AIM: To determine the effects of a low dose latrunculin (LAT)-A on dexamethasone (Dex)-induced upregulation of extracellular matrix proteins fibronectin (FN) in cultured human trabecular meshwork (HTM) cells. METHODS:...AIM: To determine the effects of a low dose latrunculin (LAT)-A on dexamethasone (Dex)-induced upregulation of extracellular matrix proteins fibronectin (FN) in cultured human trabecular meshwork (HTM) cells. METHODS: HTM cells were cultured to confluent and incubated with 0.4 mu mol/L Dex and/or 0.05 mu mol/L LAT-A. FN expression in HTM cells was evaluated by Western blot and immunofluorescence microscopy. RESULTS: Dex up-regulated FN production in HTM cells, failed to do so when co-incubated with LAT-A. LAT-A decreased production of FN in cultured HTM cells. CONCLUSION: This study indicated that LAT-A may modulate the expression of fibronectin in trabecular meshwork to achieve treatment for steroids and other types of glaucoma. It has an important prospect as an intraocular pressure-lowering drug.展开更多
Whether transforming growth factor β 2 (TGF β 2) induces apoptosis of human trabecular meshwork cells was investigated in vitro . Cultured 3 5 passage human trabecular meshwork cells were treated with 0 (con...Whether transforming growth factor β 2 (TGF β 2) induces apoptosis of human trabecular meshwork cells was investigated in vitro . Cultured 3 5 passage human trabecular meshwork cells were treated with 0 (control), 0.32, 1, 3.2 ng/ml TGF β 2 for 48 h and divided into control group and experimental group. The apoptosis of human trabecular meshwork cells was examined by transmisson electron microscopy, TUNEL technique and flow cytometry. The results showed characteristic morphologic changes of apoptotic cells were observed under transmission electron microscopy. DNA fragmentation of human trabecular meshwork cells was found by TUNEL technique. Quantitative analysis of flow cytometry showed that percentages of apoptotic human trabecular meshwork cells were (2.79±0.44) %, (4.43 ±1.17) % and (9.60±2.05) % respectively with different concentrations [1 ng/ml ( P< 0.05), 3.2 ng/ml ( P< 0.01)] of TGF β 2 with the difference being significant between experimental group and control group[(1.41±0.34) %]. It was concluded that TGF β 2 can induce apoptosis of human trabecular meshwork cells in vitro and may be involved in the decrease of trabecular meshwork cells in the patients with primary open angle glaucoma and aging of normal people.展开更多
Whether tranilast had antagonistic effect on proliferation inhibition and collagen synthesis promotion induced by TGF-β 2 in cultured human trabecular meshwork cells was investigated. Suspension of 1×104 cultur...Whether tranilast had antagonistic effect on proliferation inhibition and collagen synthesis promotion induced by TGF-β 2 in cultured human trabecular meshwork cells was investigated. Suspension of 1×104 cultured human trabecular meshwork cells of 3—5 passage was distributed in each well of a 96-well disk and divided into control group and experimental group. After 24 h, 0 μg/ml (control), 12.5 μg/ml, 25 μg/ml, 50 μg/ml tranilast with 3.2 ng/ml TGF-β 2 were added into the incubation medium. Another 24 h later, proliferation and collagen synthesis in cultured human trabecular meshwork cells were examined respectively by using tetrazolium-based semiautomated colormetric (MTT) assay and 3H-proline incorporation with liquid scintillation technique. The results showed absorbance (A) values of the experimental groups were 0.9036±0.3017, 1.1361± 0.1352, 1.2457±0.1524 according to the different concentrations of tranilast, and 0.8956± 0.1903 of the control group. In comparison with the control group, 25 μg/ml (q'=3.23, P< 0.05), 50 μg/ml (q'=4.70, P<0.01) tranilast significantly antagonized the decrease of the A values induced by TGF-β 2 in the cultured human trabecular meshwork cells. In comparison with the control group [817.37±124.21 cpm/104 cells], 12.5 μg/ml (620.33±80.46 cpm/104 cells, q'= 4.26, P< 0.05), 25 μg/ml (594.58±88.13 cpm/104 cells, q'=4.81, P<0.01), 50 μg/ml (418.64±67.90 cpm/104 cells, q'=8.62, P<0.01) tranilast significantly inhibited the incorporation of 3H-proline into the cultured human trabecular meshwork cells promoted by TGF-β 2 in a dose-dependent manner. It was concluded that tranilast had the antagonistic effect on the proliferation inhibition and collagen synthesis promotion induced by TGF-β 2 in the cultured human trabecular meshwork cells.展开更多
