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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:

35卷

期数:

2015年5期

页码:

424-428

栏目:

实验研究

出版日期:

2015-05-05

文章信息/Info

Title:

 Effective mechanism of IKBa and MMP-2 in human ciliary muscle cells

作者:

 张弛; 赵刚平; 陈俊杰; 许扬扬; 余建洪; 虞林丽; 胡丹

 528000　广东省佛山市，佛山市第一人民医院眼科

Author(s):

 ZHANG Chi ; ZHAO Gang-Ping ; CHEN Jun-Jie ; XU Yang-Yang; YU Jian-Hong; YU Lin-Li; HU Dan

 Department of Ophthatmology ,the First People ’ s Hospital of Foshan, Foshan 528000 , Guangdong Province , China

关键词:

 ; 人睫状肌细胞; 葡萄膜巩膜通道; RNA干扰; 核转录因子-κB; 基质金属蛋白酶-2

Keywords:

 ; human ciliary muscle cells ;  uveoscleral outflow;  RNA interference;  nuclear factor-kappa B ; matrix metalloproteinase-2

DOI:

10.13389/j.cnki.rao.2015.0115

文献标志码:

A

摘要:

 目的　探讨在人睫状肌（ｈｕｍａｎｃｉｌｉａｒｙｍｕｓｃｌｅ，ＨＣＭ）细胞中，基质金属蛋白酶-２（ｍａｔｒｉｘｍｅｔａｌｌｏｐｒｏｔｅｉｎａｓｅ-２，ＭＭＰ-２）的表达和核转录因子-κＢ（ｎｕｃｌｅａｒｆａｃｔｏｒ-ｋａｐｐａＢ，ＮＦ-κＢ）信号转导通路之间的关系。方法　合成针对人核转录因子抑制因子-α（ｉｎｈｉｂｉｔｏｒαｏｆｎｕｃｌｅａｒｆａｃｔｏｒ-ｋａｐｐａＢ，ＩκＢα）ｓｉＲＮＡ片段，并将其转染入ＨＣＭ中，分别于转染后２４ｈ、４８ｈ、７２ｈ利用ＲＴ-ＰＣＲ和Ｗｅｓｔｅｒｎｂｌｏｔ法检测ＩκＢα、ＮＦ-κＢｐ６５和ＭＭＰ-２ｍＲＮＡ和蛋白水平的表达情况。Ｚｙｍｏｇｒａｐｈｙ技术检测酶原形式的ＭＭＰ-２（ｐｒｏ-ＭＭＰ-２）和活性形式的ＭＭＰ-２（ａｃｔｉｖｅ-ＭＭＰ-２）的表达变化。结果　转染ＩκＢαｓｉＲＮＡ进入ＨＣＭ后２４ｈ、４８ｈ、７２ｈ，ＩκＢαｍＲＮＡ水平和蛋白水平均下降（均为Ｐ＜０．０５）。ＩκＢαｓｉＲＮＡ转染入ＨＣＭ２４ｈ、４８ｈ、７２ｈ，实验组ＮＦ-κＢｐ６５总ｍＲＮＡ水平和蛋白水平变化均无显著差异（均为Ｐ＞０．０５），但细胞核内的ＮＦ-κＢｐ６５蛋白表达却大幅度升高（均为Ｐ＜０．０５）。ＭＭＰ-２ｍＲＮＡ水平和蛋白水平的表达在ＩκＢαｓｉＲ-ＮＡ转染入ＨＣＭ细胞后各时间点均有上升，转染后７２ｈ表达最高（均为Ｐ＜０．０５）。Ｐｒｏ-ＭＭＰ-２和ａｃｔｉｖｅ-ＭＭＰ-２的表达也在转染后各时间点不同程度增加，峰值也是出现在转染后７２ｈ，分别为１７０．２％ ±１０．４％和１５１．８％ ±８．４％（均为Ｐ＜０．０５）。各时间点ｐｒｏ-ＭＭＰ-２、ａｃｔｉｖｅ-ＭＭＰ-２均较空白对照组表达增加（均为Ｐ＜０．０５）。结论　在ＨＣＭ细胞中通过抑制ＩκＢα表达，引起ＮＦ-κＢｐ６５的激活，转位入ＨＣＭ细胞核，促进了ＭＭＰ-２分泌和活化，从而降解ＨＣＭ细胞外基质，增加葡萄膜巩膜房水外流。

Abstract:

 Objective To investigate the relationship between matrix metalloproteinase-2 ( MMP-2) expression and nuclear factor kappa B ( NF-Kb) signal pathway in human ciliary muscle ( HCM) cells. Methods The small interfering RNA ( siRNA) targeting inhibitor of nuclear factor kappa B ( IKBe/ ) was transfected into HCM cells. The mRNA and protein levels of IKBa , NF-KB p65 ,MMP-2 in HCM cells were measured at 24 hours,48 hours and 72 hours after IKBa siRNA transfection by real-time reverse transcription polymerase chain reaction ( RT-PCR) and Western blot. The secretion and activity of MMP-2 were detected by gelatin zymography. Results Transfection of IKBe/ siRNA led to gradual down-regulation of IKBe/ both at the mRNA and protein level at 24 hours ,48 hours and 72 hours ( all P < 0. 05 ) . Either mRNA or protein levels of total NF-KBp65 (P> 0. 05 ) had no obviously changed at 24 hours.48 hours and 72 hours after IKBcC siRNA transfection ( all P > 0. 05 ) ,but the protein level of NF-KB p65 in the nucleus increased dramatically ( all P < 0. 05 ) . The MMP-2 mRNA and protein levels were all increased at each time point after IKBe/ siRNA transfection, which peaked at 72 hours ( all P < 0. 05 ) . The expressions of pro-MMP-2 and active-MMP-2 were all increased at each time point after IKBe/ siRNA transfection, and peaked at 72 hours. which were 170. 2% + 10. 4% and 151. 8% + 8. 4% ( all P < 0. 05 ) . Compared with the control group , the expressions of pro-MMP-2 and active-MMP-2 were all increased at each time point after IKBa siRNA transfection ( all P < 0. 05 ) . Conclusion IKBe/ siRNA enhance the expression and activity of MMP-2 in HCM cells through the activation of NF-KB signal pathway. IKBe/ may therefore be a potential target for controlling the uveoscleral outflow pathway in glaucoma.

备注/Memo

备注/Memo:

 广东省医学科研基金资助（编号：Ｂ２０１４３８５）

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更新日期/Last Update: 2015-04-27