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AIM: To investigate the effects of bovine pituitary extract on the proliferation of keratocytes and maintaining the keratocyte phenotype in vitro.METHODS: Single keratocytes were isolated by enzyme digestion for in vitro culture. Three groups were designed according to the different culture media:a bovine pituitary extract (BPE) group, a fetal bovine serum (FBS) group and the control group. The phenotypes and proliferation of cultured cells were evaluated by morphology, immunofluorescent staining and mRNA expression of CD34, Lumican, VSX1, α-SMA and proliferating cell nuclear antigen (PCNA). In the BPE group, cells underwent serial subcultivation, and their phenotypes were identified by immunofluorescent staining. To analyze the proliferation of keratocytesin differentconcentrations of BPE, six differentconcentrations were designed to ascertain the most appropriate amount.RESULTS: In the BPE group, the cells spread out and presented dendritic morphology, and their dendrites connected to one another to form networks. On the third passage, most cells maintained their phenotype. In the FBS group, the cells exhibited a dendritic appearance in early cultured stages, but their morphology subsequently changed into a fibroblast-like shape. The number of dendritic cells in BPE group was more than FBS and control groups. Immunofluorescent staining and real-time polymerase chain reaction (PCR) confirmed that few keratocytes underwent fibroblastic transformation in the BPE and control groups, and that proliferation was higher in the BPE group than in the control group. Although the proliferation was higher in the FBS group, many keratocytes underwent fibroblastic transformation.Theanalysis of cell morphology and mRNA expressions of CD34, PCNA and VSX1 in six group showed that different concentrations of BPE affected the proliferation obviously but didn’t affect the keratocyte phenotype, and the concentration of 40μg/mL was the most appropriate one.CONCLUSION: BPE can improve the proliferation of keratocytes and maintain their phenotype in vitro. Many keratocytes can be harvested rapidly and provide seeds for the construction of corneal stroma. 相似文献
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Cytotoxic effects of betaxolol on healthy corneal endothelial cells both in vitro and in vivo 
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下载免费PDF全文 AIM: To demonstrate the cytotoxic effect of betaxolol and its underlying mechanism on human corneal endothelial cells(HCE cells) in vitro and cat corneal endothelial cells(CCE cells) in vivo, providing experimental basis for safety anti-glaucoma drug usage in clinic of ophthalmology.METHODS: In vivo and in vitro experiments were conducted to explore whether and how betaxolol participates in corneal endothelial cell injury. The in vitro morphology, growth status, plasma membrane permeability, DNA fragmentation, and ultrastructure of HCE cells treated with 0.021875-0.28g/L betaxolol were examined by light microscope, 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay, acridine orange (AO)/ethidium bromide (EB) double-fluorescent staining, DNA agarose gel electrophoresis, and transmission electron microscope (TEM). The in vivo density, morphology, and ultrastructure of CCE cells, corneal thickness, and eye pressure of cat eyes treated with 0.28g/L betaxolol were investigated by specular microscopy, applanation tonometer, alizarin red staining, scanning electron microscope (SEM), and TEM.RESULTS: Exposure to betaxolol at doses from 0.0875g/L to 2.8g/L induced morphological and ultrastructural changes of in vitro cultured HCE cells such as cytoplasmic vacuolation, cellular shrinkage, structural disorganization, chromatin condensation, and apoptotic body appearance. Simultaneously, betaxolol elevated plasma membrane permeability and induced DNA fragmentation of these cells in a dose-dependent manner in AO/EB staining. Furthermore, betaxolol at a dose of 2.8g/L also induced decrease of density of CCE cells in vivo, and non-hexagonal and shrunk apoptotic cells were also found in betaxolol-treated cat corneal endothelia.CONCLUSION: Betaxolol has significant cytotoxicity on HCE cells in vitro by inducing apoptosis of these cells, and induced apoptosis of CCE cells in vivo as well. The findings help provide new insight into the apoptosis-inducing effect of anti-glaucoma drugs in eye clinic. 相似文献
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目的：观察贝伐单抗对体外培养的人视网膜色素上皮细胞ARPE－19增殖及钙黏连蛋白（ E－cadherin ）和纤维连接蛋白（ fibronectin ）表达变化的作用,探讨贝伐单抗对ARPE－19增殖及纤维化的影响。方法：用不同浓度（0、0．625、1．25、2．5、5．0mg／mL）的贝伐单抗干预体外培养人视网膜色素上皮细胞ARPE－19,采用CCK－8法分别于24、48、72 h检测细胞活性;流式细胞仪检测细胞周期;应用免疫蛋白印迹法（ Western blotting）及逆转录聚合酶链反应（RT－PCR）检测ARPE－19中E－cadherin和fibronectin的蛋白及mRNA的表达变化。结果：浓度为2．5、5．0 mg／mL贝伐单抗能有效抑制ARPE－19细胞的增殖及细胞周期,差异有统计学意义（ P＜0.05）。2．5、5．0 mg／mL贝伐单抗能抑制E－cadherin 基因,促进fibronectin基因的转录及表达,差异有统计学意义（P＜0．05）。结论：高浓度的贝伐单抗能够抑制ARPE－19细胞的增殖,下调纤维化相关因子E－cadherin ,同时上调fibronectin的表达,提示高浓度的贝伐单抗可以引起ARPE－19细胞纤维化。 相似文献
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M. Kolko J.F. Kiilgaard J. Wang K.A. Poulsen M. la Cour S. Heegaard J.U. Prause 《Experimental eye research》2009,89(3):383-379
Calcium-independent phospholipase A2, group VIA (iPLA2-VIA) is involved in cell proliferation. This study aimed to evaluate the role of iPLA2-VIA in retinal pigment epithelium (RPE) cell proliferation and in retinal diseases involving RPE proliferation. A human RPE cell line (ARPE-19) was used to explore this role in vitro. Proliferating ARPE-19 cells had increased expression and activity of iPLA2-VIA. iPLA2-VIA was found in the nuclei of proliferating ARPE-19 cells, whereas in confluent ARPE-19 cells, with limited proliferation, iPLA2-VIA was primarily found in the cytosol. Inhibition of iPLA2-VIA decreased the rate of proliferation, whereas over expression of iPLA2-VIA increased the rate of proliferation. Using an experimental porcine model of RPE proliferation we demonstrated significant nuclear upregulation of iPLA2-VIA in proliferating RPE cells in vivo. We furthermore evaluated the expression of iPLA2-VIA in proliferative vitreoretinopathy (PVR). PVR membranes revealed nuclear expression of iPLA2-VIA in the RPE cells which had migrated and participated in the formation of the membranes. Overall, the present results point to an important role of iPLA2-VIA in the regulation of RPE proliferation suggesting that iPLA2-VIA may be considered as a possible pharmaceutical target in retinal diseases involving RPE proliferation and migration. 相似文献