转化生长因子β_1对人巩膜成纤维细胞Smad泛素化调节因子2和Ⅰ型胶原α_1影响的研究

Effect of transforming growth factor β_1 on Smad ubiquitination regulatory factor 2 and collagen type,Ⅰ α_1 in human fetal scleral fibroblasts

目的探讨Smad泛素化调节因子2(Smurf2)在人胚胎眼巩膜成纤维细胞(HFSFs)中的表达,检测转化生长因子β_1(TGF-β_1)影响下HFSFs中Smurf2和Ⅰ型胶原α_1(COLIA1)含量的变化。方法 HFSFs复苏并稳定传代后,用免疫细胞化学法检测细胞中Smurf2的表达。分别用不同浓度的TGF-β_1(0、1 ng/ml、5 ng/ml及10 ng/ml)处理HFSFs 24 h以及10 ng/ml TGF-β_1处理HFSFs不同时长(1 h、6 h、12 h及24 h)后,用实时荧光定量法检测各组Smurf2信使核糖核酸(mRNA)和COLIA1 mRNA表达水平。数据以均数±标准差(±s)描述,不同浓度和不同时长的组间比较采用单因素方差分析,当差异有统计学意义时,进一步采用SNK法两两比较。结果免疫细胞化学检测的结果显示,HFSFs中有COLIA1 mRNA和Smurf2蛋白的表达。与空白对照组比较,TGF-β_110 ng/ml组COLIA1 mRNA的表达呈上升趋势(F=3. 17,P <0. 05),TGF-β_15 ng/ml、10 ng/ml组Smurf2 mRNA表达上调(F=2. 35,2. 00; P <0. 05)。10 ng/ml TGF-β_1干预HFSFs培养1 h、6 h、12 h及24 h后,24 h组COLIA1 mRNA表达显著升高(F=29. 20,P <0. 05)。Smurf2 mRNA表达改变呈先上升后下降趋势,在6 h达到最高(F=10. 65,P <0. 05)。结论 TGF-β_1可能通过调控HFSFs Smurf2的表达,诱导HFSFs COLIA1的合成。

Objective The aim of this study was to investigate the expression of Smad ubiquitination regulatory factor 2(Smurf 2)in human fetal scleral fibroblasts(HFSFs)cells,and the changes of Smurf 2 and collagen typeⅠα1(COLIA1)in HFSFs induced by transforming growth factor-beta1(TGF-β1).Methods After recovery and stable passage of HFSFs,expression of Smurf2 in cells was detected by immunocytochemistry.HFSFs were treated with different concentrations of TGF-β1(0,1 ng/ml,5 ng/ml,10 ng/ml)for 24 hours,respectively,and co-cultured with TGF-β1(10 ng/ml)for 1 hour,6 hours,12 hours,and 24 hours.Then,the expression of COLIA1 messenger RNA(mRNA)and Smurf2 mRNA were detected by reverse transcription-polymerase chain reaction.Data was described by(x±s).One-way ANOVA was used for comparison between groups,then SNK test was used if difference was statistically significant.Results The results showed that COLIA1 mRNA and Smurf2 protein expressed in HFSFs.COLIA1 mRNA expressed an upward trend in 10 ng/ml TGF-β1 group compared with control group(F=3.17,P<0.05),as well as Smurf2 mRNA in 5 ng/ml TGF-β1 group and 10 ng/ml TGF-β1 group(F=2.35,2.00;P<0.05).After 1 hour,6 hours,12 hours,24 hours for HFSFs co-cultured with 10 ng/ml TGF-β1,the expression of COLIA1 mRNA increased significantly in 24 hours group(F=29.20,P<0.05).While,the expression of Smurf2 mRNA increased first and then decreased,reaching the highest level at 6 hours(F=10.65,P<0.05).Conclusion TGF-β1 may induce the synthesis of HFSFs COLIA1 by regulating the expression of HFSFs Smurf2.