摘要
背景 角膜损伤修复过程中细胞外基质（ECM）纤维化是角膜瘢痕形成的基础,转化生长因子-β1（TGF-β1）可引起角膜基质过度产生ECM.我们在前期的研究工作中构建了角膜基质三维培养模型,而将TGF-β1添加于该三维培养体系中是否可达到构建角膜基质ECM纤维化的体外三维培养模型有待研究.目的 评估TGF-β1对该三维培养模型中ECM纤维化相关基因表达的影响,确定该三维培养体系是否可以作为角膜基质ECM纤维化的体外三维培养候选模型.方法分离新鲜成年牛角膜,并在基础培养液（DMEM/F12＋体积分数10％胎牛血清）中进行角膜基质细胞的培养,将5＆#215;105个牛角膜基质细胞收集于15 ml离心管中构建体外三维培养模型（Pellet）,根据培养液中添加的TGF-β1质量浓度的不同分为基础培养液组（无TGF-β1）、基础培养液＋0.5 ng/ml TGF-β1组和基础培养液＋1.0 ng/ml TGF-β1组,于培养2周时用钙黄绿素-AM/碘化丙啶法（Calcein-AM/PI）法进行细胞活性测定;分别于培养后48 h、1周和2周应用实时荧光定量PCR（real-time PCR）和Western blot法检测各组Pellet中α-平滑肌肌动蛋白（α-SMA）、Ⅰ型胶原（Col Ⅰ）、ColⅢmRNA及其蛋白的表达. 结果 Pellet模型培养后48 h,角膜基质细胞开始抱团,培养后2周经Calcein AM/PI染色证实绝大多数细胞存活.培养后48 h、1周和2周,基础培养液＋0.5 ng/ml TGF-β1组和基础培养液＋1.0 ng/ml TGF-β1组角膜基质细胞中α-SMA、Col Ⅰ和ColⅢmRNA的相对表达量均明显高于基础培养液组,3个组间的总体差异均有统计学意义（F分组=696.745,P＜0.001;F分组=35.166,P＜0.001;F分组=33.677,P＜0.001）,且随着培养时间的延长,α-SMA、Col Ⅰ和ColⅢmRNA的相对表达量逐渐升高,差异均有统计学意义（F时间=5.863,P＜0.05;F时间=298.614,P＜0.001;F时间=607.472,P＜0.001）;ColⅢmRNA合成速率均大于Col Ⅰ mRNA的合成速率;Western blot检�
Background Extracellular matrix （ECM） fibrosis leads to corneal scaring during the process of cornea wound healing.Transforming growth factor-β1 （TGF-β1） is known to mediate overproduce of ECM components.Our previous study developed a three-dimensional model for corneal stromal cells culture in vitro.Objective The hypothesis of this study was to apply TGF-β1 in the three-dimensional culture system to establish a corneal stroma ECM fibrosis model.Methods Fresh bovine corneas were extracted for the culture of bovine keratocytes in constructed three-dimension culture system.The Pellets were cultured in the DMEM/F12＋ 10% fetal bovine serum （FBS） medium with 0.5 ng/ml or 1.0 ng/ml TGF-β1 or without TGF-β1,respectively.Calcein AM/（propidium iodide） PI staining was employed to assay the cell viability 2 weeks after culture.The expressions of α-smooth muscle actin （α-SMA）,type Ⅰ collagen （Col Ⅰ） and Col Ⅲ mRNA and protein in the cells were detected by real-time PCR and Western blot respectively 48 hours,1 week and 2 weeks after cultured.The results were statistically analyzed.Results Cultured for 48 hours in the Pellet system,corneal stromal cells clustered and was identified alive by Calcein-AM/PI staining in 2 weeks.The relative expression levels of α-SMA,Col Ⅰ and Col m mRNA were elevated in both the 0.5 ng/ml and 1.0 ng/ml TGF-β1 supplement groups in comparison with the only DMEM/F12＋10% FBS group,with marked difference among the three groups （Fgroup =696.745,P〈0.001;Fgroup =35.166,P〈0.001;Fgroup =33.677,P〈0.001）,and the expression levels increased with the lapse of culture time （Ftime =5.863,P〈0.05;Ftime =298.614,P〈0.001;Ftime =607.472,P〈0.001）.The synthetic rate of Col Ⅲ mRNA was obviously faster than that of Col Ⅰ mRNA.Western blot showed that only a trace of α-SMA,Col Ⅰ and Col Ⅲ were detected 48 hours and 1 week after culture.The expression levels of α-SMA,Col Ⅰ and Col Ⅲ in Pellet system in 0.5 ng/ml TGF-β1 medium were 0.395±0.208,
出处
《中华实验眼科杂志》
CAS
CSCD
北大核心
2015年第5期406-411,共6页
Chinese Journal Of Experimental Ophthalmology
基金
国家自然科学基金项目（81060076、81360144）
教育部留学回国人员科研启动基金项目（20101-561）
