[1]彭昶璠,徐路星,罗世男,等.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[J].眼科新进展,2019,39(4):311-315.[doi:10.13389/j.cnki.rao.2019.0070]
　PENG Chang-Fan,XU Lu-Xing,LUO Shi-Nan,et al.Establishment of a three-dimensional culture system for corneal stromal wound healing in vitro with dynamic spatial distribution of TGF-β[J].Recent Advances in Ophthalmology,2019,39(4):311-315.[doi:10.13389/j.cnki.rao.2019.0070]
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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/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
39卷
期数:
2019年4期
页码:
311-315
栏目:
实验研究
出版日期:
2019-04-05

文章信息/Info

Title:
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
530021　广西壮族自治区南宁市，广西医科大学第一附属医院眼科
Author(s):
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
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
Keywords:
corneal stromal cells; transforming growth factor-β; three-dimensional culture; wound healing; scarring
分类号:
R772
DOI:
10.13389/j.cnki.rao.2019.0070
文献标志码:
A
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0.50 μg·L-1TGF-β1+0.25 μg·L-1TGF-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（collagen Ⅰ，Col Ⅰ）和Col Ⅲ mRNA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，Col Ⅰ mRNA表达量分别为1.445±0.035、1.165±0.008，Col Ⅲ mRNA表达量分别为1.726±0.031、1.314±0.020，Col Ⅲ/Col Ⅰ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，Col Ⅰ mRNA表达量分别为1.261±0.011、1.258±0.029，Col Ⅲ mRNA表达量分别为1.459±0.027、1.462±0.033，Col Ⅲ/Col Ⅰ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、Col Ⅰ和Col Ⅲ mRNA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、Col Ⅰ和Col Ⅲ mRNA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）。结论　Transwell小室系统与透析袋相结合可构建模拟TGF-β空间动态分布的体外三维培养系统。
Abstract:
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 lower chambers of non-dialysis tubes group （all P＞0.05）.The relative expression levels of α-SMA，FN，Col Ⅰ and Col Ⅲ mRNA in the upper chamber of the non-dialysis tubes group were significantly different from those of the upper and lower chambers of the dialysis tubes group （all P＜0.05）.The relative expressions of α-SMA，FN，Col Ⅰ and Col Ⅲ in the lower chamber of the non-dialysis tubes group were also significantly different from those of the upper and lower chamber of the dialysis tubes group （all P<0.05）.Conclusion　The combination of the Transwell chamber system and dialysis tubes is able to establish a three-dimensional culture system which can simulate the spatial dynamic distribution of TGF-β in vitro.

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备注/Memo

备注/Memo:
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）
更新日期/Last Update: 2019-04-15