丝裂霉素C对翼状胬肉成纤维细胞膜通透性及超微结构的影响

背景 丝裂霉素C(MMC)对人翼状胬肉成纤维细胞的生长和增生有明显的抑制作用,但关于其对细胞膜影响的研究很少. 目的 探讨MMC对翼状胬肉成纤维细胞膜理化特性及超微结构改变的干预.方法 收集翼状胬肉手术切除标本15例,用组织块培养法培养人成纤维细胞,以波形蛋白免疫组织化学染色法对培养细胞进行鉴定.取传3代后对数生长期细胞以5×103个/孔的密度接种于96孔板,将不同质量浓度0、50、100、200、300、400 mg/L MMC加入培养孔中处理翼状胬肉成纤维细胞12h,用细胞计数试剂盒8(CCK-8)法检测培养细胞的活力,应用Annexin V-FICT/碘化丙啶(PI)法检测细胞凋亡的情况；利用原子力显微镜(AFM)观察不同质量浓度MMC作用后人成纤维细胞膜超微结构的改变；检测细胞上清液中丙二醛( MDA)的浓度以评价细胞的脂质过氧化程度；检测细胞外液中漏出的乳酸脱氢酶(LDH)含量以评估细胞膜的通透性变化；用Fluo-3/AM标记和流式细胞术检测培养细胞内游离钙离子的变化. 结果 组织块法原代培养贴壁1～2周可见大量长梭形细胞爬出,波形蛋白检测呈阳性反应.用MMC处理培养细胞12h后,人成纤维细胞的活力减弱,MMC对细胞增生的抑制作用呈剂量依赖性.0、50、100、200、300 mg/L MMC处理组细胞凋亡百分比分别为4.2％、4.2％、5.4％、19.3％和25.8％.AFM下可见不同质量浓度MMC作用后人成纤维细胞膜超微结构的异常,不同质量浓度MMC处理组细胞外液中LDH活性及细胞上清液中MDA浓度明显高于对照组,且均随着MMC质量浓度的增加,LDH活性及细胞上清液中MDA浓度逐渐增加,组间两两比较,差异均有统计学意义(P＜0.05).Fluo-3/AM标记和流式细胞术检测表明,培养细胞内游离钙离子增多.结论 MMC能引起翼状胬肉成纤维细胞膜的理化性质改变,其作用呈剂量依赖性,细胞膜是其药效和毒性的作用靶点之一.

Effects of mitomycin C on membrane permeability and ultrastructure changes in human pterygium fibroblasts

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 × 10^3 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 Ca^2＋ 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 Ca〉 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.