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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 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 investigate the status of the use of angiotensin Ⅱ receptor blocker(ARB)in the outpatient of our hospital,and provide reference for the clinical rational drug use. METHODS:Totally 2 460 prescriptions of hypertension treated by ARB drugs in the outpatient of our hospital in 2013 were statistically collected,and the use of such drugs were comprehensively an-alyzed. RESULTS:The ratio of ARB in the treatment of hypertension in the outpatient of our hospital in 2013 was 36.22%,and the common ARB drugs were Irbesartan tablets,Irbesartan and hydrochlorothiazide tablets,Valsartan capsules and Telmisartan tab-lets. The prescriptions of hypertension in our hospital were mostly two-drug combination,the common combination of ARB drugs were ARB+calcium channel blocker(CCB)and ARB+potassium-sparing diuretics;the three-drug combination of ARB drugs were ARB+β-receptor blockers (β-RB)+CCB and ARB+CCB+potassium-sparing diuretics. Among 2 460 prescriptions of hypertension treated by ARB drugs,1 103 prescriptions were irrational,accounting for 44.84%;77 prescriptions were ARB combined with po-tassium-sparing diuretics with no clear indication,accounting for 6.98%;39 prescriptions were ARB combined with ACEI with no clear indication,accounting for 3.54%;330 prescriptions were ARB combined with β-RB with no clear indication,accounting for 29.92%;617 prescriptions had inappropriate frequency of ARB use,accounting for 55.94%;and 40 prescriptions were other irra-tional use,accounting for 3.63%. CONCLUSIONS:ARB drugs are commonly used for hypertension patients in outpatient of our hospital. However,there are still many problems of irrational use in clinical treatment. Therefore,hospital should take appropriate interventions to promote the rational drug use through the joint efforts of physicians,pharmacists and hospitals.
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BACKROUND:Corneal hemangiogenesis occurs in 40%-60%patients after keratoplasty.Blood vessel is one of the high risk factors for corneal immunological rejection.To inhibit corneal hemangiogenesis would prolong the survival time of the grafts and promote the successful rate of the keratoplasty.OBJECTIVE:To explore the inhibitive effects of doxycycline on corneal angiogenesis after keratoplasty.DESIGN,TIME AND SETTING:A randomized controlled animal experiment was performed at the State Key Laboratory of Ophthalmology(No.2006DA105054),Zhongshan Ophthalmic Center,Sun Yat-sen University from March to August 2007.MATERIALS:A total of 48 healthy dean Sprague Dawley rats served as recipients(right eye)and 24 Wistar rats as donors(both eyes).CD31-PEfluorescent antibody was obtained from Sigma,USA.Sandwich enzyme-linked immunosorbent assay(ELISA)kit for vascular endothelial growth factor(VEGF)was brought from RapidBio,USA.METHODS:Corneal allogenic transplantation models were established in rats.Recipients were equally and randomly divided into 2 groups:saline control group and doxycycline group.Twenty minutes prior to surgery,mydriasis was performed using 1%atropine,with a diameter of 2.75 mm of implant and 2.5 mm of implant bed.In the saline control group,conjunctiva of the right eye received saline,three times a day,following surgery.In the doxycycline group,conjunctiva of the right eye received 1%doxycycline,three times a day,till 30 days following surgery.MAIN OUTCOME MEASURES:The following parameters were measured:corneal angiogenesis using immunofluorescence,expression of VEGF protein by using ELISA.RESULTS:Compared with the survival time of saline control group[(9.67±2.73)days],the mean survival time of doxycycline group[(20.67±3.01)days]was significantly prolonged(P＜0.01).The mean percentages of neovascularized corneal area in the saline control group were(4.00±1.00)%,(14.33±4.04)%,(31.33±3.51)%at 3,7 and 14 days after keratoplasty,respectively.The mean percentages of neovascularized corneal area in the doxycycline group were(1.67±1.15)%,(4.67±1.53)%,(18.33±1.53)%at the same time point respectively.Compared with the saline control group,the mean percentages of neovascularized corneal area of the doxycycline group was significantly reduced at 7 and 14 days after keratoplasty(P ＜0.05).The expression of VEGF in the saline control group was(541.00±75.44)pg/mg,(960.00±90.14)pg/mg,(976.00±130.41)pg/mg at 3,7 and 14 days after keratoplasty,respectively,while expression of VEGF in the doxycycline group was(115.33±9.29)pg/mg,(239.00±41.62)pg/mg,(361.00±65.20)pg/mg,respectively.The difference of VEGF expression at all time points between the two groups was significant (P＜0.05).CONCLUSION:Doxycycline has a significant effect in inhibiting corneal angiogenesis and prolonging survival time of implants after keratoplasty.
