ABSTRACT
Background Mitomycin C (MMC) has an inhibitory effect on the growth and proliferation of human pterygium fibroblasts,however,there is little literature about its influence on plasma membrane. Objective This study was to investigate the influence of MMC on the physical and chemical features and ultrastructures of plasma membrane. Methods Human pterygium tissues were obtained during the surgery.Human pterygium fibroblasts were primarily cultured and passaged using explant cultured method and identified by Vimentin staining.The third generation of cells were incubated to 96 well plate at a density of 5 × 103 cells/well,and 0,50,100,200,300 and 400 mg/L MMC was added in the culture well respectively to act for 12 hours.Cell viability was assayed using cell counting kit-8 ( CCK-8 ),and cellular apoptosis was detected using annexin V-FICT/PI.The changes of cell membrane structure were examined under an atomic force microscope.Malondialdehyde( MDA ) content in cell supernatant and level of lactate dehydrogenase ( LDH ) in extracellular fluid were detected to assess the lipid peroxidation level and permeability of cell membrane.Intracellular Ca2+ changes and membrane surface topography were assessed by Fluo-3/AM mark and flow cytometry( FCM ).This study was approved by Ethic Commission of Affiliated First Hospital of Jinan University.Informed consent was obtained from each patient. Results A lot of cells grew with the shape of spindle 1-2 weeks after culture.Positive response was seen in cultured cells for Vimentin.Growth and proliferation of the cells reduced 12 hours after action of MMC with the increase of MMC concentrations.The apoptosis rate of human pterygium fibroblasts was 4.2％,4.2％,5.4％,19.3％ and 25.8％ in 0,50,100,200 and 300 mg/L MMC groups respectively.Different degrees of abnormalities of cells membrane were found in various concentrations of MMC groups.The elevated contents of LDH and MDA in extracellular fluid were detected in various concentrations of MMC groups compared with the control group,and the LDH and MDA levels were gradually ascended as the increase of MMC concentrations,with a significant difference between any two groups(P＜0.05).The disturbance of intracellular Ca2+ homeostasis was also been seen after MMC acted. Conclusions MMC leads to the changes of physical and chemical features in human pterygium fibroblasts at a dose-dependent manner.Cell membrane may be the acting target of MMC.
ABSTRACT
BackgroundCorneal neovascularization (CNV) is a common eye disease.The researches on the pathogenesis and treatment of CNV are focus in Ophthalmology.ObjectiveThis study was to investigate the effects of culture supernatant from human amniotic epithelial cells (AECs) on CNV in vitro and its mechanism.MethodsHuman AECs were obtained from a placenta and cultured in serum-free medium for 48 hours,and the supernatant was collected.The levels of interleukin-1 receptor antagonist (IL-1 Ra) and pigment epithelium-derived factor (PEDF) in the human AECs culture supernatant were detected by enzyme-linked immunosorbent assay (ELISA).Rabbit corneal epithelial cells (CECs) were obtained and cultured in different concentrations of human AECs culture supernatant for 48 hours,serum-free medium was used as the control group.The expression of vascular endothelial growth factor (VEGF) mRNA and basic fibroblast growth factor (bFGF) mRNA in rabbit CECs were measured by quantitative real-time PCR (QRT-PCR).Human umbilical cord vein endothelial cells (UVECs) were cultured in the three mediums above,and the proliferation of human UVECs (absorbance value,A value) was tested by the cell counting kit 8 ( CCK8 ).Migration assay was performed by the wound healing method for the human UVECs.The membrane ultra-structure of human UVECs was examined under the atomic force microscope (AFM).ResultsCultured and passaged human AECs showed a positive response for keratin.The expression of VEGF mRNA (1.00±0.22) and bFGF mRNA (1.00±0.36) in rabbit CECs was suppressed by the human AECs culture supernatant,with a significant reduction in comparison with the serum-free DMEM group (2.98±0.46,2.55±0.48 )(P=0.001,0.002).The A value was significantly lowered in the human AECs culture group for 72 hours compared with the serum-free DMEM group ( 1.941 ± 0.036 versus 2.144 ± 0.059 ) ( P =0.000 ),and the bFGF-induced migration rate of human UVECs was strongly inhibited by the culture supernatant of human AECs in comparison with serum-free DMEM.The plasma membrane of human UVECs cultured with the human AECs culture supernatant was full of bumps,and decreased intercellular connection and cellular pseudopodia were found on the AFM image.The concentration of IL-1Ra was (153.56±0.36)ng/L and that of PEDF was (70.41 ±0.68 )μ,g/L in the human AECs culture supernatant.Nothing was deteched in serum-free DMEM group.Conclusions Human AECs culture supernatant suppressed the expression of VEGF and bFGF in CECs as well as the migration and proliferation of vascular endothelial cells.The effect may be associated with IL-1Ra and PEDF secreted by human AECs.These results suggest that human AECs may be a potential therapy for the inhibition of CNV.