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ABSTRACT Purpose: As superotemporal implantation of the Ahmed glaucoma valve is not always feasible in cases of refractory glaucoma, this study examined the characteristics and surgical outcomes of cases in which the valve was implanted in a nonsuperotemporal quadrant using a modified long scleral tunnel technique. Methods: This retrospective case-control study included 37 eyes with nonsuperotemporal quadrant--Ahmed glaucoma valve implantation in Group 1 and 69 eyes with superotemporal Ahmed glaucoma valve implantation in Group 2. The demographic characteristics of these groups, surgical outcomes, including complications, further surgical interventions, and surgical success rates were compared. Surgical success was defined as an intraocular pressure not exceeding 21 mmHg, accompanied by a minimum reduction of 20% in intraocular pressure from the baseline without any additional intraocular pressure-lowering procedures, and the absence of light perception loss or phthisis bulbi. Results: Group 1 had significantly higher numbers of eyes with secondary glaucoma and preoperative surgical procedures than Group 2 (p<0.05). Both groups had mean preoperative intraocular pressure values, and mean intraocular pressure values at the last visit of 34.2 and 27.9 months, 35.5 ± 1.5 and 35.8 ± 1.2 mmHg, and 14.5 ± 5 and 14.9 mmHg, respectively. Although both groups had 70.2% and 75.8% as their five-year cumulative probability of success, respectively, the rates of complications, revisional surgery, and additional surgical procedures did not differ significantly (p>0.05). Conclusion: The modified long scleral tunnel technique for Ahmed glaucoma valve implantation in nonsuperotemporal quadrants achieves intraocular pressure control and complication rates comparable to superotemporal implantation.
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Objective:To study the effect and mechanism of angiotensin type 1 receptor (AGTR1) blocker olmesartan (OMS) on the apoptosis of human Tenon capsule fibroblasts (HTF).Methods:Tenon capsule tissues were obtained from patients during strabismus surgery in the Second Affiliated Hospital of Xi'an Jiaotong University.Primary HTF were cultured by explant culture.Primary cells were identified by vimentin immunofluorescence staining and flow cytometry.The fibrosis model of HTF was established using 10 ng/ml transforming growth factor-β2 (TGF-β2). The cells were divided into normal control group cultured in culture medium, TGF-β2 group in culture medium containing TGF-β2, TGF-β2+ OMS group in culture medium containing TGF-β2 and OMS, and OMS group in culture medium containing OMS, and were cultured for 48 hours.Cell apoptosis was detected by flow cytometry with annexin V/PI staining.The early apoptosis, late apoptosis, and total apoptosis rates were analyzed.The protein expression of procaspase-9, cleaved caspase-9, bax and bcl-2 in the mitochondrial apoptosis pathway was detected by Western blot.The activity of lactate dehydrogenase (LDH) and superoxide dismutase (SOD) was detected by colorimetry.The study protocol was approved by the Ethics Committee of the Second Affiliated Hospital of Xi'an Jiaotong University (No.2019-014).Results:Primary HTF were successfully isolated and cultured.The cultured cells were long spindle-shaped and positive for vimentin.The expression rate of vimentin in the primary cells was greater than 99%.A statistically statistical difference was found in the early apoptosis rate, late apoptosis rate, and total apoptosis rate among the four groups ( F=24.92, 3.96, 41.82; all at P<0.05). The early and total apoptosis rates were significantly higher in TGF-β2+ OMS group than normal control group and TGF-β2 group, and the late apoptosis rate in TGF-β2+ OMS group was significantly higher than that of normal control group (all at P<0.05). There were statistically significant differences in cleaved caspase-9/procaspase-9, bax, and bax/bcl-2 among the four groups ( F=4.40, 7.98, 4.61; all at P<0.05). The bax/bcl-2 expression was significantly increased in TGF-β2+ OMS group in comparison with normal control group, and the expressions of cleaved caspase-9/procaspase-9, bax, and bax/bcl-2 were significantly elevated in TGF-β2+ OMS group compared with TGF-β2 group (all at P<0.05). LDH activity in the normal control group, TGF-β2 group, TGF-β2+ OMS group and OMS group was (783.99±79.97), (913.16±196.86), (2 529.06±240.21), and (2 134.29±138.96) μmol/(min·L), respectively, showing a statistically significant difference ( F=24.95, P<0.05). Compared with normal control group and TGF-β2 group, LDH activity in TGF-β2+ OMS group was increased, and the differences were statistically significant (both at P<0.05). SOD activity in the normal control group, TGF-β2 group, TGF-β2+ OMS group and OMS group was (50.35±0.97), (41.61±4.56), (28.88±3.26), and (37.61±4.83) μmol/(min·L), respectively, showing a statistically significant difference ( F=5.71, P<0.05). SOD activity was reduced in TGF-β2+ OMS group compared with normal control group and TGF-β2 group, reduced in OMS group compared with normal control group, and the differences were statistically significant (all at P<0.05). Conclusions:AGTR1 blocker OMS can promote the apoptosis of HTF effectively.Mitochondrial apoptosis pathway mediated by bax/bcl-2/caspase-9 and oxidative stress pathway are the potential mechanisms that OMS regulates the apoptosis of HTF.
