Objective To observe the expressions of DNA methyltransferases (DNMTs) 1, 3a and 3b in retinoblastoma (RB). Methods Sixty-two RB samples and six normal retinas were studied, including 17 poorly differentiated and 45 well differentiated samples; 16 invasive and 46 non-invasive samples. The expressions of DNMT1, 3a, and 3b, and Ki-67 were detected using immunohistochemical analysis. Brown staining of nuclei was considered to represent the positive stain for DNMT1, 3a and 3b, and ki-67, blue staining as negative. The level of high expression of nuclear staining was, positive cells in DNMT1ge;65%, in DNMT3age;60% and in DNMT3bge;40%. The correlations of DNMT1, 3a and 3b expression in RB samples, and MIB-1 labeling index were analyzed. Results Viewed under the light microscope, negative expressions of DNMT1, 3a and 3b were demonstrated in normal retinas, however, positive expression was observed in RB samples, with 100% in DNMT1, 98% in DNMT3a and 92% in DNMT3b. Comparing well differentiated RB samples with poorly differentiated samples, significant differences were found in high expression of DNMT1 (chi;2=12.57,P＜0.05) and DNMT3a (chi;2=10.54,P＜0.05); also in the positive cells of DNMT1 (U=179,P＜0.05) and DNMT3a (U=198,P＜0.05). No significant difference was found comparing high expression (chi;2=1.5,P＞0.05) and positive cells (U=307,P＞0.05) of DNMT3b. When comparing invasive tumor tissues with non-invasive tumors, significant differences were shown between high expression (chi;2=4.72,P＜0.05) and positive cells comparing DNMT1 (U=236,P＜0.05). No significant difference was shown in high expression (chi;2=3.53,0.84; P＞0.05) in DNMT3a and DNMT3b, or in comparison with positive cells (U=338,257;P＞0.05). The expression of DNMTs was positively correlated with the MIB-1 labeling index in RB tissues (R2=0.554,0.376,0.219;P＜0.05). Conclusion There are high expressions of DNMT1,3a,and 3b in RB.
Objective To observe the abnormal expression of alpha;A-crystallin protein in neural retina in type 2 diabetic rats via proteomic technique.Methods Twenty-eight male Sprague-Dawley (SD) rats were randomly divided into the normal control and the diabetic experimental groups with 14 rats in each group.A type 2 diabetes rat model (T2DM) was set up in the diabetic experimental group by feeding high fat diet combined with peritoneal injection of low dose streptozotocin (STZ);the successful diabetes model is with the randomlydetected blood glucose of ＞16.7 mmol/L.The rats in the control group underwent peritoneal injection of equivalent sodium citrate solution and were fed with normal diet.All of the animals were sacrificed by decapitation 56 days after the induction of diabetes.The eyes were enucleated and the neural retina layers were carefully peeled off and preserved.The total neural retinal proteins were extracted from the control and diabetic groups, respectively,and then subjected to two dimensional gel electrophoresis (2-DE).Some different proteins spots were identified by peptide mass fingerprinting (PMF) as well as by tandem mass spectrometric (MS/MS) measurements.Western blot and indirect immunofluorescence (IMF) were used to confirmed that alpha;A-crystallin protein expression was upregulated in diabetic retina.Results An average of （3122plusmn;37） spots in normal retinas and（2702plusmn;21）spots in diabetic group were found by 2-DE image analysis software; about 150 spots in 2-DE gel of diabetic retinae exhibited statistically significant variations (t＞2.77,P＜0.05).Compared with normal rats' retinae, diabetic ones presented 68 protein spots of up regulation expression and 82 of downregulation expression in 2DE gel.Furthermore,20 of the 150 protein spots were identified by mass spectrometry.The points of 2369 and 1048 in 2-DE gel, showing high expression in diabetic retinal tissues, were identified as alpha;A-crystallin via PMF.Western blot validated that the expression level of alpha;A-crystallin in diabetic neural retina was much higher than that in the control group. Significantly increased expression of alpha;A-crystallin in nuclear retina in diabetic group was also observed by IMF. Fluorescence was mainly seen in the retinal nuclear layer;alpha;A-crystallin aggregation was detected in the perinuclear region of neurons.Conclusion The expression of alpha;A-crystallin increases in neural retina of early T2DM rats.
