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Objective:To explore the effects of conditioned medium of human bone marrow mesenchymal stem cells (BMSCs) on the proliferation, adhesion and differentiation of immortalized human Müller cell line (MIO-M1).Methods:The differentiation was induced in the third-passage BMSCs with osteogenic, chondrogenic and adipogenic medium and identified by alizarin red, alcian blue and oil red O staining, respectively.The expression levels of mesenchymal stem cell markers CD73, CD90 and CD105 and hematopoietic cell markers CD34, CD45 and human leukocyte antigen-DR (HLA-DR) were assayed by flow cytometry.The expressions levels of Müller cell markers SOX9, glutamine synthetase (GS), vimentin and cellular retinaldehyde-binding protein (CRALBP), retinal stem cell markers SOX2, nestin and CHX10, and cell proliferation marker cyclin D3 (CCND3) in MIO-M1 cells were detected by immunofluorescence staining.The MIO-M1 cells were divided into standard medium group, 293T conditioned medium group, and BMSC conditioned medium group and were incubated in the medium according to grouping.The cellular area, circularity, elongation factor and perimeter were analyzed quantitatively.The cell cycle was detected by flow cytometry, and the cell proliferation was determined by neurospora experiment and 5-ethynyl-2'-deoxyuridine (EdU) staining.The expression of vascular cell adhesion molecule 1 (VCAM-1) at protein and mRNA levels in the culture supernatant was detected by enzyme linked immunosorbent assay (ELISA) and quantitative real-time PCR (qRT-PCR), respectively.The expression of retinal neuron markers protein kinase C (PKCα), Rhodopsin, microtubule-associated protein 2 (MAP2) and β-tubulin (Tuj1) was detected by immunofluorescence staining and qRT-PCR.Results:CD73, CD90, CD105 showed an enhanced expression, and CD34, CD45 and HLA-DR showed weakened expression in the BMSCs.The BMSCs differentiated into osteoblasts, chondrocytes and adipocytes.Expression of SOX9, GS, vimentin and CRALBP, SOX2, CHX10, nestin and CCND3 was found in the MIO-M1 cells.Compared with standard medium group and 293T conditioned medium group, MIO-M1 cells cultured in BMSC conditioned medium group changed into an elongated spindle-shaped or multipolar morphology with reduced cell area, increased elongation index and decreased circularity, showing statistically significant differences among them ( F=6.973, 12.370, 6.311; all at P<0.01). There were increased neurospheres formed by MIO-M1 cells in BMSC conditioned medium group compared with standard medium group and 293T conditioned medium group at different time points ( Fgroup=134.300, P<0.001; Ftime=82.910, P<0.001). Compared with the standard medium group and 293T conditioned medium group, the EdU-positive rate and proliferation index of MIO-M1 cells in BMSC conditioned medium group were significantly increased, with statistically significant differences ( F=6.973, 74.110; all at P<0.05); the VCAM-1 protein expression in cell supernatant and the relative expression level of VCAM-1 mRNA in BMSC conditioed medium group were significantly increased ( F=13.720, 7.896; all at P<0.05); the mRNA expression levels of PKCα, Rhodopsin, Tuj1 and MAP2 were higher in MIO-M1 cells of BMSC conditioned medium group under the condition of differentiation ( F=14.490, 5.424, 14.330, 7.405; all at P<0.05). Conclusions:BMSCs conditioned medium can change the morphology of MIO-M1 cells and promote their proliferation, adhesion and differentiation into retinal neurons.
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Müller cells are glial cells of the retina, whose major processes cross the internal and external limiting membranes of the retina, maintaining the function and metabolism of retinal photoreceptors and neurons. Their structure and function are closely related to the development of macular hole (MH). Müller cells are involved in the formation and recovery of MH from the aspect of traction and protein, and their morphology and biological function also influence the regression of MH. The current treatment modality for MH is vitrectomy combined with internal limiting membrane (ILM) peeling, in which Müller cells play a dual role after ILM peeling in different stages of MH. And its potential to re-acquire a progenitor-like state following retinal injury with the ability to proliferate and generate new neurons making it a current research hot topic, which can be a reference and inspiration for clinical treatment.
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To report a rare case of Muller cell sheen dystrophy and to describe its clinical and diagnostic aspects. A 42-year-old woman presented with unilateral defective vision. Fundus evaluation revealed bilateral glistening retinal reflexes throughout the posterior pole with a wrinkled appearance in the right. Spectral Domain-OCT in the right showed abnormal internal limiting membrane, intraretinal schisis with serous detachment at macula. Angiography revealed staining along vessels. Electroretinogram in the affected eye was negative. At 4 months of follow up, vision dropped and intraretinal schisis increased. Family screening was negative.
