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Objective:To predict potential target genes in dexamethasone-induced open-angle glaucoma via bioinformatics technology.Methods:The GEO datasets GSE16643, GSE37474, and GSE124114 were used to analyze the differentially expressed genes by GEO2R.Gene Set Enrichment Analysis (GSEA) was performed on the differentially expressed genes between GSE37474 and GSE124114.Intersection of the three datasets were displayed by Venn diagram.The annotation and enrichment analysis of the intersection genes were performed through Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and then were compared with normal tissue in GTEx Portal database.The corresponding protein interaction network was obtained by STRING.Finally, the candidate genes were searched for their transcription factors in UCSC and JASPAR.Primary human trabecular cells were divided into dexamethasone group and control group, treated with 2 ml 500 nmol/L dexamethasone and the same amount of ethanol, respectively.The expression of BDKRB1 and TAGLN in trabecular cells was detected by Western blot.Results:Differential genes between GSE37474 and GSE124114 datasets enriched in complement and coagulation cascade by GSEA.There were 89 intersecting genes of the three datasets.These genes mainly regulated the formation of extracellular matrix by GO analysis.The gene with the highest enrichment score and collagen-containing extracellular matrix was found to be associated with fibroblasts in GTEx Portal database.ACTA2, MYL9, TAGLN, and LMOD1 were closely related in STRING protein-protein interaction network.Transcription factor SP1 in UCSC and JASPAR according to related genes, BDKRB1, NID1, MFGE8 and TAGLN.The relative expression levels of BDKRB1 and TAGLN proteins were 1.32±0.14 and 0.44±0.09 in dexamethasone group, respectively, which were significantly higher than 1.00±0.00 and 0.20±0.10 in the control group, respectively ( t=-3.61, 2.89; both at P<0.05). Conclusions:Bioinformatics analysis showed that transcription factor SP1 may play a role in human trabecular meshwork cells to myofibroblasts transition after dexamethasone treatment.
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Objective:To evaluate the influence of benzalkonium chloride (BAK) in anti-glaucoma medication on intraocular pressure and ocular surface.Methods:A Meta-analysis was performed.The literature about anti-glaucoma medications published in PubMed, EMbase, Cochrane Library from January 1, 2000 to December 31, 2016 were comprehensively searched to identify eligible studies.Randomized controlled trials (RCT) for intraocular pressure and ocular surface disease in anti-glaucoma eye drops with or without BAK in various types of glaucoma or ocular hypertension patients aged >18 years were collected.EXCEL tables were used to count the relevant data and Cochrane collaboration's tool was used to assess the risk of bias system and to evaluate the quality of studies.RevMan 5.3 and Comprehensive Meta Analysis (CMA)V3 were used to perform the Meta-analysis of all RCT.Results:Thirteen RCTs were incorporated.With BAK or without BAK, anti-glaucoma eye drops could efficiently decrease intraocular pressure, meanwhile this comparison had no significant difference ( SMD: -0.00, 95% CI: -0.063-0.063, P=0.99, I2=0%). Adverse events including conjunctival hyperemia, dry eye and keratitis between the two groups were analyzed.During the follow-up period, conjunctival hyperemia, dry eye and keratitis were the main adverse reactions.A random effect model was used for conjunctival hyperemia Meta-analysis, and the fixed effect model was used for dry eye and keratitis Meta-analysis.The combined effect values of conjunctival hyperemia, keratitis and dry eye were OR=1.163, 95% CI: 0.896-1.509, P=0.257, I2=51.6%; OR=1.15, 95% CI: 0.76-1.76, P=0.50, I2=0%; OR=1.13, 95% CI: 0.79-1.61, P=0.51, I2=40%.Systematic comparison result showed that there were no statistically significant differences in the three adverse reactions between the two groups (all at P>0.05). Conclusions:This Meta-analysis indicates that the reduction in intraocular pressure achieved by BAK-free eye drops is equivalent to the effect of eye drops with BAK, and the short term use of eye drops with BAK doesn't change the incidence of conjunctival hyperemia, keratitis and dry eye.