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Purpose: To assess the efficacy and clinical outcome of Tenon’s patch graft (TPG) in corneal perforation and descemetocele. Methods: In this retrospective study, medical records of 83 patients (85 eyes) who underwent TPG for corneal perforation (58, 68%) or descemetocele (27, 32%) between July 2018 and October 2021 were reviewed. Clinical examination and anterior segment optical coherence tomography (AS?OCT) were performed on every follow?up visit. Anatomical success was considered as the restoration of the structural integrity with the formation of scar and anterior chamber (AC). Results: The mean size of the corneal lesions (corneal perforation or descemetocele) was 4.20 ± 1.01 mm. The mean follow?up period was 9.2 ± 5.48 months. The common underlying etiologies were infectious keratitis in 48% and autoimmune disorders in 35% of cases. TPG successfully restored the globe integrity in 74 (87%) eyes (83% in perforation and 96% in descemetocele). Anatomical failure occurred in 11 eyes (13%). The failures were due to graft dehiscence (8 eyes), graft ectasia (1 eye), and scarring with flat AC (2 eyes). The median time to epithelialization and scar formation were 3 and 15 weeks, respectively. Logistic regression analysis showed few predictors for a successful outcome: descemetoceles, noninfective causes, viral keratitis in infectious etiology, and paracentral or peripheral lesions. Conclusion: TPG can be considered an effective and inexpensive treatment for restoring the structural integrity in the eyes with perforations and descemetoceles, particularly when the donor tissue is unavailable. AS?OCT is a valuable noninvasive tool for monitoring the graft status
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Objective:To investigate the effect of lithium chloride (LiCl) on the gap junctional intercellular communication (GJIC) in human Tenon capsule fibroblasts (HTFs) and its underlying mechanism.Methods:The Tenon capsule tissue of a patient who underwent strabismus surgery in Dezhou People's Hospital in April 2019 was collected and cut into tissue blocks of dimensions 1 mm×1 mm×1 mm.Primary culture and subculture were carried out, and the 4th-generation HTFs were taken for experiment.HTFs were divided into the control group and LiCl treatment group and were cultured with cell medium without or with 80 mmol/L LiCl for another 48 hours according to grouping.The cell scratch and dye labeling technique were used to label the coupling index and evaluate the GJIC function.The expression and localization of Cx43 in HTFs were detected by immunofluorescence staining.The expression levels of Cx43 mRNA and protein were detected by real-time fluorescence quantitative PCR and Western blot, respectively.The study protocol was approved by an Ethics Committee of Dezhou People's Hospital (No.2019-023). Written informed consent was obtained from the subject.Results:The cultured spindle-shaped HTFs grew adhering to the wall showing radial monolayer or vortexlike, and the cytoplasm was vimentin positive.Results of dye tracer experiment of cell scratch showed that the cell coupling index of LiCl treatment group was 9.04±0.53, which was significantly higher than 4.94±0.39 of the control group ( t=-18.79, P<0.01). Immunofluorescence staining showed that the Cx43 fluorescence was dotted in the cell membrane between adjacent cells in the control group, and Cx43 staining was obviously enhanced in the LiCl treatment group.The results of real-time fluorescence quantitative PCR showed that with relative expression level of Cx43 mRNA in the control group set to 1, the relative expression level of Cx43 in the LiCl treatment group was significantly increased to 1.97±0.23, showing a statistical significance between them ( t=-14.426, P<0.01). Western blot showed that the relative expression level of Cx43 protein was 0.871±0.057 in the LiCl treatment group, which was significantly higher than 0.446±0.028 in the control group ( t=-11.682, P<0.01). Conclusions:LiCl can enhance the GJIC function between HTFs by upregulating the expression levels of Cx43 mRNA and protein, suggesting that the enhanced GJIC function by LiCl may be one of the mechanisms of its inhibition on HTFs proliferation.
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@#AIM: To explore the effects of heat shock protein 47(HSP47)siRNA on biological behaviors of human Tenon capsule fibroblasts(HTCF)cells cultured <i>in vitro</i> and the expression level of transforming growth factor-β1(TGF-β1).<p>METHODS: HTCF were cultured <i>in vitro</i> and divided into blank control group, empty vector group and transfection group. In transfection group, interfering siRNA sequences were designed and synthesized based on the HSP47 gene sequences, vectors were constructed and introduced into HTCF. The empty vector group was introduced with empty vectors. The expressions of HSP47 mRNA and protein in cells were detected by RT-PCR and Western blot. The proliferation, apoptosis, invasion and migration of cells were detected by clone formation assay, flow cytometry, Transwell method and scratch test. The expressions of proliferation, apoptosis, invasion and migration proteins, and TGF-β1 were detected by Western blot.<p>RESULTS: Compared with empty vector group, expression of HSP47 mRNA and protein, clone formation rate, cell healing rate, number of invasive cells, relative expression levels of Ki67, N-cadherin and TGF-β1 were significantly decreased in transfection group(<i>P</i><0.05), relative expression level of E-cadherin protein was significantly increased(<i>P</i><0.05), but there was no difference in apoptosis rate, and relative expression levels of Bcl-2 and Bax(<i>P</i>>0.05).<p>CONCLUSION: HSP47 siRNA can reduce proliferation, invasion and migration abilities of HTCF cells by inhibiting the expression of TGF-β1 protein, without significant effects on the apoptosis of HTCF cells.