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BACKGROUND: It has been found that salidroside can improve diabetic retinopathy. However, it is not clear whether salidroside can protect retinal Müller cells (rMC-1) against damage induced by high glucose. OBJECTIVE: To investigate the effect and mechanism of salidroside on oxidative stress and apoptosis induced by high glucose in rat rMC-1 cells. METHODS: The rMC-1 cells were divided into four groups: normal control group, high glucose group (culture medium with a final glucose concentration of 35.5 mmol/L), salidroside group (treatment with salidroside for 4 hours followed by co-culture with high glucose) and PI3K inhibitor group (treatment with salidroside and PI3K inhibitor for 4 hours followed by co-culture with high glucose). The viability of the rMC-1 cells was measured by cell counting kit-8 assay. Reactive oxygen species production was detected by DCFH-DA. The superoxide dismutase and catalase activities were tested by specific kits. Cell apoptosis was detected by flow cytometry. Western blot was used to detect the expression levels of Cleaved caspase-3, PI3K, AKT and phosphorylated AKT. RESULTS AND CONCLUSION: Compared with the normal control group, the cell viability and the activities of antioxidant enzymes (superoxide dismutase, catalase) in the high glucose group were significantly decreased, the production of reactive oxygen species, apoptotic rate and the level of Cleaved caspase-3 were significantly increased, and the phosphorylated AKT/AKT ratio was down-regulated (P < 0.05). Compared with the high glucose group, salidroside significantly enhanced the cell viability and increased the activities of antioxidant enzymes (superoxide dismutase, catalase), decreased the production of reactive oxygen species, reduced the apoptotic rate of the cells and downregulated the expression level of Cleaved caspase-3 (P < 0.05). Salidroside also activated the phosphorylation of Akt (P < 0.05). Whereas, the addition of LY294002, a pharmacological inhibitor of PI3K, showed similar results in the high glucose group (P < 0.05). To conclude, salidroside can protect rMC-1 cells through inhibiting apoptosis and oxidative stress induced by high glucose. The main mechanism responsible for the inhibition of oxidative stress is the activation of the PI3K/Akt pathway.
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In the central nervous system (CNS), three types of myelin-associated inhibitors (MAIs) have major inhibitory effects on nerve regeneration. They include Nogo-A, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein. MAIs possess two co-receptors, Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PirB). Previous studies have confirmed that the inhibition of NgR only results in a modest increase in regeneration in the CNS; however, the inhibitory effects of PirB with regard to nerve regeneration after binding to MAIs remain controversial. In this study, we demonstrated that PirB is expressed in primary cultures of retinal ganglion cells (RGCs), and the inhibitory effects of the three MAIs on the growth of RGC neurites are not significantly decreased after direct PirB knockdown using adenovirus PirB shRNA. Interestingly, we found that retinal Müller cells expressed PirB and that its knockdown enhanced the regeneration of co-cultured RGC neurites. PirB knockdown also activated the JAK/Stat3 signaling pathway in Müller cells and upregulated ciliary neurotrophic factor levels. These findings indicate that PirB plays a novel role in retinal Müller cells and that its action in these cells may indirectly affect the growth of RGC neurites. The results also reveal that PirB in Müller cells affects RGC neurite regeneration. Our findings provide a novel basis for the use of PirB as a target molecule to promote nerve regeneration.
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In the central nervous system (CNS), three types of myelin-associated inhibitors (MAIs) have major inhibitory effects on nerve regeneration. They include Nogo-A, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein. MAIs possess two co-receptors, Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PirB). Previous studies have confirmed that the inhibition of NgR only results in a modest increase in regeneration in the CNS; however, the inhibitory effects of PirB with regard to nerve regeneration after binding to MAIs remain controversial. In this study, we demonstrated that PirB is expressed in primary cultures of retinal ganglion cells (RGCs), and the inhibitory effects of the three MAIs on the growth of RGC neurites are not significantly decreased after direct PirB knockdown using adenovirus PirB shRNA. Interestingly, we found that retinal Müller cells expressed PirB and that its knockdown enhanced the regeneration of co-cultured RGC neurites. PirB knockdown also activated the JAK/Stat3 signaling pathway in Müller cells and upregulated ciliary neurotrophic factor levels. These findings indicate that PirB plays a novel role in retinal Müller cells and that its action in these cells may indirectly affect the growth of RGC neurites. The results also reveal that PirB in Müller cells affects RGC neurite regeneration. Our findings provide a novel basis for the use of PirB as a target molecule to promote nerve regeneration.
