UCP-2C (P=0.003). The protein levels of Bcl-2 in the UCP-2T and UCP-2C group were significantly greater than that in the NC group (F=425.679,P=0.000), and the Bcl-2 expression of UCP-2T was greater than that of UCP-2C (P=0.002). The Bcl-2 density in the UCP-2T and UCP-2C group were greater than that in the NC group (F=11.827,P=0.008), while there was no difference between UCP-2T and UCP-2C group (P=0.404). Conclusion The variants of UCP-2 rs660339 may influence HUVEC proliferation and apoptosis, and UCP-2T showed a stronger effect of inhibiting apoptosis than UCP-2C.