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Objective To investigate the effects of hepatocyte growth factor (HGF) on proliferation and transdifferentiation of human Tenon capsule fibroblasts induced by transforming growth factor-β1(TGF-β1) in vitro.Methods Human Tenon capsule fibroblasts were cultured and divided into blank control group,TGF-β1 treated group and different concentrations HGF+TGF-β1 groups.The TGF-β1 at 10 μg/L was added into culture medium of the TGF-β1 treated group,and different concentrations of HGF (25,50,100,200 μg/L) and 10 μg/L TGF-β1 were added into culture medium of the HGF25 μg/L +TGF-β1,HGF50 μg/L +TGF-β1,HGF100 μg/L +TGF-β1,HGF200 μg/L +TGF-β1 group respectively,Methyl thiazolyl tetrazolium (MTT) was employed to measure the cell proliferation (absorbance at 560 nm).Immunofluorescence staining was used to evaluate and locate the expression of α-smooth muscle action (α-SMA) in the cells.The expression of α-SMA protein in the cells was detected by Western blot assay.Results Cultured cells showed fusiform in shape with the positive response for vimentin.The proliferation value of the cells was 0.203±0.025,0.497 ± 0.101,0.426 ± 0.062,0.354 ± 0.040,0.272 ± 0.084,0.241 ± 0.011 in the blank control group,TGF-β1 treated group,HGF25 μg/L +TGF-β1 group,HGF50 μg/L +TGF-β1 group,HGF100 μg/L +TGF-β group and HGF200 μg/L + TGF-β1 group,respectively,showing a significant difference among the groups (F =9.21 0,P =0.003).Compared with the TGF-β1 treated group,the proliferation values of the cells were significantly reduced in the blank control group and HGF+TGF-β1 group (all at P＜0.05).Immunofiuorescence staining showed that α-SMA protein mainly located in cytoplasm with the strong red fluorescence in the cells of the TGF-β1 treated group and weak red fluorescence in HGF+TGF-β1 group,and the expression of α-SMA was absent in the blank control group.The percentage of α-SMA-positive cells was (60.0±4.7)％ in the TGF-β1 treated group and (14.3±3.1)％ in the HGF+TGF-β1 group,with significant difference between the two groups (t =19.856,P＜0.001).The relative expression levels of the α-SMA protein in the cells were 0.642 ±0.032,1.330± 0.069 and 0.884 ±0.040 in the blank control group,TGF-β1 group and HGF100μg/L +TGF-β1 group,respectively,showing a significant difference among the groups (F =13.370,P＜ 0.001),and relative expression levels of the α-SMA protein in the cells were significantly lower in the blank controlgroup and HGF100 μg/L+TGF-β1 group than that in the TGF-β1 treated group (all at P＜0.05).Conclusions HGF can inhibit the proliferation of human Tenon capsule fibroblasts,down-regulate the expression of α-SMA protein induced by TGF-β1 and arrest the phenotype transformation of fibroblasts in vitro.
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@#AIM: To investigate the effect of connective tissue growth factor(CTGF)on the proliferation and migration and transformation in Tenon's capsule fibroblasts(Tfb)of primary open angle glaucoma(POAG)patients.<p>METHODS: Tfb were cultured from Tenon's tissues in POAG patients <i>in vitro</i>. The free-serum DMEM-F12 containing 1.0, 10.0, 100.0ng/mL of CTGF was added into medium for 24h and 48h in different experimental group respectively, and only equal volume of free-serum DMEM-F12 was added in the negative control group. After 24h, the cell proliferation was analyzed through MTT assay, and migration was evaluated by crutch method. After 48h, Semi-quantitative RT-PCR was used to observed the mRNA expression of α-smooth muscl actin(α-SMA), and expression of α-SMA protein was examined by immunochemistry.<p>RESULTS: The proliferation values <i>A</i> of the cells in 1.0, 10.0, 100.0ng/mL of CTGF group respectively were 0.436±0.009, 0.643±0.009, 0.679±0.006, and 0.423 ±0.156 in the negative control group. The migrated cell number was 34.600±3.507, 70.400±2.074, 80.000±2.915 in different concentrations of CTGF group respectively, and 31.000±3.536 in the negative control group. And also in different experimental groups respectively, the absorbance ratio of α-SMA/β-actin was 0.873±0.161, 1.213±0.312, 1.352±0.376, and 0.851±0.158 in the negative control group, the expressing levels <i>A</i> of α-SMA protein in Tfb were 0.110±0.026, 0.141±0.017, 0.175±0.027, and 0.108±0.020 in the negative control group. The statistics of the above experimental data showed that, comparing with the negative control group, the 10.0 and 100.0ng/mL CTGF groups was statistically significant different(<i>P</i><0.05), but there was no statistical different between the 1.0ng/mL CTGF group and the negative control group(<i>P</i>>0.05). <p>CONCLUSION: The proliferation, migration, and phenotypic transformation of Tfb can be promoted in CTGF group in POAG patients. These findings suggest that CTGF may play a role in the development of filtering bleb scarring.
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Objective To investigate the effects of matrine on proliferation and apoptosis of human Tenon capsule fibroblasts (HTFs) in.vitro.Methods After treated with 0,0.3,0.6 and 0.9 g/L matrine in vitro,cell counting kit-8 (CCK-8) method was used to assay the proliferation of HTFs at 24,48 and 72 hours,Western blot and PCR were performed to evaluate the expression of apoptosis-associated factor caspase-3 on both protein and RNA levels.Results The activity of human Tenon capsule fibroblast at 48 hours and 72 hours after treated with 0.3,0.6,0.9 g/L matrine was significantly inhibited when compared with the 0 g/L matrine group,and the inhibitory effect was dose-dependent and time-dependent (F ion =1 019.51,P =0.00;Ftime =5 848.66,P =0.00;Fi ion =147.45,P=0.00).After treated with 0,0.3,0.6 and 0.9 g/L matrine,the early apoptosis rate of HTFs was (2.68±0.30)％,(5.08±0.47)％,(6.97±0.69)％ and (10.30±1.20)％,the grey value ofcaspase-3 protein was 1.00±0.13,1.90±0.19,2.50±0.30 and 2.67±0.30,the relative expression of caspase-3 mRNA was 0.98 ±0.12,2.01 ±0.34,6.15 ± 0.60 and 11.40 ± 1.12,respectively,with significant differences among them (F =55.74,66.01,154.50;all at P＜0.01),the early apoptosis rate of HTFs,the grey value of caspase-3 protein and the relative expression of caspase-3 mRNA were all increased significantly as the concentration of matrine increased,with significant differences between any two groups (all at P＜0.05).Conclusions Matrine can inhibit the proliferation of HTFs and induce the apoptosis of HTFs in a time-and dose-depended manner.