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Objective To study the correlation between pterygium area and the clinical manifestation and signs of primary pterygium obtained from OCULUS Keratograph.Methods A prospective case observation study was performed.Thirty-nine (55 eyes) primary pterygium patients were selected from June to September 2016 in Zhongshan People's Hospital.The area of the pterygium invaded cornea and duration of pterygium were recorded.The ocular surface condition was detected by corneal fluorescein staining.The break up time of tear film (BUT) and the gland function score were measured with OCULUS Keratograph.This study was approved by the Ethics Committee of Zhongshan People's Hospital (2015 [13]).All operations followed the Helsinki Declaration and all patients signed informed consent forms.Results The areas of pterygium invaded cornea was 2-20 mm2,the mean size was 5 (3,10) mm2;the duration of pterygium was 3-8 years,the mean duration was 5 (4,6)years;the BUT was 2.1-15.0 seconds,the mean BUT was (6.3±3.0) seconds.The mean gland function score was 2 (1,3).The area of pterygium was not significantly correlated with the duration of pteryguim (r =0.197,P =0.148),while it was negatively correlated with BUT (r=-0.711,P＜0.001) and positively correlated with the tarsal gland score (r =0.554,P＜0.001).What's more,82％ (45/55 eyes) of the patients' tear film rupture appeared firstly near pterygium's head.Conclusion OCULUS Keratograph can directly evaluate the ocular surface condition of pterygium patients in a non-contact and non-invasive method.Assessing the ocular surface damage by observing the area of pterygium invaded cornea may provide a prospective treatment for pterygium patients.
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Objective To investigate the effects of transient receptor potential melastatin (TRPM) 7 on dexamethasone (Dex)-mediated cytoskeleton remodeling in human trabecular meshwork.Methods Human trabecular meshwork cells (HTMs) were primarily cultured and the cells of generation 3 to 6 were used in this study.The expression of TRPM7 protein in the cells was located using immunofluorescence technology.Dex at the dose of 0.2 mg was added into culture medium for 4 days with the final concentration of 1×10-5,1×10-6 and 1×10-7 mol/L,respectively.Western blot assay was employed to detect the relative expression level of TRPM7 protein.Cultured cells were divided into non-transfected group,siRNA transfected group,TRPM7-siRNA1 transfected group and TRPM7-siRNA2 transfected group,and the expressions of TRPM7 protein and p-cofilin protein in the cells were assayed by Western blot method.Cultured cells were divided into normal control group,Dex-treated group,siRNA transfected group and TRPM7-siRNA transfected group,and the expression of phalloidin (a cytoskeletal protein) and Vinculin (focal adhesion protein) was detected by immunofluorescence staining.In addition,cultured cells were divided into normal control group,Dex-treated group,2-APB (a Ca2+ inhibitor) treated group,ethylene glycol tetraacetic acid (EGTA) (a calcium chelator)-treated group,TRPM7-siRNA transfected group and TRPM7-siRNA+Dex group,and the [Ca2+] i in the cells was observed by Fluo-3AM immunofluorescence staining.Western blot assay was used to detect the expression of p-cofilin in the cells.Results TRPM7 was positively expressed on the cell membrane.The relative expression of TRPM7 was gradually reduced with an increase of Dex dose (F=4.210,P＜0.05),and the expression of TRPM7 was significantly decreased in 1 × 10-5 mol/L Dex group compared with the normal control group (P＜ 0.05).Western blot assay revealed that the relative expression levels of TRPM7 in the TRPM7-siRNA1 and TRPM7-siRNA2 group were significantly lower than those of non-siRNA transfected group and siRNA transfected group (all at P＜0.05).In the Dex-treated group and TRPM7-siRNA transfected group,the cells were enlarged in size with the lessened processes in comparison with the normal control group.Immunofluorescence staining showed that the actin fiber and vinculin increased in the Dex-treated group,and more spread but depolymerized actin fiber was seen in the TRPM7-siRNA transfected group.Compared with the normal control group,the fluorescence intensity of [Ca2×] i was weak in the Dex-treated group and TRPM7-siRNA transfected group.The relative expression levels of p-confilin protein was lower in the TRPM7-siRNA transfected group than that in the siRNA transfected group (0.317 ±0.031 vs.0.092±0.071) (t =5.030,P =0.007).Conclusions Dex induces the downregulation of TRPM7 expression in HTMs.The downregulation of TRPM7 probably participates in Dex-induced cytoskeletal remodeling by causing the depolymerization of actin cytoskeleton and reduction of [Ca2+] i in HTMs.