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Ischemic retinopathy,resulting in multiple lesions like microvasculature damage,inflammation and neovascularization,is a major contributor of vision damage.In these pathological changes,retinal glia cannot be ignored in the development of retinopathy.They constitute a highly versatile population that interacts with various cells to maintain homeostasis and limit disease.Therefore,glial activation and gliosis are strikingly ubiquitous responses to almost every form of retinal disease.Both of microglial cells and Müller cells are major intrinsic retinal glial cells and they are in close relationship,which means they can influence each other,make joint action or even become interdependent.They exhibit morphological and functional changes to have an impact on degree of retinal injury through different responses,which mediated by glial cells are important not only for course of disease progression,but also for the maintenance of neuronal and photoreceptor survival.Thus,defining the mechanisms that underlie communications between microglial cells and Miller cells could enable the development of more selective therapeutic targets,with great potential clinical applications.
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Objective To observe the expression ofprobucol on high glucose-induced specificity protein 1 (SP 1),kelchlike ECH associated protein 1 (Keap 1),NF-E2-related factor 2 (Nrf2) and glutamatecysteine ligase catalytic (GCLC) in the cultured human müller cells and preliminary study the antioxidation of the probucol on müller cells.Methods Primary cultured human müller cells were randomly divided into four groups:normoglycaemia group (5.5 mmol/L glucose),normoglycaemia with probucol group (5.5 mmol/L glucose+100 μmol/L probucol),hyperglycemia group (25.0 mmol/L glucose),hyperglycemia with probucol group (25.0 mmol/L glucose + 100 μmol/L probucol).Immunofluorescence staining was used to assess distribution of SP1,Keapl,Nrf2,GCLC in human Müller cells.SP1,Keapl,Nrf2 and GCLC messenger RNA (mRNA) expression was evaluated by quantitative real-time RT-PCR (qRT-PCR).Independent sample t test was used to compare the data between the two groups.Results All müller cells expressed glutamine synthetase (＞ 95％),which confirmed the cultured cells in vitro were the purification of generations of müller cells.The expressions of SP 1,Keap 1,Nrf2,and GCLC protein were positive in human müller cells.qRT-PCR indicated that SP1 (t=28.30,P＜0.000),Keap1 (t=5.369,P=0.006),and Nrf2 (t=10.59,P=0.001) mRNA in the hyperglycemia group increased obviously compared with the normoglycaemia group;GCLC (t=4.633,P=0.010)mRNA in the hyperglycemia group decreased significantly compared with the normoglycaemia group.However,SP1 (t=12.60,P=0.000) and Keapl (t=4.076,P=0.015) in the hyperglycemia with probucol group decreased significantly compared with the hyperglycemia group;Nrf2 (t=12.90,P=0.000) and GCLC (t=l 5.96,P＜0.000)mRNA in the hyperglycemia with probucol group increased obviously compared with with the hyperglycemia group.Conclusion Probucol plays an antioxidant role by inhibiting the expression of SP 1,Keap 1 and upregulating the expression of Nrf2,GCLC in müller cells induced by high glucose.
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Objective: To investigate the molecular mechanism of Salvia miltiorrhiza (SM) in the treatment of retinitis pigmentosa (RP) by interfering with the expression of characteristic genes and key protein in Müller cells (MC) based on the methods of network pharmacology and bioinformatics. Methods: Retrieval and screening of active ingredients and therapeutic targets of SM in blood was performed by Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). Differentially expressed genes of MC in normal and RP mice were obtained by searching GEO database. RP-related gene targets were retrieved through disease database. Cytoscape was used to construct protein-protein interaction networks of differentially expressed MC genes, disease targets and component targets and the intersection was extracted. Gene Ontology and KEGG signaling pathway analysis of characteristic genes were carried out by DAVID. CytoHubba was used to analyze and screen the key protein targets. Results: A total of 202 chemical constituents related to SM were retrieved, 65 active ingredients were screened according to ADME parameters, of which 13 were active ingredients in blood. A total of 117 possible targets were obtained by further searching and matching. A total of 242 differentially expressed genes in MC of normal and RP mice were obtained from chip data. A total of 206 targets closely related to RP were obtained from disease databases. A total of 85 characteristic genes of SM affecting MC in RP pathological process were extracted and intersected. These genes were mainly involved in transcriptional regulation, apoptotic signaling pathway regulation, DNA nuclear replication regulation and other biological processes. Molecular functions mainly include transcriptional coactivator activity, protein kinase activity, core promoter binding, etc. They were enriched in nuclear, nucleoplasm, transcription factor complex, Rb-E2F complex and other regions. The signaling pathways involved include splicer signaling pathway, actin cytoskeleton signaling pathway, cell cycle signaling pathway and so on. A total of eight key protein targets of SM on MC in RP pathological process were analyzed and screened. Conclusion: The substance basis of the pharmacodynamics of SM is 13 chemical constituents, such as cryptotanshinone, luteolin, tanshinone IIA, etc. The MC characteristic genes involved in the pathological process of RP intervened by SM are related to spliceosome signaling pathway, actin cytoskeleton signaling pathway, cell cycle regulation pathway, etc. The key targets include eight protein such as RB1, E2F1, TFDP1, etc.
