血清在构建角膜基质细胞体外三维培养模型中的作用  

Effect of serum in constructing pellet three-dimensional model for keratocytes culture in vitro

作　　者：范梓晰[1] 罗世男 靳荷[1] 李杰[1] 李霞[1] 

机构地区：[1]广西医科大学第一附属医院眼科,南宁530021 [2]广西医科大学第三附属医院眼科,南宁530021

出　　处：《眼科》2017年第5期328-332,共5页Ophthalmology in China

基　　金：国家自然科学基金(81360144;81060076);教育部留学回国人员科研启动基金(2010-1561)

摘　　要：目的研究血清在构建角膜基质细胞体外三维培养模型中对细胞生长情况及胶原纤维生成的影响,为体外研究角膜基质细胞外基质(ECM)纤维化提供新的培养模型。设计实验研究。研究对象牛角膜基质细胞。方法分离原代牛角膜基质细胞,取第一代细胞作为种子细胞构建Pellet模型,实验分为对照组(DMEM/F12)和血清组(DMEM/F12+10%胎牛血清FBS)。Pellet模型培养3周后,肉眼观察细胞生长状态,同时进行HE染色及Calcein AM/PI染色测定细胞活性,并应用MASSON染色及透射电子显微镜(TEM)观察胶原纤维的形成、走向及排列情况。主要指标细胞生长状态、细胞活性、胶原纤维走向及排列情况。结果Pellet模型培养3周后,对照组未观察到角膜基质细胞抱团生长现象,且细胞易松散,难以进行后续实验;血清组Pellet模型中细胞明显成簇生长,呈粒状,结合牢固。Calcein AM/PI染色及HE染色可见血清组大多数细胞存活且密集生长,细胞结构完整。MASSON染色示血清组较多胶原纤维显色,并于TEM下观察到胶原纤维密集分布于细胞外且走向紊乱。结论含血清的培养环境能成功构建牛角膜基质细胞体外Pellet三维培养模型,且能生成含有胶原纤维的ECM。Objective To investigate the influence of serum in establishing a corneal stroma Pellet three-dimensional model by detecting cell growth and collagen fibers production,in order to offer a new culture pattern for extracellular matrix（ECM） fibrosis study in vitro.Design Experimental study.Participants Bovine keratocytes.Methods The primary bovine keratocytes were isolated and cultured,and the first generation cells were used for building Pellet model.The constructions in Pellet systems were cultured in the DMEM/F12 without serum（control group） or with 10% fetal bovine serum（FBS）（group serum）,respectively.After 3 weeks cultured,the Pellets were observed.The state of cell growth and cell activity was assayed by HE and Calcein AM/PI staining.And then,MAS-SON staining and transmission electron microscopy（TEM） were used to detect the formation of collagen arrangement.Main Outcome Measures Cell growth,cell activity,collagen arrangement.Results Cultured for 3 weeks in Pellet model,cells in the control group didn＇t assemble and could be broken up easily.It was difficult to do sequential experiment.Inversely,in group serum Pellet huddled growing and was in grain appearance,combining firmly.The majority of living cells in Group serum growed closely and remained struc-tural integrity through the HE and Calcein AM/PI staining.MASSON staining showed many collagen fibers.TEM showed that extracel-lular collagen deposited and densely distributed.Conclusions Serum could assist in-vitro three-dimensional culture of keratocytes,and generate the ECM rich of collagen fibers.

关 键 词：Pellet三维培养模型 角膜基质细胞 血清 细胞外基质 胶原纤维 

分 类 号：R77[医药卫生—眼科]