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Objective To study the correlation between pterygium area and the clinical manifestation and signs of primary pterygium obtained from OCULUS Keratograph. Methods A prospective case observation study was performed. Thirty.nine ( 55 eyes ) primary pterygium patients were selected from June to September 2016 in Zhongshan People's Hospital. The area of the pterygium invaded cornea and duration of pterygium were recorded. The ocular surface condition was detected by corneal fluorescein staining. The break up time of tear film ( BUT) and the gland function score were measured with OCULUS Keratograph. This study was approved by the Ethics Committee of Zhongshan People's Hospital ( 2015 [ 13 ] ) . All operations followed the Helsinki Declaration and all patients signed informed consent forms. Results The areas of pterygium invaded cornea was 2-20 mm2,the mean size was 5(3, 10)mm2;the duration of pterygium was 3-8 years,the mean duration was 5(4,6)years;the BUT was 2. 1-15. 0 seconds,the mean BUT was (6. 3±3. 0) seconds. The mean gland function score was 2(1,3). The area of pterygium was not significantly correlated with the duration of pteryguim (r=0.197,P=0.148),while it was negatively correlated with BUT (r=-0. 711, P<0. 001 ) and positively correlated with the tarsal gland score (r=0. 554,P<0. 001). What's more,82％ (45/55 eyes) of the patients' tear film rupture appeared firstly near pterygium's head. Conclusion OCULUS Keratograph can directly evaluate the ocular surface condition of pterygium patients in a non. contact and non.invasive method. Assessing the ocular surface damage by observing the area of pterygium invaded cornea may provide a prospective treatment for pterygium patients.
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Background Selective laser trabeculoplasty (SLT) can increase the outflow of aqueous humor and reduce the intraocular pressure of patients with open angle glaucoma,but its mechanism is unknown.To investigate the mechanism of SLT would improve the therapeutic effect of SLT.The aqueous outflow resistance in trabecular meshwork was affected by the extracellular matrix (ECM).Matrix metalloproteinase-3 (MMP-3) and MMP-9 were closely related to ECM degradation in trabecular meshwork.Objective This study was to observe the effects of SLT on the expressions MMP-3 and-9 in human trabecular ceils in vitro.Methods Immortallized human trabecular cells were cultured with pigment particles mixed suspension for 16 hours and incubated overnight.Then the cells were irradiated with Q switch frequency doubling 532 nm Nd:YAG laser to establish SLT-effective cells with the energy of 0.2 mJ,spot diameter of 400 μm and pulse duration of 3 ns.The expressions of MMP-3 mRNA and MMP-9 mRNA in the cells were detected by fluorescence quantitative real time PCR before and 1 hour and 4,8,12,28 hours after exposure of laser.The concentrations of MMP-3 and MMP-9 in the medium were assayed using ELISA 1 hour and 4,8,12,28 hours after exposure of laser and compared between the non-irradiation group and the irradiation group.Results The relative expressing levels of MMP-3 mRNA were 1.00,1.32±0.12,2.08±0.05,2.34±0.04,2.77± 0.05 and 2.49±0.27 in the non-irradiation group and irradiation for 1 hour and 4,8,12,28 hours after exposure of laser SLT,showing a significant difference among the groups (F =15.966,P=0.007),and relative expressing levels of MMP-3 mRNA were significantly higher in various time points after laser irradiation than those of the non-irradiation group (all at P＜0.05).The relative expressing levels of MMP-9 mRNA were 1.00,0.91 ±0.10,1.27 ± 0.07,1.46±0.07,1.69±0.09 and 0.87±0.09 in the non-irradiation group and irradiation for 1 hour and 4,8,12,28 hours after exposure of SLT,which was considerably different among the groups (F =30.526,P =0.005),and significant increased values were seen in the 4,8 and 12 hours after irradiation compared with the non-irradiation group (all at P＜0.05),with highest expression in the irradiation for 12-hour group.The concentrations of MMP-3 and MMP-9 proteins in medium were significantly increased in various time points after laser exposure in comparison with the control group (all at P＜0.05).Conclusions The expressions of MMP-3 and MMP-9 in human trabercuolar cells upregulate and the secretion ability of human trabecular cells to MMP-3 and MMP-9 proteins improves in early stage of SLT in vitro.However,these procedures gradually diminish with the lapse of time.