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PURPOSE: To investigate the role of hydrogen sulfide in the survival and collagen gel contraction of cultured human Tenon's capsule fibroblasts (HTCFs). METHODS: Primarily cultured HTCFs were exposed to 0, 100, 200, or 300 µM hydrogen sulfide (sodium hydrogen sulfide, NaHS) for 2 days. Cellular survival was assessed by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay. Degree of apoptosis was assessed with flow cytometry using annexin-V/propidium iodide double staining. To evaluate the effect of NaHS on cellular transdifferentiation, HTCFs were stimulated with 5 ng/mL TGF-β1 and the level of expression of α-smooth muscle actin (SMA) mRNA was assessed using reverse-transcription polymerase chain reaction. The cells were embedded in collagen gel, and the amount of gel contraction was measured. RESULTS: NaHS at 300 µM reduced HTCF survival (p = 0.013); NaHS at both 200 and 300 µM increased apoptosis in a dose-dependent manner (p = 0.013 and p = 0.016). TGF-β1 increased the expression of α-SMA mRNA (p = 0.041); co-treatment with 100 µM NaHS decreased TGF-β1-induced α-SMA mRNA expression (p = 0.039) and inhibited collagen gel contraction. CONCLUSIONS: NaHS at high concentration reduced cellular survival and increased HTCF apoptosis. NaHS decreased TGF-β 1-induced increases in α-SMA mRNA expression and collagen gel contraction. Thus, hydrogen sulfide may suppress scar formation by inhibiting HTCF transdifferentiation and contraction of collagen gels.
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Humans , Actins , Apoptosis , Cicatrix , Collagen , Fibroblasts , Flow Cytometry , Gels , Hydrogen Sulfide , Hydrogen , Polymerase Chain Reaction , RNA, Messenger , Tenon CapsuleABSTRACT
ABSTRACT Purpose: To evaluate the response to sub-Tenon's triamcinolone injection in patients with uveitis. Methods: We studied 28 eyes with macular edema associated with controlled uveitis. We administered sub-Tenon's injection of triamcinolone and followed the patients for 180 days to analyze the positive effects (improvement of macular edema and visual acuity) and monitor the possible adverse effects. This prospective study was conducted at the Department of Ophthalmology, Hospital de Clínicas de Porto Alegre, Brazil. Results: We observed improvement in macular edema in 86% of patients. The mean central macular thickness at each time point of assessment was 432.22, 298.80, 286.37, 267.49, 253.87, and 253.49 mm at baseline (before sub-Tenon's injection of triamcinolone), 15 days after the procedure, at 30 days, at 60 days, at 90 days, and at 180 days, respectively. The mean reduction in retinal thickness was 30.8%, 33.7%, 38.11%, 41.2%, and 41.35% at 15, 30, 60, 90, and 180 days of follow-up, respectively. Visual acuity also improved in 85.7% of patients, with a mean improvement of 1.36, 1.93, 2.23, 2.26, and 2.30 lines gained on the Early Treatment Diabetic Retinopathy Study chart at 15, 30, 60, 90, and 180 days of follow-up, respectively. No statistically significant increases in intraocular pressure and conjunctival abnormalities were caused by the procedure, and no other adverse effects were observed. Overall, the results of this study were similar to those described in the literature. Conclusions: Sub-Tenon's injection of triamcinolone provides reduced macular thickness and improvement in visual acuity with no significant adverse effects and is therefore an effective and safe procedure for the treatment of sequelae of uveitis.
RESUMO Objetivos: Avaliar os efeitos da injeção subtenoniana de triancinolona em pacientes com uveítes. Métodos: Foram incluídos na avaliação 28 olhos com edema macular associado à uveíte. Esses pacientes foram submetidos à injeção subtenoniana de triancinolona e acompanhados ao longo de 180 dias, para analisar os efeitos em relação à melhora do edema macular, da acuidade visual e acompanhamento de possíveis efeitos adversos. Trata-se de um estudo prospectivo, realizado no Serviço de Oftalmologia do Hospital de Clínicas de Porto Alegre. Resultados: Foi verificada melhora do edema macular em 86% dos pacientes, sendo uma redução média da espessura retiniana de 30,8% aos 15 dias, 33,7% aos 30 dias, 38,11% aos 60 dias, 41,2% aos 90 dias e 41,35% aos 180 dias de seguimento. Também foi observado melhora da acuidade visual em 85,7% dos pacientes e ganho de linhas na tabela de acuidade visual, sendo 1,36 linhas aos 15 dias de seguimento, 1,93 linhas aos 30 dias, 2,23 linhas aos 60 dias, 2,26 linhas aos 90 dias e 2,30 linhas aos 180 dias. Não houve significância estatística em relação ao aumento da pressão intraocular e às alterações conjuntivais causadas pelo procedimento, sem detecção de qualquer outro efeito colateral. Foi concluído que os resultados encontrados nesse estudo são similares aos descritos na literatura. Conclusões: A injeção subtenoniana de triancinolona é um procedimento eficaz e seguro para o tratamento das sequelas por quadros de uveítes, proporcionando redução da espessura macular e melhora da acuidade visual, sem relação com efeitos adversos significativos.
