转化生长因子-β(TGF-β)空间动态分布的角膜基质创伤修复体外三维培养体系的构建  

Establishment of a three-dimensional culture system for corneal stromal wound healing in vitro with dynamic spatial distribution of TGF-β

作　　者：彭昶璠 徐路星 罗世男 王霜宁 袁检宝 李霞[1] PENG Chang-Fan;XU Lu-Xing;LUO Shi-Nan;WANG Shuang-Ning;YUAN Jian-Bao;LI Xia(Department of Ophthalmology,the First Affiliated Hospital of Guangxi Medical University,Nanning 530021,Guangxi Zhuang Autonomous Region,China)

机构地区：[1]广西医科大学第一附属医院眼科,广西壮族自治区南宁市530021

出　　处：《眼科新进展》2019年第4期311-315,共5页Recent Advances in Ophthalmology

基　　金：国家自然科学基金资助(编号:81360144;81060076);广西自然科学基金资助(编号:2017-GXNSFAA198250)~~

摘　　要：目的研究构建模拟角膜基质创伤修复过程中转化生长因子-β(transforming growth factor-β,TGF-β)空间动态分布的体外三维培养系统。方法从新鲜牛眼中分离角膜基质细胞,用含体积分数10%胎牛血清(fetal bovine serum,FBS)的DMEM/F12培养基进行培养,而后构建Pellet体外三维培养模型进行培养。将Pellet分为有透析袋组和无透析袋组,置入Transwell小室系统上下室中培养48 h后换液,上室为0.50μg·L^(-1)TGF-β1+0.25μg·L^(-1)TGF-β2+体积分数10%FBS,下室为体积分数10%FBS,分别于培养后72 h观察Pellet培养模型的形态变化并采用Real-time PCR法分别检测2组上下室Pellet中α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、纤维连接蛋白(fibronectin,FN)、Ⅰ型胶原(collagenⅠ,ColⅠ)和ColⅢmRNA相对表达量。结果培养后72 h Pellet均呈团状生长。有透析袋组Transwell小室系统上、下室α-SMA mRNA表达量分别为1.595±0.025、1.148±0.009,FN mRNA表达量分别为1.090±0.011、0.844±0.015,ColⅠmRNA表达量分别为1.445±0.035、1.165±0.008,ColⅢmRNA表达量分别为1.726±0.031、1.314±0.020,ColⅢ/ColⅠ比值分别为1.126±0.019、0.957±0.013,上下室各项指标比较差异均有统计学意义(均为P=0.000)。无透析袋组Transwell小室系统上、下室α-SMA mRNA表达量分别为1.363±0.018、1.360±0.002,FN mRNA表达量分别为0.946±0.017、0.952±0.012,ColⅠmRNA表达量分别为1.261±0.011、1.258±0.029,ColⅢmRNA表达量分别为1.459±0.027、1.462±0.033,ColⅢ/ColⅠ比值分别为1.157±0.029、1.163±0.090,上下室各项指标比较,差异均无统计学意义(均为P>0.05)。无透析袋组上室细胞中α-SMA、FN、ColⅠ和ColⅢmRNA的相对表达量与有透析袋组上室、有透析袋组下室比较,差异均有统计学意义(均为P<0.05)。无透析袋组下室细胞中α-SMA、FN、ColⅠ和ColⅢmRNA的相对表达量与有透析袋组上室、有透析袋组下室比较,差异均有统计学意义(均为P<0.05)Objective To establish a three-dimensional culture system simulating the spatial dynamic distribution of transforming growth factor-β(TGF-β) during corneal matrix healing in vitro . Methods Corneal stromal cells were isolated from fresh bovine eyes and cultured in DMEM/F12 culture medium containing volume fraction of 10%FBS.Then a three-dimensional culture model of Pellet was established for culture.Pellets were divided into dialysis tubes group and non-dialysis tubes group.After 48 h of culture in the upper and lower compartments of Transwell chamber system,the fluid in the upper chamber was changed to 0.50 μg·L^-1 TGF-β1+0.25 μg·L^-1 TGF-β2+volume fraction of 10% FBS,and volume fraction of 10%FBS in the lower chambers.The morphological changes of Pellets’ culture model were observed 72 h after culture.Real-time PCR was used to detect the relative expressions of α-smooth muscle actin (α-SMA),fibronectin (FN),type Ⅰ collagen (Col Ⅰ) and type Ⅲ collagen (Col Ⅲ) mRNA in each group. Results After 72 hours of culture,Pellets grew into clusters.In the upper and lower chambers of the dialysis tubes group,the expression level of α-SMA mRNA was 1.595±0.025 and 1.148±0.009,respectively;FN mRNA was 1.090±0.011 and 0.844±0.015,respectively;Col I mRNA was 1.445±0.035 and 1.165±0.008,respectively;Col Ⅲ mRNA was 1.726±0.031 and 1.314±0.020,respectively;and the ratio of Col Ⅲ/Col Ⅰ was 1.126±0.019 and 0.957±0.013,respectively.The differences between the upper and lower chambers were statistically significant (all P=0.000).In the upper and lower chambers of the non-dialysis tubes group,the expression level of α-SMA mRNA was 1.363±0.018 and 1.360±0.002,respectively;FN mRNA was 0.946±0.017 and 0.952±0.012,respectively;Col Ⅰ mRNA expression was 1.261±0.011 and 1.258±0.029,respectively;Col Ⅲ mRNA was 1.459±0.027 and 1.462±0.033,respectively;and the ratio of Col Ⅲ/Col Ⅰ was 1.157±0.029 and 1.163±0.090,respectively.There was no significant statistical difference between upper and

关 键 词：角膜基质细胞 转化生长因子β 三维培养 创伤修复 瘢痕化 

分 类 号：R772[医药卫生—眼科]