[关键词]
[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。
[Key word]
[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-β
2 (TGF-β
2).
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.
[中图分类号]
[基金项目]
国家自然科学基金项目(No.81300765)