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In mammalian retina,Müller cells are dominating macroglial cells and span the entire retina.These cells perform a variety of physiological roles to maitain the normal function of retina.However,Müller cells become ‘reactivity'in response to every pathological changes in the retina.Reactive Müller cells play an important role in retinal damage and repair.Reactive gliosis is a complex process that is considered to represent a cellular response to protect the retina from further damage and to promote its repair following pathological insult in the early stage of retina injury.Reactive Müller cells protect the tissue and preserve tissue function by releasing neurotrophic factors,and may contribute to retinal regeneration by generating neural progenitor.However,continued proliferation of Müller cells can also lead to cell dysfunction and damage of photoreceptors and neurons.What's more,Müller cell gliosis may result in the formation of glial scars,which can inhibit retinal remodeling and reprograming of the injured retina.A better understanding of the role and mechanism of Müller cells in retinopathy is essential for the efficient therapeutic strategies of retina diseases.
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@#AIM: To investigate the protective effects of berberine on Sprague-Dawley(SD)rat retinal Müller cells with high concentration glucose <i>in vitro</i>.<p>METHODS: The retinal Müller cells of SD rats were primary cultured by enzyme digestion. The second generation of Müller cells were randomly divided into 7 groups. They were normal glucose concentration(5mmol/L glucose)group, high glucose concentration(25mmol/L glucose)group, high glucose+ berberine group(5, 10, 25, 50 and 100μmol/L). After cultured for 24h, 48h and 72h, the cell proliferative viability was measured by CCK-8 method.<p>RESULTS: After cultured for 24h, 48h and 72h, compared to the normal glucose concentration group, the absorbance of cells in the high glucose concentration group reduced significantly(All <i>P</i><0.01). Compared to the high glucose concentration group, the absorbance of cells in different concentration berberine(10, 25, 50 and 100μmol/L)groups increased significantly(All <i>P</i><0.05). It showed a dose-dependent effect. There was no statistically significant difference on the cells absorbance between high glucose+5μmol/L berberine group and high glucose group(<i>P</i>>0.05).<p>CONCLUSION: Berberine could reduce the inhibitory effect of high glucose on the proliferative viability of Müller cells to some extent. The intensity of effect was positively correlated with the berberine concentration.
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Objective To evaluate the effect of adenosine receptor antagonist SCH442416 on the expression of Kir2.1,Kir4.1 and TASK-1 in rat Müller cell at an elevated hydrostatic pressure in vitro.Methods Thirty SPF Sprague Dawley rats were purchased from Shanghai Slack Laboratory Animals Ltd.Cultured Müller cells were divided into normal control group (n =6),40 mmHg/24 hours (1 mmHg =0.133 kPa) group (n =6) and adenosine + SCH442416 intervention group (n =6).Müller cells were treated with 40 mmHg pressure for 24 hours in 40 mmHg/24 hours group,and Müller cells were treated with 40 mmHg pressure for 24 hours + 10 μ mol/L adenosine + 100 nmol/L SCH442416 in adenosine + SCH442416 intervention group.The real-time PCR,Western blot,whole-cell patch-clamp recordings and immunohistochemistry were used to detect Kir2.1,Kir4.1 and TASK-1 expression and Müller cells Kir currents.The experimental procedures were in accordance with the National Institutes of Health (NIH) guidelines for the Care and Use of Laboratory,and follow the 3R principle.Results Western blot assay showed that,following 40 mmHg pressure cultured for 24 hours,the expression of Kir4.1 and TASK-1 protein in Müller cell were significantly decreased by 38.6％ and 52.6％ compared with the normal control group,with significant differences between the two groups (both at P =0.000);Kir2.1 protein expression decreased by 14.7％,with insignificant difference between the two groups (P =0.082).Kir4.1 and TASK protein expression in adenosine + SCH442416 intervention group was increased by 60.7％ and 61.4％ compared with the 40 mmHg/24 hours group,with significant differences between the two groups (both at P =0.000);Kir2.1 protein expression in adenosine + SCH442416 intervention group was increased by 8.8％ compared with the 40 mmHg/24 hours group,with insignificant difference between them (P =0.354).Real-time PCR assay showed that,following 40 mmHg pressure cultured for 24 hours,Kir2.1,Kir4.1 and TASK-1 mRNA expression in Müller cells were significantly decreased compared with the normal control group,with significant differences between the two groups (P =0.047,0.001,0.000);Kir4.1 and TASK-1 mRNA expression in Müller cells in the adenosine + SCH442416 intervention group was significantly increased compared with the 40 mmHg/24 hours group,with significant differences between the two groups (P =0.038,0.030);however,there is no significant change in Kir2.1 mRNA expression (P =0.612).Conclusions SCH442416 upregulates the expression of Kir4.1 and TASK-1 mRNA and protein,but weakly affects the expression of Kir2.1.