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Humans , Male , Female , Adult , Middle Aged , Aged , Young Adult , Uveitis/drug therapy , Triamcinolone Acetonide/administration & dosage , Anti-Inflammatory Agents/administration & dosage , Uveitis/complications , Visual Acuity , Macular Edema/etiology , Prospective Studies , Treatment Outcome , Intravitreal Injections , Intraocular PressureABSTRACT
PURPOSE: To evaluate the anti-fibrotic effects of nilotinib on the survival of cultured human Tenon's capsule fibroblasts (HTFs). METHODS: HTF primary cultures were obtained from samples following glaucoma surgery. Primarily cultured HTFs were exposed to 1, 5, 10, and 20 µM nilotinib for 24 hours. The effects of nilotinib on HTF proliferation and cell viability were determined using the 3-(4,5-dimethylthiazone-2-yl)-2,5-diphenyl tetrazolium (MTT) assay, and apoptosis was determined by flow cytometry using annexin-V/propidium iodide (PI) double staining. Apoptosis-related proteins were detected by western blotting. RESULTS: The MTT assay showed that nilotinib induced an inhibition of HTF proliferation at concentrations of 10 and 20 µM (p < 0.001 and p < 0.001, respectively). Annexin V/PI double staining showed significantly increased apoptosis in cells treated with nilotinib. Nilotinib activated caspase-3, -9, and poly adenosine diphosphate ribose polymerase cleavage, and downregulated the expression of B-cell lymphoma-extra large and Bax, which indicated that nilotinib-induced apoptosis was partly mediated through the mitochondrial pathway. In addition, treatment with nilotinib decreased the expression of α-smooth muscle actin and transforming growth factor-β. CONCLUSIONS: Nilotinib decreased cell survival of cultured HTFs and induced mitochondria-mediated apoptosis. The results suggested that nilotinib may exert antiproliferative effects on HTFs, making it a possible agent to control postoperative fibrosis in patients undergoing glaucoma surgery.
Subject(s)
Humans , Actins , Apoptosis , B-Lymphocytes , Blotting, Western , Caspase 3 , Cell Survival , Fibroblasts , Fibrosis , Flow Cytometry , Glaucoma , In Vitro Techniques , Poly Adenosine Diphosphate Ribose , Tenon CapsuleABSTRACT
Objective To investigate the effects of hepatocyte growth factor ( HGF ) on proliferation and transdifferentiation of human Tenon capsule fibroblasts induced by transforming growth factor .β1(TGF.β1) in vitro. Methods Human Tenon capsule fibroblasts were cultured and divided into blank control group, TGF.β1 treated group and different concentrations HGF+TGF.β1 groups. The TGF.β1 at 10 μg/L was added into culture medium of the TGF.β1 treated group,and different concentrations of HGF (25,50,100,200 μg/L) and 10μg/L TGF.β1 were added into culture medium of the HGF25μg/L+TGF.β1 ,HGF50μg/L+TGF.β1 ,HGF100μg/L+TGF.β1 ,HGF200μg/L+TGF.β1 group respectively,Methyl thiazolyl tetrazolium ( MTT) was employed to measure the cell proliferation ( absorbance at 560 nm) . Immunofluorescence staining was used to evaluate and locate the expression of α.smooth muscle action (α.SMA) in the cells. The expression ofα.SMA protein in the cells was detected by Western blot assay. Results Cultured cells showed fusiform in shape with the positive response for vimentin. The proliferation value of the cells was 0. 203±0. 025,0. 497±0. 101,0. 426±0. 062,0. 354±0. 040,0. 272±0. 084,0. 241±0. 011 in the blank control group, TGF.β1 treated group,HGF25μg/L+TGF.β1 group,HGF50μg/L+TGF.β1 group,HGF100μg/L+TGF.β1 group and HGF200μg/L+TGF.β1 group,respectively,showing a significant difference among the groups (F=9. 210,P=0. 003). Compared with the TGF.β1 treated group,the proliferation values of the cells were significantly reduced in the blank control group and HGF+TGF.β1 group ( all at P<0. 05 ) . Immunofluorescence staining showed that α.SMA protein mainly located in cytoplasm with the strong red fluorescence in the cells of the TGF.β1 treated group and weak red fluorescence in HGF+TGF.β1 group,and the expression of α.SMA was absent in the blank control group. The percentage of α.SMA.positive cells was ( 60. 0 ± 4. 7 )％ in the TGF.β1 treated group and ( 14. 3 ± 3. 1 )％ in the HGF+TGF.β1 group, with significant difference between the two groups (t=19. 856,P<0. 001). The relative expression levels of the α.SMA protein in the cells were 0. 642±0. 032,1. 330±0. 069 and 0. 884±0. 040 in the blank control group,TGF.β1 group and HGF100μg/L+TGF.β1 group, respectively, showing a significant difference among the groups ( F=13. 370, P<0. 001),and relative expression levels of the α.SMA protein in the cells were significantly lower in the blank control group and HGF100μg/L+TGF.β1 group than that in the TGF.β1 treated group (all at P<0. 05). Conclusions HGF can inhibit the proliferation of human Tenon capsule fibroblasts, down.regulate the expression of α.SMA protein induced by TGF.β1 and arrest the phenotype transformation of fibroblasts in vitro.