Whether cultured human trabecular meshwork cells express transforming growth factor-β 2 (TGF-β 2) messenger RNA (mRNA) and protein was investigated. Total RNA of 10 6 cultured human trabecular meshwork cells was ...Whether cultured human trabecular meshwork cells express transforming growth factor-β 2 (TGF-β 2) messenger RNA (mRNA) and protein was investigated. Total RNA of 10 6 cultured human trabecular meshwork cells was extracted with TRIZOL reagent, reverse transcriptase-polymerase chain reaction (RT-PCR) were used for detection of TGF-β 2 messenger RNA, and the PCR product was verified by sequencing. Immunohistochemical staining was used to detect TGF-β 2 protein. The results showed that a single RT-PCR amplified product was obtained, and the sequence was homologous to the known sequence. TGF-β 2 immunostaining was positive. It was concluded that trabecular meshwork cells could produce TGF-β 2 and contribute to the presence of TGF-β 2 in trabecular meshwork microenvironment as well as aqueous humor. Trabecular meshwork cells were affected by TGF-β 2 not only through paracrine, but also autocrine action. Whether abnormal changes in TGF-β 2 production contribute to the pathogenesis of primary open-angle glaucoma is worth further investigation.展开更多
Background:Loss of cells in the human trabecular meshwork(TM)has been reported with ageing and in glaucoma.This study aims to identify,quantify and determine the age-related changes of human TM stem cells(TMSCs).Metho...Background:Loss of cells in the human trabecular meshwork(TM)has been reported with ageing and in glaucoma.This study aims to identify,quantify and determine the age-related changes of human TM stem cells(TMSCs).Methods:Isolation of TM cells/paraffin sectioning was carried out using human corneoscleral rings and whole globes.The TM cells/sections were immunostained for the stem cell markers ATP-binding cassette protein G2(ABCG2),nerve growth factor receptor p75 and AnkyrinG(AnkG).Images were acquired using Leica SP8 confocal microscope.The isolated cells were analyzed for two parameters-ABCG2 expression and nucleus to cytoplasmic ratio(N/C ratio).The total number of TM cells and those positive for ABCG2 and p75 in each section were quantified.Spearman rank order correlation was used to determine the association between age and the cell counts.Results:The TMSCs were identified based on two parameters-high ABCG2 expression and high N/C ratio>0.7.These stem cells were also positive for p75 and AnkG.The TMSC content based on the two parameters was 21.0±1.4%in<30 years age group,12.6±6.6%in 30–60 years and 4.0±3.5%in>60 years.The stem cells with high ABCG2 and p75 expression were restricted to the Schwalbe’s line region of the TM.A significant correlation was observed between the reduction in TMSC content and TM cell count during ageing.Conclusion:The human TMSCs were identified and quantified based on two parameter analysis.This study established a significant association between age-related reduction in TMSC content and TM cell loss.展开更多
基金National Natural Science Foundation of China (No.30772379)Education Department Project of Hunan Province, China(No.04A049)
文摘AIM: To determine the effects of a low dose latrunculin (LAT)-A on dexamethasone (Dex)-induced upregulation of extracellular matrix proteins fibronectin (FN) in cultured human trabecular meshwork (HTM) cells. METHODS: HTM cells were cultured to confluent and incubated with 0.4 mu mol/L Dex and/or 0.05 mu mol/L LAT-A. FN expression in HTM cells was evaluated by Western blot and immunofluorescence microscopy. RESULTS: Dex up-regulated FN production in HTM cells, failed to do so when co-incubated with LAT-A. LAT-A decreased production of FN in cultured HTM cells. CONCLUSION: This study indicated that LAT-A may modulate the expression of fibronectin in trabecular meshwork to achieve treatment for steroids and other types of glaucoma. It has an important prospect as an intraocular pressure-lowering drug.
文摘Whether transforming growth factor β 2 (TGF β 2) induces apoptosis of human trabecular meshwork cells was investigated in vitro . Cultured 3 5 passage human trabecular meshwork cells were treated with 0 (control), 0.32, 1, 3.2 ng/ml TGF β 2 for 48 h and divided into control group and experimental group. The apoptosis of human trabecular meshwork cells was examined by transmisson electron microscopy, TUNEL technique and flow cytometry. The results showed characteristic morphologic changes of apoptotic cells were observed under transmission electron microscopy. DNA fragmentation of human trabecular meshwork cells was found by TUNEL technique. Quantitative analysis of flow cytometry showed that percentages of apoptotic human trabecular meshwork cells were (2.79±0.44) %, (4.43 ±1.17) % and (9.60±2.05) % respectively with different concentrations [1 ng/ml ( P< 0.05), 3.2 ng/ml ( P< 0.01)] of TGF β 2 with the difference being significant between experimental group and control group[(1.41±0.34) %]. It was concluded that TGF β 2 can induce apoptosis of human trabecular meshwork cells in vitro and may be involved in the decrease of trabecular meshwork cells in the patients with primary open angle glaucoma and aging of normal people.