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Objective To observe the effect oftert-Butylhydroquinone (tBHQ) on the expression of nuclear factor erythroid 2-related factor 2 (Nrf2),heme oxygenase (HO)-1 and phosphatidylinositol 3-kinase (PI3K) in high glucose cultured retinal Müller cells;and to investigate the anti-oxidative stress and anti-apoptotic effects oftBHQ.Methods Retinal Müller cells were divided into normal glucose group (5.5 mmol/L,N group),high glucose group (45 mmol/L,HG group) and tBHQ intervention group (HG+tBHQ group).After retinal Müller cells were cultured with high glucose for 48 hours,the pretreatment with tBHQ (20 μmol/L) induced the expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) and HO-1.The Müller cells were identified by immunofluorescence staining.The expressions of Nrf2,HO-1,PI3K,B-cell lymphoma-2 (Bcl-2) and Bax were detected by Western blot and real-time fluorescence quantitative PCR.Flow cytometry was used to detect the apoptosis of retinal Müller cells in rats.Results Müller cytoplasm and nucleus GS showed strong positive,large cell body,abundant cytoplasm,uniform green fluorescence;nuclear DAPI staining round or oval,clear boundary.The expression of Nrf2 protein (t=4.114,P=0.006),HO-1 protein (t=9.275,P=0.000),Nrf2 mRNA (t=7.292,P=0.000) and HO-1 mRNA (t=15.014,P=0.000) in the HG group were higher than those in the N group.The expressions of Nrf2 protein (t=7.847,P=0.000),HO-1 protein (t=7.947,P=0.000),PI3K protein (t=5.397,P=0.002),Bcl-2 protein (t=6.825,P=0.000),Nrf2 mRNA (t=18.046,P=0.000),HO-1 mRNA (t=39.458,P=0.000),PI3K mRNA (t=4.979,P=0.003) and Bcl-2 mRNA (t=9.535,P=0.000) in the HG+tBHQ group were significantly higher than those in the HG group.The protein and mRNA expressions of Bax protein in the HG+tBHQ group were significantly lower than those in the HG group (t=14.998,16.520;P=0.000,0.000).Flow cytometry showed that the apoptosis rate of Müiller cells in the HG group was significantly higher than that in the N group (t=39.905,P=0.000).The apoptosis rate of Müller cells in the HG+tBHQ group was significantly lower than that in the HG group (t=21.083,P=0.000).Conclusion tBHQ can inhibit the apoptosis of retinal Müller cells by up-regulating the expression ofNrf2,HO-1 and PI3K.
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The retinal neurons can not repair or regenerate once they are injured.Repairing the injured neurons by stem cell therapy is a hotspot in current study field.Endogenous stem cells exist in retina,which have the potential to restore retinal neural cells.Stimulating the endogenous retinal stem cells and using them to repair the injured retina has important research value and broad application prospects.It draws attention from neurobiology and ophthalmology researchers all over the world.The retinal regeneration ability of fish and amphibians is strong,while it is quite limited in birds and mammals.The retina regrowth character varies in different species.Ciliary marginal zone,retinal pigmented epithelial cells,and Müller cells are all potential cell sources for retinal regeneration.In fish,birds and mammals,the regenerated retinal neurons derived from Müller cells,while in amphibians they derived from RPE cells.The endogenous retinal stem cells need to be activated before they generated into retinal neurons.Researchers reported several methods to activate those cells,including using excitatory amino acid,growth factor,transcription factor,and intracellular signals.Here we reviewed the recent advances about retinal regeneration in different species including fish,amphibians,birds,and mammals;also the different source of endogenous retinal stem cells,including ciliary marginal zone,retinal pigmented epithelial cell,and Müller cell.Also,we reviewed the activation factors which could activate the endogenous retinal stem cells to proliferate and differentiate.
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Objective:To observe the effects of different doses of advanced glycosylation end products ( AGEs ) on bFGF expression of cultured rabbit M üller cells in vitro.Methods:Immunocytochemistry and transmission electron microscopy methods were used identified cultured M üller cell.Immunocytochemistry method was used to semi-quantitate bFGF expression of retinal Müller cells at 640 μl/2 000 μl AGEs conditions.We observed effects of AGEs and PKC inhibitor Calphstion C on bFGF mRNA expression .Results:640 μl/2 000 μl AGEs stimulate bFGF expression of retinal Müller cell.Calphostin C inhibits bFGF mRNA increase stimulated by AGEs,and inhibition achieves strongest at concentration 50 nmol/L.Conclusion:AGEs can stimulate bFGF expression of Müller cell to exert the role of angiogenesis .bFGF mRNA expression may be regulated by activation of PKC pathway .
