人巩膜成纤维细胞体外三维胶原基质培养系统的构建及其生物力学特性

Establishment of human scleral fibroblasts-seeded collagen matrix and its mechanical properties

背景眼轴的过度伸长和巩膜组织的扩张是高度近视发病机制研究的热点之一，建立合理的人巩膜成纤维细胞（HSFs）培养体系模型有助于进一步探讨HSFs在高度近视形成过程中与胶原基质相互作用的生物学机制。 目的构建HSFs与Ⅰ型胶原的三维培养系统，微观模拟在体巩膜，建立巩膜重塑模型。 方法采集新鲜供体巩膜组织，采用组织块培养法分离和培养HSFs，采用免疫荧光技术检测细胞中波形蛋白和角蛋白的表达以鉴定细胞；获取8周龄SPF级雄性SD大鼠鼠尾腱制备鼠尾胶原，400 μl胶原加入50 μl NaOH（0.1 mol/L）溶液中混匀，加入80 μl 10倍培养液，混匀，溶液pH值约为7。加入1 100 μl终浓度1×106/ml细胞悬液混合形成凝胶培养系统，于倒置相差显微镜下观察HSFs的增生情况和细胞形态，采用IPP-5软件对三维培养的含细胞胶原收缩面积进行测量以分析三维胶原系统的物理特性；采用生物力学试验机对三维胶原系统的生物力学特性进行测定。 结果培养的HSFs波形蛋白呈阳性表达，角蛋白表达阴性。未接种培养细胞的无成纤维细胞SD鼠鼠尾胶原凝胶透明，实验期间性状无明显变化，而含HSFs的三维胶原凝胶随着细胞培养时间的延长细胞数量增加，倒置相差显微镜下可观察到数十层成纤维细胞相互交错呈网状排列，培养后7～14 d凝胶块呈乳白色，凝胶面积保持在正常的10%左右，三维培养系统中的HSFs接种后24 h细胞产生多向性突起，呈双极形或纺锤形。HSFs胶原凝胶复合物的弹性材料蠕变曲线呈非线性（0～100 s）部分与线性（100～600 s）部分，前者是胶原凝胶在短暂应力的作用下本身的弹性变化，后者是胶原凝胶在定力的作用下随拉伸时间的延长而出现的蠕变变化。 结论鼠胶原凝胶对培养的HSFs有良好的生物相容性，HSFs胶原凝胶可形成三维复合物，可呈现机械性生物学特性，是研究成纤维细胞与细胞外基质之间以及细胞之间相互作用的良好模型，可用于巩膜重塑的模拟研究。

BackgroundExcessive elongation of axis and expansion of sclera is one of the hot topics in the study of the pathogenesis of high myopia.To establish a human scleral fibroblasts （HSFs）-collagen matrix culture model is helpful for understanding the reciprocal and adaptive interactions between HSFs and the collagen matrix in tissue. ObjectiveThe aim of this study was to establish a HSFs-seeded collagen three-dimension culture system that may mimic the sclera remolding in myopia. MethodsHSFs were isolated and cultured from donor eyes by explant culture and purified by passages culture in vitro.The expressions of vimentin and keratin in the cells were detected by immunofluorescence technique to identify the cells.Rat tail tendon was obtained from 8-week-old SPF SD rats to prepare the collagen matrix.The mixed solution of 400 μl collagen matrix and 1 100 μl PBS, 200 μl nutrient medium, 50 μl NaOH and HSF suspension were mixed to prepare the collagen gel three-demension culture system.The growth and morphology of the cells in the culture system were observed under the inverted phase contrast microscope, and IPP-5 software was used to measure the contraction area of collagen gel, and the mechanical creep properties of the HSFs-seeded collagen matrix were measured by a biomechanics test instrument. ResultsHSFs emigrated from tissue 7 days after culture and passage could be performed 14 days after culture.The expression of keratin was absent in HSFs, while vimentin was positively expressed.The free-cell collagen gel was clear and unchanged in the experimental duration.However, the cells were obviously increased on the three-demension culture system and showed a tissue-like structure of net-like arrangement on dozens of layers.In 7-14 days after culture, the collagen gel area in a three-demension collagen matrix revealed a decrease of 90%.Duotriode-like and fusiform cells were seen 24 hours after culture.The biomechanical creep curve of HSFs-seeded collagen matrix consisted of the nonlinear section （0-100 seconds） and linear section （100-600 seconds）, and the former appeared to be an elastic change of the gel under the temporal stress, and the latter was the creeping of the gel with the time. ConclusionsRat tail collagen appears to have a good biocompatibility to HSFs.HSFs-seeded collagen matrix can retain the mechanical creep properties, and it may be a good tool for the study on the relationship between HSFs and extracellular matrix or intercellular biological behaviour for scleral remodeling.