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Background Selective laser trabeculoplasty (SLT) plays lowing-intraocular pressure (IOP)effect by irradiating pigmented trabecular cells selectively using 532 nm Nd:YAG laser.However,its affect pattern is not fully known.Objective This study was to establish a human trabecular cells (HTCs) model of SLT effect,and to observe the morphological changes of HTCs after they were exposed to different energies of SLT.Methods Immortalized HTCs were incubated in DMEM/F12 with pigmental granules for 16 hours (overnight) to prepare pigmented trabecular cells.The cells were irradiated with modified laser lens with the spot of 400 μm and emitting duration of 3 ns.The energy scale of 0.2 mJ was set for standardized energy and 0.1 mJ was used as the low energy.The morphology and ultrastructure of the cells were examined under the light microscope and transmission electron microscope 1 hour,4,8,12,24 hours after irradiation.Trypan blue staining and TUNEL fluorescence staining were used to evaluate the death and apoptosis of the cells after laser irradiation.Results The brown pigment particles were seen in cytoplasm around nuclei of trabecular cells after cultured for 16 hours.After laser irradiation,there was no obvious change in the shape of nonpigmented trabcular cells,but a 400 μm spot was found in the pigmented trabecular cells under the light microscope,and the pigmented particles released as the cell disruption.After 0.2 mJ laser irradiation,cell loss zone was seen at the center of the laser spot,and the positive cells for trypan blue and TUNEL staining were found;while the abnormal manifestations were slight after the 0.1 mJ irradiation.With the lapse of the time,the number of positive staining cells was gradually decreased.Twenty-four hours after laser irradiation,proliferative trabecular cells emerged and migrated to the center area of laser spot.Statistical analysis showed that the numbers of the positive cells for trypan blue and TUNEL staining were significantly reduced in the lower energy group in comparison with the standardized energy group at various time points (all at P＜0.001),and those of both energy groups were gradually reduced with lapse of time with the significant differences between any two time points (all at P＜0.01).Conclusions An in vitro laser effect model of HTCs can be established by exposing pigmented HTCs to SLT laser.After exposed to SLT,the gasification,necrosis-like death,apoptosis-like change appear at the laser center.The laser destroyed cells can be replaced by proliferative cells with the lapse of time.Low-energy laser irradiation cause a similar morphological change of the cells to high-energy irradiation,but the number of abnormal cells is much less.
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The cultivation of clinical skills is essential for graduate education in modern ophthalmology. In order to improve the quality of graduate education and cultivate qualified ophthalmologist, postgraduates should be trained of specific purpose, coordinate the relationship between project research and clinical work, a complete clinical training system and quality control system should be established and the construction of the tutor team should also be strengthened.
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Background Researches have demonstrated that dexamethasone (Dex) can induce the changes of the function and structure of trabecular meshwork cells,and latrunculin A (Lat A) can enhance the outflow of aqueous humour and therefore low the intraocular pressure.Objective The aim of the present study is to investigate the effects of Dex and Lat A on the expression of protein in human trabecular meshwork cells.Methods Human trabecular meshwork cells were primarily cultured in DMEM using expand culture method and the fifth generation of cells were used to this experiment.Dex and/or Lat A were added in medium as 10~(-6)mol/L Dex group(Dex treating for 24 hours),Dex+Lat A group(10~(-6)mol/L Dex+2mmol/L Lat A for 24 hours),Lat A group(2mmol/L Lat A for 24 hours) and DMEM culture group.Two dimensional gel electrophoresis(2 DE) was used to compare the protein expressions among these four groups.Subsequently protein spots with different intensity were selected for mass spectrometry analysis.Results Four gel patterns of two dimensional gel electrophoresis of human trabecular meshwork cells from Dex,Dex+Lat A,Lat A and control groups were obtained.A good isolated result for majority of proteins in human trabecular meshwork cells was found in all of the four groups.An obvious expression difference of proteins in human trabecular meshwork cells was seen among the different culture conditions.Twenty four kinds proteins were identified by GDPiMALDI TOF MS,including cytoskeleton related proteins,heat shock proteins,redox related proteins,and proteins participating in carbohydrate metabolism.The expressions of aldehyde dehydrogenase(ADLH)and Rab were increased in Lat A group and decreased in Dex group,but HSP27 and hCRMP2 showed the contrary outcome.Conclusion This study construct the pattern of protein expression in human trabecular meshwork cells by using 2 DE.Dex and Lat A impact the protein expressions in human trabecular meshwork cells.
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Filtration surgeries,especially trabeculectomy,are the most common surgeries for glaucoma.Although trabeculectomy has excellent efficacy for reducing intraocular pressure(IOP),glaucoma filtering blebs have been frequently associated with various postoperative complications including filtering bleb failure and ocular surface dysfunctions.Surgical treatments of the natural aqueous outflow system,in and around Schlemm's canal,to restore normal function without penetration of the intraocular space has been considerable interest in recent years as glaucoma surgeons continue their quest for a procedure that reduces IOP without the risk related with filtering bleb.As a result,more blebless glaucoma surgeries have developed gradually in the past years.