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Background Intravitreal injection with triamcinolone acetonide (TA) may cause complications,including increase of intraoculapressure (IOP),cataracand endophthalmitis.Ketorolatromethamine (Ketorolac) inew,lesadverse reactionof non-steroidal anti-inflammatory drug.The action mechanism of Ketorolaisimilato TA.Therefore,Ketorolamay be completely opartly replace Tin the treatmenof retinal edema.Objective The purpose of thistudy wato investigate the effectof Tand Ketorolaon the expressionof aquaporin-4 (AQP4) and vasculaendothelial growth facto(VEGF) in hypoxiretinal Müllecellin vitro and to explore the mechanism of treating retinal edemwith Tand Ketorolac.MethodThe propose of research and use of the animalwere approved by Animal ExperimenResearch Review Committee of Hubei University of Medicine.Twenty eyeof New Zealand albino rabbitwere extracted and the retinal tissue waisolated.The Müllecellwere cultured and passaged using the enzymatidigestion method and Müllecellwere identified using glial fibrillary acidiprotein (GFAP),vimentin and α-smooth muscle actin (α-SMA) by immunofluorescence staining.The hypoxicell modelwere established by culturing the cellin DMEM with 500 μmol/L CoCl2 fo0,6,12,24 hours.The cellof hypoxifo24 hourwere divided into normal control group,hypoxicontrol group,hypoxia+50,100,200 mg/L To50,100,200 mg/L Ketorolagroups.Corresponding drugwere added into the medium in the differengroups.The expressionof AQP4 mRNand VEGF mRNin Müllecellwere detected by semi-quantitative reverse transcription PC(RT-PCR).ResultThe cellgrew well and reached 80％ confluence with the irregulashape and ovoid nuclei 14-15 dayaftecultured.More than 95％ primary cellshowed positive reaction to GFAP,vimentin and α-SMA.The expressing levelof AQP4 mRNand VEGF mRNin Müllecell(values) were significantly differenin varioutime point(AQP4 mRNA:F=18.70,P＜0.01 ; VEGF mRNA:F =53.20,P＜0.01),and those of 6,12 and 24 houraftecultured with CoCl2were increased than those withouCoCl2 (P＜0.05).The expressing levelof AQP4 mRNand VEGF mRNin Müllecell(values) were significandifferenamong the normal control group,hypoxicontrol group,hypoxia+50,100,200 mg/L ToKetorolagroup(AQP4 mRNA:F =27.98,P ＜ 0.01 ; VEGF mRNA:F =10.03,P ＜0.01).Compared with the hypoxicontrol group,the expressing levelof AQP4 mRNand VEGF mRNin the Müllecellwere declined in the hypoxia+ 100,200 mg/L Tgroup and the hypoxia+100,200 mg/L Ketorolagroup (P＜0.05).The expressing levelof AQP4 mRNand VEGF mRNwere found statistically insignificandifference between hypoxia+ 100 mg/L Tgroup and hypoxia+ 100 mg/L Ketorolagroup,obetween hypoxia+ 200 mg/L Tgroup and hypoxi+200 mg/L Ketorolagroup (P＞ 0.05).ConclusionTand Ketorolacan downregulate the expressionof AQP4 and VEGF in Müllecellundehypoxiconditions,inferring thathey have similamechanism in the impacon AQP4 function in retinal edematoueye.