基金ThisprojectwassupportedbyagrantfromtheNationalNaturalSciencesFoundationofChina (No .38970 75 8) .
文摘Whether tranilast had antagonistic effect on proliferation inhibition and collagen synthesis promotion induced by TGF-β 2 in cultured human trabecular meshwork cells was investigated. Suspension of 1×104 cultured human trabecular meshwork cells of 3—5 passage was distributed in each well of a 96-well disk and divided into control group and experimental group. After 24 h, 0 μg/ml (control), 12.5 μg/ml, 25 μg/ml, 50 μg/ml tranilast with 3.2 ng/ml TGF-β 2 were added into the incubation medium. Another 24 h later, proliferation and collagen synthesis in cultured human trabecular meshwork cells were examined respectively by using tetrazolium-based semiautomated colormetric (MTT) assay and 3H-proline incorporation with liquid scintillation technique. The results showed absorbance (A) values of the experimental groups were 0.9036±0.3017, 1.1361± 0.1352, 1.2457±0.1524 according to the different concentrations of tranilast, and 0.8956± 0.1903 of the control group. In comparison with the control group, 25 μg/ml (q'=3.23, P< 0.05), 50 μg/ml (q'=4.70, P<0.01) tranilast significantly antagonized the decrease of the A values induced by TGF-β 2 in the cultured human trabecular meshwork cells. In comparison with the control group [817.37±124.21 cpm/104 cells], 12.5 μg/ml (620.33±80.46 cpm/104 cells, q'= 4.26, P< 0.05), 25 μg/ml (594.58±88.13 cpm/104 cells, q'=4.81, P<0.01), 50 μg/ml (418.64±67.90 cpm/104 cells, q'=8.62, P<0.01) tranilast significantly inhibited the incorporation of 3H-proline into the cultured human trabecular meshwork cells promoted by TGF-β 2 in a dose-dependent manner. It was concluded that tranilast had the antagonistic effect on the proliferation inhibition and collagen synthesis promotion induced by TGF-β 2 in the cultured human trabecular meshwork cells.
基金This project was supported by the National Natural ScienceFoundation of China ( Serial No.3 8970 75 8)
文摘Whether cultured human trabecular meshwork cells express transforming growth factor-β 2 (TGF-β 2) messenger RNA (mRNA) and protein was investigated. Total RNA of 10 6 cultured human trabecular meshwork cells was extracted with TRIZOL reagent, reverse transcriptase-polymerase chain reaction (RT-PCR) were used for detection of TGF-β 2 messenger RNA, and the PCR product was verified by sequencing. Immunohistochemical staining was used to detect TGF-β 2 protein. The results showed that a single RT-PCR amplified product was obtained, and the sequence was homologous to the known sequence. TGF-β 2 immunostaining was positive. It was concluded that trabecular meshwork cells could produce TGF-β 2 and contribute to the presence of TGF-β 2 in trabecular meshwork microenvironment as well as aqueous humor. Trabecular meshwork cells were affected by TGF-β 2 not only through paracrine, but also autocrine action. Whether abnormal changes in TGF-β 2 production contribute to the pathogenesis of primary open-angle glaucoma is worth further investigation.
基金supported by the Science and Engineering Research Board(Project reference number:EMR/2016/002351).
文摘Background:Loss of cells in the human trabecular meshwork(TM)has been reported with ageing and in glaucoma.This study aims to identify,quantify and determine the age-related changes of human TM stem cells(TMSCs).Methods:Isolation of TM cells/paraffin sectioning was carried out using human corneoscleral rings and whole globes.The TM cells/sections were immunostained for the stem cell markers ATP-binding cassette protein G2(ABCG2),nerve growth factor receptor p75 and AnkyrinG(AnkG).Images were acquired using Leica SP8 confocal microscope.The isolated cells were analyzed for two parameters-ABCG2 expression and nucleus to cytoplasmic ratio(N/C ratio).The total number of TM cells and those positive for ABCG2 and p75 in each section were quantified.Spearman rank order correlation was used to determine the association between age and the cell counts.Results:The TMSCs were identified based on two parameters-high ABCG2 expression and high N/C ratio>0.7.These stem cells were also positive for p75 and AnkG.The TMSC content based on the two parameters was 21.0±1.4%in<30 years age group,12.6±6.6%in 30–60 years and 4.0±3.5%in>60 years.The stem cells with high ABCG2 and p75 expression were restricted to the Schwalbe’s line region of the TM.A significant correlation was observed between the reduction in TMSC content and TM cell count during ageing.Conclusion:The human TMSCs were identified and quantified based on two parameter analysis.This study established a significant association between age-related reduction in TMSC content and TM cell loss.
