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中华眼科医学杂志(电子版) ›› 2019, Vol. 09 ›› Issue (01) : 14 -20. doi: 10.3877/cma.j.issn.2095-2007.2019.01.003

论著

转化生长因子β1对人巩膜成纤维细胞Smad泛素化调节因子2和Ⅰ型胶原α1影响的研究
陈丽娟1, 陈婷2, 陈雪兰2, 叶文文2, 黄丽娟2, 胡建民2,†()   
  1. 1. 362000　泉州，福建医科大学附属第二医院眼科　视障辅助技术福建省高校工程研究中心；363000　漳州卫生职业学院眼视光技术教研室
    2. 362000　泉州，福建医科大学附属第二医院眼科　视障辅助技术福建省高校工程研究中心
  • 收稿日期:2019-01-27 出版日期:2019-02-28
  • 通信作者: 胡建民
  • 基金资助:
    福建省自然科学基金资助项目(2015J01382); 福建省医学创新课题(2011-cxb-23); 福建省卫生系统中青年骨干人才培养项目(2015-ZQN-ZD-25)

Effect of transforming growth factor β1 on Smad ubiquitination regulatory factor 2 and collagen type Ⅰ α1 in human fetal scleral fibroblasts

Lijuan Chen1, Ting Chen2, Xuelan Chen2, Wenwen Ye2, Lijuan Huang2, Jianmin Hu2,†()   

  1. 1. Department of Ophthalmology, the Second Affiliated Hospital of Fujian Medical University, Quanzhou 362000, China; Department of Optometry, Zhangzhou Health Vocational College, Zhangzhou 363000, China
    2. Department of Ophthalmology, the Second Affiliated Hospital of Fujian Medical University, Quanzhou 362000, China
  • Received:2019-01-27 Published:2019-02-28
  • Corresponding author: Jianmin Hu
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引用本文：

陈丽娟, 陈婷, 陈雪兰, 叶文文, 黄丽娟, 胡建民. 转化生长因子β1对人巩膜成纤维细胞Smad泛素化调节因子2和Ⅰ型胶原α1影响的研究[J]. 中华眼科医学杂志(电子版), 2019, 09(01): 14-20.

Lijuan Chen, Ting Chen, Xuelan Chen, Wenwen Ye, Lijuan Huang, Jianmin Hu. Effect of transforming growth factor β1 on Smad ubiquitination regulatory factor 2 and collagen type Ⅰ α1 in human fetal scleral fibroblasts[J]. Chinese Journal of Ophthalmologic Medicine(Electronic Edition), 2019, 09(01): 14-20.

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β1(TGF-β1)影响下HFSFs中Smurf2和Ⅰ型胶原α1(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-β1(0 、1 ng/ml、5 ng/ml及10 ng/ml)处理HFSFs 24 h以及10 ng/ml TGF-β1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-β1 10 ng/ml组COLIA1 mRNA的表达呈上升趋势(F＝3.17，P<0.05)，TGF-β1 5 ng/ml、10 ng/ml组Smurf2 mRNA表达上调(F＝2.35，2.00；P<0.05)。10 ng/ml TGF-β1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-β1可能通过调控HFSFs Smurf2的表达，诱导HFSFs COLIA1的合成。

关键词: Smad泛素化调节因子2, 人胚胎眼巩膜成纤维细胞, 转化生长因子β1 Smad通路, 近视
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 type Ⅰ α1 (COLIA1) in HFSFs induced by transforming growth factor-beta 1 (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 (±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.

Key words: Smad ubiquitination regulatory factor 2 (Smurf2), Human fetal scleral fibroblasts, Transforming growth factor-β1 (TGF-β1) Smad pathway, Myopia
表1 人Smurf2、COLIA1信使核糖核酸检测用引物序列
引物 引物序列(5′-3′)
Smurf2 上游：5′-TTGCTAAGGTGGTGGTTG-3′
下游：5′-AAGCGTATTCTTCACAGTATCT-3′’
COLIA1 上游：5′-CGGCTCCTGCTCCTCTTA-3′
下游：5′-GGTGGGATGTCTTCGTCTT-3′
β肌动蛋白 上游：5′-TGACGTGGACATCCGCAAAG-3′
下游：5′-CTGGAAGGTGGACAGCGAGG-3′
图3 不同时长转化生长因子β1干预对人胚胎眼巩膜成纤维细胞Ⅰ型胶原α1信使核糖核酸和Smad泛素化调节因子2信使核糖核酸表达的影响　3A图示实时逆转录-聚合酶链反应检测不同时长转化生长因子β1干预对人胚胎眼巩膜成纤维细胞Ⅰ型胶原α1信使核糖核酸相对表达量的影响；3B图示实时逆转录-聚合酶链反应检测不同时长转化生长因子β1干预对人胚胎眼巩膜成纤维细胞Ⅰ型胶原α1信使核糖核酸相对表达量影响。*与对照组比较，P<0.05。
表2 不同浓度TGF-β1干预对HFSFs COLIA1 mRNA和Smurf2 mRNA表达的影响
组别 COLIA1 mRNA相对表达量 Smurf2 mRNA相对表达量
空白对照组 1.00±0.02 1.00±0.02
1 ng/ml组 0.80±0.01 0.87±0.10
5 ng/ml组 1.20±0.06 1.35±0.02*
10 ng/ml组 2.40±0.06* 1.19±0.02*
F值 2.45 2.34
P值 <0.05 <0.05
表3 不同时长TGF-β1干预对HFSFs COLIA1 mRNA和Smurf2 mRNA表达的影响
组别 COLIA1 mRNA相对表达量 Smurf2 mRNA相对表达量
空白对照组 1.00±0.31 1.00±0.48
1 h组 1.51±0.18 0.92±0.16
6 h组 1.48±0.13 1.60±0.18*
12 h组 1.90±0.21 1.36±0.11
24 h组 2.42±0.18* 1.12±0.17
F值 4.45 3.32
P值 <0.05 <0.05
图4 Smad泛素化调节因子2降解通路的作用底物　图示Smad泛素化调节因子2对TGF-β Smad信号通路中负向调节蛋白的泛素化降解机制Smad泛素化调节因子2蛋白在TGF-β1 Smad信号通路中的可能作用
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