ILK SiRNA对TGF-β2诱导的人Tenon囊成纤维细胞增殖和凋亡的影响
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国家自然科学基金项目(No.81300765)


Effect of ILK SiRNA on the proliferation and apoptosis of human Tenon fibroblasts induced by transforming growth factor-β2
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National Natural Science Foundation of China(No.81300765)

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    摘要:

    目的:探讨小干扰RNA(the small interference RNA,SiRNA)沉默整合素链接激酶(integrin-linked kinase,ILK)基因对转化生长因子-β2(transforming growth factor beta 2,TGF-β2)诱导的人Tenon囊成纤维细胞(human Tenon fibroblasts,HTFs)增殖和凋亡的影响。

    方法:体外培养HTFs细胞,波形蛋白(Vimentin)和角蛋白(keratin)免疫荧光染色进行鉴定。实验分组包括NC组:正常对照HTFs; H+T组:HTFs+5μg/L TGF-β2; H+T+NC SiRNA组:HTFs+5μg/L TGF-β2+50nmol/L阴性对照SiRNA; H+T+ILK SiRNA组:HTFs+5μg/L TGF-β2+50nmol/L ILK SiRNA。50nmol/L ILK SiRNA转染HTFs细胞,5μg/LTGF-β2刺激后,分别利用Western-blot检测细胞内ILK的蛋白表达,CCK-8检测细胞增殖能力,Hoechst 33342/PI双染检测细胞凋亡。

    结果:Western-blot结果显示,TGF-β2可以明显上调HTFs细胞内ILK的表达,ILK SiRNA可以明显抑制TGF-β2诱导的ILK蛋白表达(P<0.05)。CCK-8检测结果显示,5μg/L TGF-β2可以促进HTFs细胞增殖,ILK SiRNA转染后48h,细胞的增殖明显受到抑制,且低于正常对照组(P<0.05)。Hoechst 33342/PI双染结果显示,TGF-β2并不诱导HTFs细胞发生凋亡(P>0.05),但ILK SiRNA可以诱导HTFs细胞发生明显凋亡,并出现少量坏死细胞(P<0.05)。

    结论:ILK SiRNA可以抑制TGF-β2诱导的HTFs增殖,并诱导HTFs细胞发生凋亡。

    Abstract:

    AIM: To investigate the role of small interference RNA interference targeted Integrin-linked kinase(ILK SiRNA)on the proliferation and apoptosis of human Tenon fibroblasts(HTFs)induced by transforming growth factor-β2 (TGF-β2).

    METHODS: The HTFs were identified by immunofluorescence analysis with Vimentin and keratin. HTFs with no other addiction was as normal control; H+T group: HTFs+5μg/L TGF-β2; H+T+NC SiRNA group: HTFs+5μg/L TGF-β2+50nmol/L negtive SiRNA; H+T+ILK SiRNA group: HTFs+5μg/L TGF-β2+50nmol/L ILK SiRNA. The ILK SiRNA were transfected into HTFs by lipofectamine 2000, then the cells were stimulated with 5μg/L TGF-β2. The protein expression of ILK were analyzed by Western Blot. The proliferation levels of HTFs were analyzed by CCK-8, the apoptosis of HTFs were analyzed by Hoechst 33342/PI double staining.

    RESULTS: The protein ILK were expressed in both TGF-β2 treated and control groups, and TGF-β2 up-regulated the expression of ILK, ILK SiRNA inhibited the protein expression of ILK(P<0.05). CCK-8 analysis showed that compared with the normal control group, the cell proliferation rate of HTFs in TGF-β2 treated group increased, and in ILK SiRNA group the cell proliferation rate was suppressed after exposured to ILK SiRNA for 48h(P<0.05). Hoechst 33342/PI double staining showed that there was no change on the apoptosis of TGF-β2 stimulated group(P>0.05), compared with the normal control group, however in the ILK SiRNA group, we found lots of apoptosis cells and a few of necrotic cells(P<0.05).

    CONCLUSION: The ILK SiRNA attenuates the abnormal proliferation of HTFs induced by TGF-β2, thereby enhancing the apoptosis of HTFs.

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王群,崔丽珺,刘思伟,等. ILK SiRNA对TGF-β2诱导的人Tenon囊成纤维细胞增殖和凋亡的影响.国际眼科杂志, 2018,18(4):621-625.

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  • 收稿日期:2017-11-24
  • 最后修改日期:2018-03-13
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  • 在线发布日期: 2018-03-26
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