Objective To investigate if insulin can affect the expression of vascular endothelial growth factor (VEGF) in the retina of streptozotocin-induced diabetic rats. Methods A total of 60 male SpragueDawley rats were randomly divided into sodium citrate buffer control group (CIT-CON, n=30) and STZ-induced diabetic group (STZ-DM, n=30). At the 16th week, 24 rats from CIT-CON group at random were randomly divided to group A (sodium citrate buffer control group, n=12) and group B (sodium citrate buffer plus insulin group, n=12). The remaining 6 rats from as CIT-CON group served as negative control. At the same time, 24 rats from STZDM group at random were randomly divided to group C (STZinduced diabetic group, n=12) and group D (STZ-induced diabetic plus insulin group, n=12). The remaining 6 rats from STZ-DM group also served as negative control. 4 IU of insulin was injected subcutaneously to rats of group B and D. Immunohistochemistry, Western blot and Real-time polymerase chain reaction (RT-PCR) were used to measure the expression level of VEGF protein and mRNA respectively. RESULTS Insulin significantly increased the VEGF mRNA (7.71plusmn;0.25 vs 5.36plusmn;0.37, t test Plt;0.05) and protein expression (0.4925plusmn;0.0122 vs 0.4272plusmn;0.0110, t test Plt;0.05) in the retina of CITCON rats. However, in retina of STZDM rats, insulin had no effect on VEGF mRNA (8.92plusmn;0.27 vs 9.05plusmn;0.28, t test, Pgt;0.05) and protein expression (0.5152plusmn;0.0109 vs 0.5099plusmn;0.0100, t test Pgt;0.05). Conclusions Insulin had no effect on VEGF expression in the retina of STZ-DM rats.
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 effect of celecoxib on the expression vascular endothelial growth factors (VEGF) in diabetic rats. Methods Thirty-six wistar rats were used to establish the diabetic models by intraperitoneal injection with streptozotocin. The diabetic rats were divided into 2 groups: diabetic group (n=18) and celecoxib group (n=18). Celecoxib (50 mg/kg) was administered orally to the rats in celecoxib group and the physiological saline with the same volume was given orally to the rats in diabetic group. Eighteen else rats were in normal control group. All of the rats were executed 3 months later. The expression of VEGF protein was detected by immunohistochemistry method. Reverse transcription-polymerase chain reaction（RT-PCR） analysis was used to examine the expression of retinal VEGF mRNA and cyclooxygenase-2 mRNA. Results Lower positive expression of VEGF mRNA and cyclooxygenase-2 mRNA, weakly positive action of immunohistochemistry of VEGF, and lower expression of VEGF protein were detected in normal control group; in the diabetic group, the expression of VEGF mRNA and cyclooxygenase-2 mRNA increased obviously comparing with which in the control group (Plt;0.05), and the bly positive action of immunohistochemistry of VEGF and increased expression of VEGF protein were detected (Plt;0.01); in celecoxib group, the expression of VEGF mRNA was lower than that in the diabetic group (Plt;0.05), the expression of cyclooxygenase-2 mRNA didnprime;t decrease much (Pgt;0.05), the positive action of immunohistochemistry of VEGF decreased, and the expression of VEGF protein decreased (Plt;0.01). Conclusion By inhibiting the activation of cyclooxygenase-2, celecoxib can inhibit the expression of retinal VEGF mRNA and protein in diabetic rats induced by streptozotocin. (Chin J Ocul Fundus Dis,2007,23:265-268)
Objective To observe the changes of expression of glutamine synthetase (GS) in early diabetic ratsprime; retina and investigate its possible mechanism. Methods Three groups of streptozotocininduced diabetic models of SpragueDawley (SD) rats with different diseased courses, ie, 1 month, 2 months, and 3 months, respectively, with 8 rats in each group, and 8 normal ones as control were examined. The expressions of GS, interleukin-1beta; (IL-1beta;) and c-Jun in retina in the 4 groups were detected by indirect immunofluorescence and western blotting techniques. Meanwhile, different doses of IL-1beta;（0,100,500,and 1000 ng/ml） were injected into the vitreous cavities of 32 normal rats (8 rats in each group), and 24 hours later, the expressions of GS and c-Jun in retina were detected by the same methods. Results The expression of GS in retina did not changed in control group or in 1 month and 2 months group, but decreased obviously in 3 months group comparing with which in the control group (Plt;0.01).The expressions of c-Jun and IL-1beta; in retina in control group were very low, but increased gradually in diabetic rats in 1-3 months group, which significantly differed from which in the control group (Plt;0.01). Vitreous injection with IL-1beta; (500 and 1000 ng/ml) down regulated the expressions of GS, and the expression of c-Jun increased in a dose-dependent way after injection with IL-1beta; at the concentration of 100 ng/ml. Conclusions In early diabetic ratsrsquo; retina, IL-1beta; may down regulate the expression of GS. The possible mechanism may be the activation of c-Jun by IL-1beta;. (Chin J Ocul Fundus Dis,2007,23:260-264)