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Objective To investigate the effects of colchicine on the proliferation and apoptosis of human Tenon's capsule fibroblasts (HTCFs) in vitro.Methods HTCFs were cultured in vitro,and the subcultured cells were identified.MTS assay was used to observe the inhibitory actions of colchicine on HTCFs at different concentrations.The apoptotic rates of the cells were detected by flow cytometry.The apoptosisrelated proteins,including PARP,Caspase-9,Caspase-3 and active-Caspase-3,were detected by Western blot.Results MTS assay showed that colchicine inhibited the proliferation of HTCFs in a dose-and time-dependent manner.Flow cytometry showed that colchicine induced a dose-dependent apoptosis of HTCFs for 48 hours,the apoptotic rates of 5 μmol · L-1,10 μmol · L-1 and 20 μmol · L-1 were (18.37 ±4.26)％,(33.80 ±5.02)％ and (52.00 ± 2.00)％,respectively,there were significant differences compared with the control (F =91.59,P ＜ 0.001).Western blot showed the activation of Caspase-3 and PARP followed by colchicine treatment.Conclusion Colchicine significantly inhibits the proliferation of HTCFs in vitro,and induced apoptosis,which may be associated with the activation of mitochondrial apoptotic pathways.
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Objective To investigate the effects of artesunate (Art) on cell proliferation and apoptosis of human Tenon's capsule fibroblasts (HTFs),and discuss the countermeasures of bleb scarfing in glaucoma.Methods In vitro,HTFs were cultivated and applicated by different concentrations (50 μg · mL-1,100 μg · mL-1,150 μg ·mL-1,200 μg · mL-1) of Art for 48 hours.The effect of Art on cell proliferation was assessed by MTT method.The rate of apoptosis induced by Art was determined by flow cytometry.Western Blot was performed to detect the relative expression levels of Bax and Bcl-2 after Art was treated.Results After treated with Art for 48 hours,compared with blank control group,Art (50 μg · mL-1,100 μg · mL-1,150 μg · mL-1,200 μg · mL-1) group exhibited notable anti-proliferative effect on HTFs with concentration-dependence (all P ＜ 0.05).The results of flow cytometry showed that the apoptosis rates (8.80％ ±0.88％,11.60％ ±0.56％,16.30％ ±1.03％,23.40％ ±1.62％) of HTFs were significantly enhanced with the increase of Art concentration (all P ＜ 0.05).The relative expression levels of Bax were obviously high with the increase of Art concentration,while Bcl-2 levels were significantly low with the increase of Art concentration (all P ＜ 0.05).Conclusion Art can inhibit the proliferation and induce cell apoptosis of HTFs possibly by enhancing the expression of Bax and reducing the expression of Bcl2.Art may be a potential drug in preventing fibrous scar formation after glaucoma filtration surgery.
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AIM:To research the effects of lithium chloride on transforming growth factor beta (TGF-β) and connective tissue growth factor (CTGF) in cultured human Tenon capsule fibroblasts (HTFs) and explore its mechanism.METHODS:HTFs were cultured and identified by vimentin staining with immunofluorescence and the morphological characteristics.The experimental group was processed 48h with LiCl in concentration of 80mmol/L, the control group without LiCl.The mRNA expression of TGF-β and CTGF in two groups were analyzed with real-time fluorescent quantitative polymerase chain reaction (real time-qPCR) and the protein expression was detected with Western blot.RESULTS:The cultured HTFs expressed TGF-β and CTGF.The mRNA expression of TGF-β and CTGF significantly decreased compared with the control group(t=20.042, 14.995, P<0.05).the protein expression of TGF-β and CTGF also decreased significantly compared with the control group(t=46.058、12.452, P<0.05)CONCLUSION:The cultured HTFs can express TGF-β and CTGF in mRNA and proteins' level.LiCl can reduce the expression of TGF-β and CTGF both in gene and proteins' level.LiCl has the potential to modulate wound healing for glaucoma filtration surgery.
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Background Researches showed that transforming growth factor-β2 (TGF-β2) promotes the activity of human Tenon capsular fibroblasts (TFs),which plays a role in the scarring of filtering blebs after antiglaucoma surgery.However,its mechanism is not fully clear.Lysyl oxidases (LOXs) are important extracellular matrix proteases which can catalyze the cross-linking of collagen and elastin.Investigating the impact of TGF-β2 on the expression of LOXs has a great significance for the understanding of the pathogenesis of filtering bleb scarring and its prevention.Objective This study was to investigate the effect of TGF-β2 on the expression of LOXs in cultured human TFs.Methods The TFs at 4-8 generations were divided into normal control group and different concentrations of TGF-β2 treated-groups,and 100,200,400,800 μ1 of TGF-β2 with the final concentration of 2,4,8 and 16 ng/ml was added into the medium to treat human TFs respectively for 24 hours.The LOXs in the cells were detected by Western blot to determine the optimal dose of TGF-β2.The 4 ng/ml TGF-β2(200 μ1) was used to treat human TFs for 6,12,24 and 48 hours respectively,and the change of LOXs expression in the cells over time was assayed by Western blot.The expression and distribution of LOX protein in the normal cells and TGF-β2-treated cells was detected by using immunofluorescence technique.This study was approved by Daping Hospital of Third Military Medical University Ethic Commission.The guardians of the patients who offered the specimen knew the purpose of the study and signed informed consent.Results Western blot assay showed that the expressions of LOX,LOXL1,LOXL2,LOXL3 and LOXL4 in the cells were gradually elevated from the normal control group and 2,4,8,16 ng/ml TGF-β2-treated groups,showing significant differences among the groups (F =37.338,13.438,31.067,11.767,15.167,all at P＜0.01).The expression of LOXL2 protein in the cells was 0.68±0.07,1.09±0.10,1.32±0.07,1.50± 0.06 and 1.89±0.12 in the normal control group and 6-,12-,24-and 48-hour groups respectively after 4 ng/ml TGF-β2 treatment,with a significant increase over time (F =82.832,P=0.000).The expression of LOX was weak in the normal cultured TFs,while the fluorescence intensity of LOX expression was evidently enhanced in the cytoplasm of the cells in the TGF-β2-treated group.Conclusions TGF-β2 upregulates the expressions of LOXs in human TFs in a dose-and time-dependent manner,which probably offers a basis for the further study on the prevention of filtering bleb scarring after glaucoma surgery.
