ABSTRACT
Objective Although vascular endothelial growth factor(VEGF)is a primary mediaor in diabetic retinopathy(DR).VEGF inhibition alone is insufficient for preventing retinal neovascularization.Some studies showed that erythropoietin(EPO)is a potent retinal angiogenic factor of independent of VEGF in DR.The present study is to investigate the effect of high glucose on the expression of mRNA and protein of the erythropoietin receptor(EPOR)in cultured human umbilical vein endothelial cells(UVECs)in vitro.MethodsHuman UVECs from the cell center of the hospital were cultured in vitro and passaged in DMEM containing 10% neonatal bovine serum with 22mmol/L of glucose for 12,24,48 and 72 hours in the experimental group.Cells cultured in 5.5mmol/L glucose were used as control group Ⅰ and mannitol + 22mmol/L of glucose(isotope)as control group Ⅱ.The expression of EPOR mRNA in Human UVECs were detected by semi-quantitative reverse transcriptase(RT)-polymerase chain reaction(PCR)and detected at A_(260mm)/A_(280mm).The PCR product was calculated as the A value of EPOR mRNA amplification/the A value of GAPDH mRNA amplification.The expression of EPOR protein in Human UVECs was detected by immunocytochemistry.ResultsThe A_(260mm)/A_(280mm) value of EPOR mRNA receptor expression was 0.32±0.02 in the 5.5mmol/L glucose group,and 0.34±0.02 in the mannitol+22mmol/L glucose group(P>0.05).In 12 hours,24 hours and 72 hours after the experiment,the A260mm/A280mm value of EPOR mRNA was 0.82±0.01,0.96±0.02 and 1.02±0.01,respectively,indicating a significant increase in comparison with the 5.5mmol/L glucose group.The expression of Human UVECs protein was gradually increased with passage in the experimental group.Expression of Human UVECs protein was stronger in various time points in the 22mmol/L glucose group than in the 5.5mmol/L glucose group.ConclusionHigh glucose elevates the expression of EPOR mRNA and protein in Human UVECs in a time-dependent manner.The effect of high glucose(22mmol/L glucose)on the expression of EPOR mRNA and protein in Human UVECs is not related to osmotic pressure.
ABSTRACT
Objective To determine the signal pathway of specifically expressed oncostatin M(OSM) in lens inducing retinal degeneration in transgenic mice. Methods A sequence-truncated OSM cDNA (661 bp) of mice was linked to ?A-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/OSMR? 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. Results Expression of gp130/OSMR? mRNA 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. Conclusions specifically expressed OSM in lens may act on gp130/OSMR? receptor in retinae, activate STAT-3, and cause the release of cytochrome C from mitochondria, which eventually induces widespread retinal degeneration.
ABSTRACT
Objective To determine the effects of lensspecific overexpression of OSM on the eye development. Methods A truncated mouse OSM cDNA (661 bp) was linked to the ?A-crystallin promoter. Transgenic mice were characterized by routine histological and immunohistochemical techiniques. TUNEL assays were used to detect cell death. The mRNA expression of caspase-3 was detected by in situ hybridization, Rabbit anti-cleavage caspase-3 antibody was used to detect active capase-3. Results At embryonic day (E) 14.5 and 17.5, expression of the OSM transgenic protein was detected specifically in lens fiber cells. The onset of retinal degeneration in the mid portion of the transgenic retinae was observed started from E17.5. By the time of birth 50% or more of the retinal cells were missing. The OSM transgenic retinal cells underwent apoptosis indicated by TUNEL assays. Most strikingly, activation of caspase-3 protein were observed throughout the transgenic retinas. Conclusions Lens-specific overexpression of OSM activate caspase-3, leading to abnormal eye development, apoptosis and widespread retinal degeneration.