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Background Research showed that exposure of 530 nm monochromatic light can induce myopia in animal,and retinal Müller cells participate in the formation of myopia.However,the effect and mechanism of retinal Müller cells during the formation of monochromatic light induced-myopia is below understood.Objective This study was to investigate biologic characteristics of rat retina Müller cells and the expression of cell factors in Müller cells after being illuminated by the 530 nm monochromatic light,and discuss the role of the retina Müller cells in myopia induced by monochromatic light.Methods Immortalized rat retinal Müller cells were cultured with DMEM containing 10％ fetal bovine serum in a self-made cell incubator with monochromatic light by adjusting luminance of 530 nm LED source.The cells were exposed to 125,250 and 500 lx luminance respectively for 6,12 and 24 hours,and the cells without light-irradiation were used as control.The growth of the cells under the different light time and different illuminations was described by MTT as the absorbance at the wavelength 570 nm (A570),and cell cycle analysis of Müller cells was performed by flow cytometry 48 hours after cultured,and the expression of transforming growth factor-beta 1 (TGF-β1),tyrosine hydroxylase(TH),inducible nitric oxide synthase(iNOS) and basic fibroblast growth factor(bFGF)in the cells were detected by reverse transcription PCR(RT-PCR),respectively.Results The Müller cells were uniform in size with polygonal shape and defined edges.No statistically significant difference was found in the A570 value in the cells of the 125 lx and 250 lx illuminated groups compared with the control group in various time points(P＞0.05).However,significant lowing was seen in the A570 value in the cells of the 500 lx illuminating for 12 hours and 24 hours in comparison with the control group (P =0.013,0.001).Compared with the control group,the ratio of the number between G2 and G1 phase was not significantly declined in 125 lx,250 lx illuminating for 48 hours (P =0.073,0.330),and the ratio in the 500 lx illuminating group was significantly lower than those in the 250 lx illuminated group and the control group (P =0.028,0.038).RT-PCR revealed that the expression of TGF-β1 mRNA in the cells was higher in the 250 lx illuminated group than that of the 500 lx illuminated group (P=0.006).The expression of iNOS mRNA was gradually upregulated in the 250 lx illuminated group compared with the control group (P =0.001),but that in the 500 lx illuminated group was downregulated (P =0.000).The expression of bFGF mRNA was raised in the 125 lx and 250 lx groups but reduced in the 500 lx group when compared with the control group(P=0.002,0.000,0.005).Also,the expression of TH mRNA was significantly increased in the 250 lx group(P=0.000),but decreased in the 500 lx group(P=0.000,P=0.001).Conclusions The monochromatic light of 530 nm can inhibit the growth of rat Müller cells and downregulate the expression of myopia-related cell factors and therefore exert effect in the formation of myopia.
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Background Retinal Muller cells participate in the pathological process of diabetic retinopathy (DR) through expressing vascular endothelial growth factor (VEGF).It is reported that FK506 inhibits the expression of VEGF in solid tumors and experimental corneal neovascularization,but whether FK506 exerts its role on retinal Müller cells or not is still unclear.Objective This study aimed to investigate how FK506 affects the expression vascular endothelial growth factor (VEGF) in rat retinal Müller cells under the condition of high glucose.Methods Immortalized rat retinal Müller cell line was regularly cultivated and logarithmic phase of cells were incubated in 96-well plate with the cell density of 1 × 104/ml.Different concentrations of FK506 (800.00,400.00,200.00,100.00,75.00,50.00,25.00,12.50 and 6.25 pg/ml) (100 μl/well) were added into the culture medium to determine the half maximal inhibitory concentration (IC50) of FK506 by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).The cell lines were cultured with DMEM medium (containing D-glucose of 5.5 mmol/L) or high glucose DMEM (containing D-glucose of 50 mmol/L),and 75 pg/ml FK506 were added into DMEM,respectively,and the cells were divided into the normal control group,FK506 group,high glucose culture group and high glucose + FK506 group.ELISA was employed to assay the content of VEGF protein in the cell supernatant.The expressions of VEGF mRNA and protein in the cells were detected by reverse transcription PCR (RT-PCR) and Western blot,respectively.Results The cells grew well in the normal control group,FK506 group,high glucose culture group and high glucose+FK506 group in 12,24 and 48 hours after culture with the polygon-like shape.The IC50 of FK506 was 75 pg/ml.The contents of FK506 in the cell supernatant were (966.46± 13.59) pg/ml,(1 059.42±67.43) pg/ml,(16 243.11 ±3 926.38) pg/ml and (9 467.25± 1 525.56) pg/ml in the normal control group,FK506 group,high glucose culture group and high glucose+FK506 group,respectively,showing a significant difference among the four groups (F =20.51,P =0.00).The VEGF levels in cell supernatant were significantly higher in the high glucose group than those of the normal group and the high FK506 group (P =0.00,P =0.02),but no significant difference was found in the VEGF level in cell supernatant between the control group and FK506 group (P =0.08).The expressions of VEGF mRNA and protein in the cells were significantly different among the four groups (F=126.06,P=0.00;F=5.44,P=0.01),and the relative expressing values of VEGF mRNA and protein in the cells of the high glucose group were significantly higher than those of the control group and the high+ FK506 group (all at P＜0.01).The relative expressing values of VEGF mRNA and protein were 0.64±0.09 and 0.68±0.18 in the FK506 group,which were lower than those of the normal control group (0.84±0.07 and 0.75± 0.03).However,no significant differences were seen between the two groups (P =0.05,0.07).Conclusions The expression of VEGF in rat retinal Müller cells up-regulates under the high glucose condition.FK506 plays inhibitory effects on VEGF expression to certain extent in vitro.
