[小干扰RNA沉默结缔组织生长因子基因对p27蛋白表达的影响]

[Effects of small interfering RNA approach to silence the connective tissue growth factor gene on the expression of the protein p27].

作者信息

Chen Hong, Zhang Ming-chang, Zhang Hong-xu

机构信息

Department of Ophthalmology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.

出版信息

Zhonghua Yan Ke Za Zhi. 2010 Jul;46(7):631-4.

PMID:21054972

Abstract

OBJECTIVE

To investigate the effects of the small interfering RNA (siRNA) approach to silence the expression of connective tissue growth factor (CTGF) gene expression on the expression of the protein p27.

METHODS

Experimental research. The specific siRNA of CTGF was designed and synthesized, then transfected into cultured bovine corneal endothelial cells by liposomes. The blank control group was only treated with the culture medium and not be transfected with siRNA. Reverse transfection-polymerase chain reaction (RT-PCR) was applied to show the effect of gene-silencing, and western blot was used to observe the protein p27 expression of bovine corneal endothelial cells, which were treated with 1.00 µg/L transforming growth factor β(2) (TGF-β(2)). The results were analyzed by one-factor analysis of variance.

RESULTS

CTGF siRNA was successfully transfected into cultured bovine corneal endothelial cells and effectively silence the expression of CTGF mRNA. The CTGF mRNA expression decreased to 17.3%, 24.4% and 41.7% of control values at 24, 48 and 72 h after transfection, there was significant difference between these groups (F = 389.9, P < 0.05). Significant decrease of protein p27 expression was detected at 36 h after transfection (F = 299.3, P < 0.05).

CONCLUSION

CTGF-specific siRNA was able to inhibit the expression of CTGF mRNA effectively and down-regulate the expression of p27.

摘要

目的

探讨小干扰RNA（siRNA）沉默结缔组织生长因子（CTGF）基因表达对蛋白p27表达的影响。

方法

实验研究。设计并合成CTGF特异性siRNA，然后通过脂质体转染培养的牛角膜内皮细胞。空白对照组仅用培养基处理，不转染siRNA。采用逆转录聚合酶链反应（RT-PCR）显示基因沉默效果，并用蛋白质印迹法观察用1.00μg/L转化生长因子β(2)（TGF-β(2)）处理的牛角膜内皮细胞中蛋白p27的表达。结果采用单因素方差分析。

结果

CTGF siRNA成功转染至培养的牛角膜内皮细胞，并有效沉默CTGF mRNA的表达。转染后24、48和72小时，CTGF mRNA表达分别降至对照值的17.3%、24.4%和41.7%，各组间差异有统计学意义（F = 389.9，P < 0.05）。转染后36小时检测到蛋白p27表达显著降低（F = 299.3，P < 0.05）。