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Background Scarring of filtration channel following glaucoma filtering surgery is a main cause of the failure of the surgery.The proliferation,epithelial-mesenchymal transition and extracellular matrix remodeling of fibroblasts are thought to be the primary pathological mechanism of scarring.Connective tissue growth factor (CTGF) plays a promoting role in the formation of scar.Whether CTGF participates in mesenchymal-epithelial transition of human Tenon capsule fibroblasts (HTFs) is not clear yet.Objective This study attempted to investigate the effect of CTGF on the mesenchymal-epithelial transition of HTFs in vitro.Methods HTFs were cultured and passaged in high glucose DMEM medium with 10％ fetal bovine serum,and the cells of generation 3-6 were used in this experiment.The cells were divided into the blank control group and CTGF-treated group and were routinely cultured in the blank control group.CTGF was added in the medium in the CTGF-treated group,with the final concentration 50 ng/ml CTGF.Immunofluorescence staining was used to identify and locate the expression of E-cadherin protein in the cells,and Western blot assay was employed to quantitatively analyze the expression level of E-cadherin protein in 48 hours after culture.Results The HTFs grew well with the spindle-like shape and vortex-like arrangement.The red fluorescence (E-cadherin protein) in the cytoplasm and blue fluorescence in the cellular nucleus were seen in the CTGF-treated group,but only nucleus with blue fluorescence were obtained in the blank control group.Western blot assay showed that the E-cadherin protein expression was absent in the blank control group,however,the relative expression level of E-cadherin protein in the cells was 0.63± 0.08.Conclusions E-cadherin protein is not expressed in fibroblast derived from mesenchymal tissue.However,CTGF can induce the expression of E-cadherin in HTFs.This study suggests that CTGF promotes the mesenchymal-epithelial transition of HTFs in vitro.
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Background Studies confirmed that hydroxycamptothecin cause the apoptosis of human Tenon capsule fibroblasts (HTFs) by protein kinase R-like endoplasmic reticulum stress kinase (PERK) single pathway.Autophagy and apoptosis are programmed cell death following stress reaction,so they remain a close association.However,the effect of hydroxycamptothecin on the autophagy of HTFs and its mechanism are still unclear.Objective This study was to explore the promoting effect of PERK signal pathway on hydroxycamptothecin inducing the autophagy of HTFs.Methods This study procedure was approval by Ethic Committee of Nanjing Medical University.Human Tenon capsule tissue was obtained from fresh adult donors.HTFs were cultured and passaged by explant-culture method and identified by immunofluorescence for vimentin and keratin.pLVX-PERK lentiviral packed by 293T cells was transfected into HTFs to obtain stable PERK-knockout cell line by puromycin selection.Then the HTFs were treated with 0.10 g/L of hydroxycamptothecin for 5 minutes and consecutively cultivated for 24 hours,and the untreated cells were used as the control group.Western blot assay was used to detect the expressions of autophagy specific proteins in the cells,including autophagy related gene 5 (ATG-5),Beclin-1,light chain 3 (LC-3).Cyto-ID staining was used to identify the autophagosome in the cells.The experimental results were analyzed and compared between different treating groups.Results The gray scales for the expressions of Beclin 1,ATG-5,LC-3-Ⅰ and LC-3-Ⅱ proteins in HTFs were 0.365:±0.045,0.765 ±0.055,0.120±0.030 and 0.215 ±0.035 in the control group,and those in the hydroxycamptothecin treated group were 0.980±0.070,1.495±0.095,0.585±0.025 and 0.785±0.055,showing a significant decline in the hydroxycamptothecin treated group(P=0.018,0.022,0.007,0.013).The green fluorescence of the autophagosome was stronger in the hydroxycamptothecin treated group compared with the control group.Western blot revealed that the gray scale of PERK expression in the cells was 0.130±0.030 in the PERK-knockout group,with a significant reduce in comparison with 0.765 ±0.055 of the control group (P =0.010).However,no obvious distinctions were seen in the band intensities of the expressions of Beclin-1,ATG-5 and LC-3 proteins between the two groups.Western blot indicated that the grey scale of the PERK expression in the cells was 1.790± 0.060 in the 0.10 g/L hydroxycamptothecin group,which was significantly higher than 0.880 ± 0.070 of the control group (P =0.010).Expression levels (gray scales) of Beclin-1,ATG-5,LC-3-Ⅰand LC-3-Ⅱ in the PERK-knockout+ 0.10 g/L hydroxycamptothecin group were 0.475 ± 0.045,0.390 ± 0.040,0.055 ± 0.015 and 0.075 ± 0.025,which were significantly lowed in comparison with 0.955 ± 0.065,0.765 ± 0.055,0.155 ± 0.015 and 0.280 ± 0.030 of the control+ 0.10 g/L hydroxycamptothecin group (P =0.026,0.031,0.042,0.034).In addition,the fluorescence intensity of autophagosomes was weaker in the PERK-knockout+0.10 g/L hydroxycamptothecin group compared with the control+0.10 g/L hydroxycamptothecin group.Conclusions Hydroxycamptothecin induces the autophagy of HTFs by PERK signal pathway.
