Effects of mitomycin C on membrane permeability and ultrastructure changes in human pterygium fibroblasts / 中华实验眼科杂志

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.