ABSTRACT
Background Oxidative damage plays an important role in pathogenesis of age-related macular degeneration( AMD ),and its mechanism is the destroy of blood-retinal barrier.Müller cells is a primary component to stabilize the inner barrier of the blood-retina.Researches showed that epidermal growth factor(EGF) can promote the proliferation and migration of animal Müller cells,but less study was found in the effect of EGF on human Müller cells. Objective The present study was to investigate the effects of EGF on the proliferation and migration of human Müller cells and its molecular mechanism. Methods Human Müller cell line MIO-M1 cells were cultured and incubated,and cultured cells were identified using glial fibrillory acidic protein (GFAP),factor Ⅷ,α-smooth muscle actin( α-SMA ),keratin and S-100.Different concentrations of EGF( 0,1,10,30,100 mg/L)was added in freeserum DMEM,and the positive rate of the cells was calculated using 5-bromo-2-deoxyuridine(BrdU) method.The cells were divided into EGF group,H2 O2 group,EGF + H2 O2 group,glucose oxidase ( GO ) group,GO + EGF group,EGF + LY294002+H2O2 group according to the different intervention,and the effects of LY294002 on the proliferation of Müller cells (A590 )were detected by colorimetric assay for cellular growth and survival( MTT assay).The scratch test of Müller cells was used to assess the influence of EGF(0,1,10,30,100 mg/L)on H2 O2-induced damage of human Müller cell.Western blot was used to detect the cell proliferation under the protection of EGF on co-cultured cells using LY294002 and H2O2 and the activation of Akt signal pathways. Results The proliferative rates of the cells were 28.0％,32.9％,39.0％ in 10,30,100 mg/L EGF groups respectively and obviously higher than those in 0,1 mg/L EGF groups (24.5 ％,26.2 ％ ).Under the H2O2 culture,GO culture,respectively,the A570 value of the Müller cell in high concentrations of EGF groups was significantly increased in comparison with lower concentrations EGF groups with the statistical significance among the groups( F=23.582,P=0.000).Compared with EGF+H2O2 group,the A570value of the Müller cells was lowed in EGF+LY294002+H2O2 group.The maximum migration rate of Müller cells was found in 10 mg/L EGF group.Western blot revealed that the presence of H2O2 reinforced the expression of Akt in Müller cells,however,pretreatment with 100 mg/L EGF antagonized the harmful effect of H2O2 on Müller cells.Meanwhile,pretreatment with EGF and LY294002 reduced the expression of Akt in Müller cells. Conclusions EGF can induce the proliferation and migration of human Müller cells with the strongest effect in 10 mg/L.100 mg/L exogenous EGF has a stronger protection to the Müiller cells against H2O2-induced cell damage by activating the PI3KAkt cell survival pathway.
ABSTRACT
Background Retinal Müller cells can offer nutrient and maintain the normal structure of retina.Researches showed that the abnormality of Müiller cells leads to retinal vascular disease.To explore the effect of high glaucoma on retinal Müller cells is of a very important significance for the study on diabetic retinopathy (DR).Objective This study was to investigate the effects of different concentrations of glucose on retinal Müller cells in vitro.Methods Retinal tissue was isolated from 1 10-day-oM clean SD rat.Mtiller cells were cultured by explant culture method and passaged in DMEM containing 20％ fetal bovine serum.The third generation of cells were obtained and identified using glial fibrillary acidic protein (GFAP) staning.Then,5.5,30.0 and 40.0 mmol/L glucose were added into the culture medium for 4 days respectively.The proliferation (A570) of Müller cells was detected by MTT,and apoptosis rate of Müller cells was calculated by flow cytometer to evaluate the effects of 5.5,30.0 and 40.0 mmol/L glucose to cell vitality.Results Cultured and passaged cells grew well with the spindle shape.The positive reactive cells were ＞95％ for GFAP.The A570 value of Müller cells was 0.24±0.01,0.21±0.03 and 0.20±0.02 in 5.5,30.0 and 40.0 mmol/L glucose group respectively,showing a significant difference among the three groups(F=6.755,P＜0.05).Compared with 5.5 mmol/L glucose group,As70 values were significantly lower in 30.0and 40.0 mmol/L glucose group (q =0.645,0.486,P ＜ 0.05).Apoptosis rates of Miiller cells were (26.40 ±0.25)％,(30.19±0.16)％ and (36.23±0.19)％ in 5.5,30.0 and 40.0mmol/L glucose groups,with a significant difference among them (F =294.530,P＜0.05),and those in 30.0 and 40.0 mmol/L glucose groups were significantly reduced in comparison with 40.0 mmol/L glucose group (q =0.754,0.484,P ＜ 0.05).Conclusions High concentration of glucose inhibits the viability and promote the apoptosis of retinal Müller cells at a concentrationdependent manner.