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Background The fibrosis of filtering area caused by proliferation of human Tenon fibroblasts (HTFs) is one of failure causes following glaucoma surgery.Researches revealed that hydroxycamptothecin can induce the apoptosis of HTFs,but its influence on autophagy of HTFs is unclear.Objective This study attempted to investigate whether hydroxycamptothecin can cause an alteration of autophagic activity in HTFs.Methods Human Tenon capsular tissue was obtained from 3 patients during strabismus correction surgery under the informed consent of patients and their parents for the primary culture and passaged of HTFs in DMEM containing 10％ fetal bovine serum.The generation 3 to 6 cells then were incubated with 0.0,0.5,1.0,4.0,10.0 mg/L hydroxycamptothecin for 24 hours,respectively.A cell counting kit-8 (CCK-8) was used to detect the cell viability in different treated groups.The autophagic activity of HTFs was evaluated by a Cyto-ID autophagy detection kit,and then the autophagic flux was evaluated by counting the Cyto-ID positive cells under a fluorescence microscope,and the green fluorescence intensity was determined by flow cytometry.Quantitative reverse transcriptase PCR (qRT-PCR) and Western blot analysis were employed to assay the relative expressions of autophagic-associated genes and their proteins in HTFs,including Beclin-1,autophagy related gene 5 (ATG-5) and light chain 3 (LC-3).Results The cell viability of HTFs in the 0.0,0.5,1.0,4.0 and 10.0 mg/L hydroxycamptothecin groups were (100.00 ± 6.44) ％,(91.70 ± 6.36) ％,(81.47 ± 6.00) ％,(68.43 ± 6.69) ％ and (59.97 ± 6.98) ％ respectively,showing a gradually declining trend with the increase of hydroxycamptothecin doses,with a significant difference among them (F=19.040,P＜0.001),and the viability of HTFs in the 1.0,4.0 and 10.0 mg/L hydroxycamptothecin groups were significantly decreased than the control group (P＜0.05,P＜0.01,P＜0.01).qRT-PCR analysis revealed that the relative expression levels of Beclin-1 mRNA,ATG-5 mRNA and LC-3 mRNA in 4.0 mg/L hydroxycamptothecin group were (3.225 ±0.346),(2.839 ±0.418) and (3.761±0.224) folds higher than those of the control group.The expressions of Beclin-1 and ATG-5 proteins were significantly increased in the 4.0 mg/L hydroxycamptothecin group in comparison with the control group,and the expression intensity ratio of LC-3-Ⅱ/Ⅰ was 0.965±0.159 in the hydroxycamptothecin group,which was significantly higher than 0.275 ±0.860 of the control group (P =0.003).Cyto-ID staining showed that the percentage of autophagic cells increased dramatically from (11.333±4.010) ％ to (55.000±9.013) ％ upon the exposure of HTFs to 4.0 mg/L hydroxycamptothecin (P=0.002).Flow cytometry analysis showed that the green fluorescence intensity in the 4.0 mg/L hydroxycamptothecin group was (3.037 ±0.513) fold relative to that in the control group,showing a significant difference between the two groups (P =0.003).Conclusions Hydroxycamptothecin can induce autophagy in HTFs in vitro.
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Background Conjunctivochalasis is an age-related inflammatory ocular surface disease manifesting redundant,loose conjunctiva folds.Studies showed that conjunctivochalasis often accompanies with inflammatory response,indicating that a variety of inflammatory cytokines are involved in pathogenesis of conjunctivochalasis.Objective This study attempted to investigate the expressions of pentraxin-3 (PTX-3) and tumor necrosis factor-α stimulated gene-6 (TSG-6) proteins in fibroblasts of conjunctivochalasis.Methods The protocol was approved by Ethic Committee of Putuo Hospital affiliated Shanghai University of Traditional Chinese Medicine.Thirteen eyes of 13 patients with conjunctivochalasis,16 eyes of 16 patients with age-related cataract and 16 eyes of 15 patients with pterygium were included in Putuo Hospital Affiliated to Shanghai University of Traditional Medicine from January,2011 to June,2012.The samples of conjunctival tissue were collected in the patients during surgery under the informed consent,and fibroblasts were cultured and passaged by explant culture method.Content of PTX-3 and TSG-6 proteins in cell supernatant was detected by ELISA.Results Translucent poor,dark black area was around the cell overflow.Cultured cells of third generation showed long shuttle type,uniform size,radiated out in all directions,and oval nuclei was sited in the cytoplasm,surrounded by varying the length of cross-linking of cell processes.The concentrations of PTX-3 protein in cell supernatant were (2 182.33 ± 738.68) pg/ml in the conjunctivochalasis group,which were higher than (738.32±335.00) pg/ml in the age-related cataract group and (981.37±416.04)pg/ml in the pterygium group,showing a significant difference among the three groups (F =6.474,P=0.032).The contents of TSG-6 proteins in cell supernatant were (59.10±6.52) pg/ml,(53.99± 11.16) pg/ml and (35.01±5.33)pg/ml in the conjunctivochalasis group,pterygium group and age-related cataract group,with a significant difference among the three groups (F =7.421,P =0.024),and contents of TSG-6 proteins in the agerelated cataract group was lowest,however,no significant difference was found in the TSG-6 contents between the conjunctivochalasis group and the pterygium group (P＞0.05).Conclusions Inflammatory reaction participates in the pathogenesis and development of conjunctivochalasis,up-regulation of PTX-3 and TSG-6 expression in fibroblasts might partakes in the pathogenesis of conjunctivochalasis.