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BACKGROUND: Cervical lymph nodes are draining region of cornea. It is believed that aqueous fluid goes through a minor pathway named uveoscleral drainage, which will allow passage of antigen-presenting cells (APC) directly to the draining lymph nodes and induce allograft rejection after keratoplasty.OBJECTIVE: To explore the inhibitory effects of cervical lymphadenectomy in alkali induced high-risk corneal transplantation.DESIGN: A randomized controlled animal experiment.SETTING: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University.MATERIALS: The experiment was performed in the State Key Laboratory of Ophthalmology (No. 2006DA105054), Zhongshan Ophthalmic Center, Sun Yat-sen University from May 2005 to February 2007. 144 male animals (1-2 months old) including 104 SD rats and 40 Wistar rats were provided by the animal experimental center of Sun Yat-sen University. Sandwich enzyme-linked immunosorbent assay (ELISA) kits for interleukin-2 (IL-2) and interferon-γ (IFN-γ) were brought from BioSource International company (USA). The animal treatment in the experiment was accorded with the statement in Association for Research in Vision and Ophthalmology (ARVO) for animals.METHODS: With the SD rats as recipients, and Wistar rats as donors, all rats were subjected to corneal allografting. The recipient rats were randomly divided into 4 groups (n=20): group A (control group) which underwent corneal transplantation; group B which was subjected to bilateral cervical lymphadenectomy; group C, corneal transplantation 21 days after the alkali burn injury; group D, cervical lymphadenectomy following group C. The immune rejection of grafts was evaluated by detecting the expression of IFN-γ and IL-2 using ELISA. The time when allograft rejection occurred was recorded and mean survival time (MST) was compared among the groups. The development of corneal inflammation and new vessels was examined by slit lamp microscope and histopathological examination.MAIN OUTCOME MEASURES: ①The development of corneal inflammation after corneal alkaline burns. ②MST of rats in each group following transplant. ③The expression of IL-2 and IFN-γ in grafts of each group. RESULTS: ①Normal rat cornea was transparent without inflammation or neovascularization. There were many inflammatory cells invading to stroma on day 3 after burn. Then, the inflammation of cornea resolved gradually 3 weeks after the burn, but corneal neovascularization reached the peak at that time. Corneal blood vessels regressed completely at the end of 8 weeks after the burn. ②The MST of group A, B, C, and D was (10.40±1.14), (46.30±9.46), (7.00±1.58), and (15.00±3.39) days, respectively. Compared with the group A, the MST of group B was significantly longer (P < 0.05), and the MST of grafts in group D was also significantly longer than group C (P < 0.05). ③The expression of IFN-γ and IL-2 proteins was absent in group B. Compared with group C, the expression of IL-2 and IFN-γ proteins in group D significantly decreased on days 3, 7, 10, and 14 after keratoplasty (P < 0.05). CONCLUSION: Cervical lymphadenectomy therapy can effectively inhibit corneal allograft rejection in normal and high-risk corneal beds after alkali burn injury.
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Objective To investigate the blood conservation techniques during orthotopic liver transplantation(OLT). Methods Thirty-eight patients undergoing OLT without veno-venous bypass under general anesthesia were studied. During operation, the blood conservation measures such as controlled blood loss and blood coagulation, etc. were implemented. The input and output of liquid, blood component transfusion in preanhepatic, anhepatic and reperfusion phases was analyzed. The blood samples from central vein were collected, plasma creatinine, hemoglobin and albumin were determined after anesthesia, 1 h after preanhepatic phase, 20 min after anhepatic phase and 1 h after reperfusion phase, and the central venous pressure and Sonoclot data were measured.Results The total volume of whole blood transfusion was about ( 816? 86.3) ml and that of red blood cells ( 962? 55.3) ml during operation. Two patients had no blood transfusion. The normal urinary output was maintained during preanhepatic, anhepatic and reperfusion phases. The central venous pressure at three phases was significantly decreased compared to that after anesthesia (P 0.05). The plasma creatinine was significantly increased at reperfusion phase (P