人羊膜匀浆上清液对兔角膜上皮细胞中bFGF表达的影响

Impact of human amniotic homogenate supernatant on bFGF expression in rabbit corneal epithelial cells

目的:观察不同浓度人羊膜匀浆上清液对体外培养的兔角膜上皮细胞碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)表达的影响,探讨人羊膜匀浆上清液在促进角膜上皮损伤修复中的作用。方法:将兔角膜上皮细胞离体培养并传代,然后分组。实验组分别在含100mL/L胎牛血清的DMEM培养液中分别加入终浓度为40,80,160μg/mL的人羊膜匀浆上清液,对照组仅用100mL/L胎牛血清的DMEM培养液。分别在培养24,48和96h后,采用甲基噻唑基四唑(methyl thiazolyl tetrazolium,MTT)比色法观察人羊膜匀浆上清液对兔角膜上皮细胞增殖的影响。同时,采用RT-PCR技术检测三个不同等级浓度对兔角膜上皮细胞bFGF-mRNA表达的影响。结果:培养24h后,与对照组(0.087±0.013)比较,加入终浓度为40,80,160μg/mL人羊膜匀浆上清液的实验组角膜上皮细胞增殖数量(分别为0.185±0.010,0.318±0.015,0.501±0.014)明显增加,差异具有显著性(P<0.05);同时,随着作用时间的延长,差异愈加明显;加入40μg/mL和80μg/mL浓度羊膜匀浆上清液组兔角膜的bFGF-mRNA(分别为0.750±0.007,0.785±0.006)和空白对照组(0.708±0.013)比较,差异无显著性意义(P>0.05),但160μg/mL浓度羊膜匀浆上清液组(1.013±0.120)与其它两组及对照组(0.708±0.013)比较,差异均具有显著性(P<0.05)。结论:人羊膜匀浆上清液具有促进兔角膜上皮细胞增殖的作用,并随着人羊膜匀浆上清液所含蛋白浓度的增加,其促进作用增强;人羊膜匀浆上清液具有促进体外培养的兔角膜上皮细胞bFGF-mRNA表达的作用,但与羊膜匀浆上清液的浓度有关。

AIM：To investigate the expression level of rabbit corneal epithelial cells＇ basic fibroblast growth factor （bFGF） in vitro under the condition of using different concentration of human amniotic homogenate supernatant, and to discuss the function of human amniotic homogenate supernatant in the process of corneal epithelial repair.METHODS：The rabbit corneal epithelial cells were cultured in vitro and they were divided into several groups. Then the experiment group was treated in DMEM culture liquid of 100mL/L fetal blood serum mixed with different concentration of human amniotic homogenate supernatant （the amniotic homogenate protein concentration was 40,80,160μg/mL respectively）, with the control group treated in DMEM culture liquid of 100mL/L fetal blood serum only. By using the method of methyl thiazolyl tetrazolium（MTT） colorimetry, we aim to investigate the impact of human amniotic homogenate supernatant on rabbit corneal epithelial cells proliferation at different times （24, 48, 96 hours）. At the same time, we tested the bFGF-mRNA expression level in each group by RT-PCR.RESULTS：Rabbit corneal epithelial cells proliferated at different concentrations of human amniotic homogenate supernatant （40, 80, 160μg/mL）, the amount of corneal epithelial cells in each experimental group was increased significantly （0.185±0.010, 0.318±0.015, 0.501±0.014 respectively, P0.05）. As the culture time prolonged, the difference became more apparent; There was no significant difference about the expression of bFGF-mRNA between the control（0.708±0.013） and the amniotic homogenate supernatant with the concentration of 40μg/mL and 80μg/mL （0.750±0.007, 0.785±0.006 respectively）, but the differences about the expression of bFGF-mRNA between the 160μg/mL group（1.013±0.120） and the 40, 80μg/mL, control group were significant respectively.CONCLUSION：Human amniotic homogenate supernatant can significantly stimulate the proliferation of rabbit corneal epithelial cells and its effect shows dose-dependant; Human amniotic homogenate supernatant could promote the expression of the bFGF-mRNA in rabbit corneal epithelium cells in vitro, which is concentration related.