[通过短发夹RNA抑制bcl-2以诱导人晶状体上皮细胞凋亡]

[Inhibition of bcl-2 by shRNA to induce apoptosis in human lens epithelial cells].

作者信息

Wu Xin-Hua, Lu Yi, Fang Yan-Wen, Jiang Yong-Xiang, Tian Jie

机构信息

Department of Ophthalmology, Eye and ENT Hospital, Fudan University, Shanghai 200031, China.

出版信息

Zhonghua Yan Ke Za Zhi. 2009 Jul;45(7):636-40.

PMID:19957693

Abstract

OBJECTIVE

To investigate whether apoptosis of human lens epithelial cells (HLEC) can be induced by inhibition of bcl-2 shRNA.

METHODS

It was an experimental study. Two pairs of oligonucleotides were synthesized and inserted into plasmid PGCsi to generate shRNA eukaryotic expression vectors, named P1 and P2. At 48 hours after transfection in HLEC (SRA01/04) with Lipofectamine 2000, the whole cell protein was extracted and detected by Western blot. The bcl-2 mRNA level was detected by real-time PCR. The percentage of HLECs undergoing apoptosis was measured by Annexin V-PI staining. The activity of caspase-3 was analyzed by Western blotting.

RESULTS

The shRNA eukaryotic expression vectors were constructed successfully. At 48 hours after transfection, the rate of transfection of P1 and P2 was about 44.1% and 47.2% respectively. The protein and mRNA level of bcl-2 was 0.435 and 0.476, greatly downregulated (F = 1672.4, P < 0.05). The percentage of HLEC undergoing apoptosis was 42.3% and 45.4%, greatly improved (F = 1756.2, P < 0.05). The activity of caspase-3 was greatly improved.

CONCLUSION

P1 and P2 can both down-regulate the expression of bcl-2, and induce the apoptosis of HLEC.

摘要

目的

研究抑制bcl-2的短发夹RNA（shRNA）能否诱导人晶状体上皮细胞（HLEC）凋亡。

方法

本研究为实验性研究。合成两对寡核苷酸并插入质粒PGCsi以构建shRNA真核表达载体，命名为P1和P2。用脂质体2000转染人晶状体上皮细胞（SRA01/04）48小时后，提取全细胞蛋白并进行蛋白质免疫印迹检测。采用实时荧光定量聚合酶链反应检测bcl-2 mRNA水平。用膜联蛋白V-碘化丙啶染色法检测HLEC凋亡率。通过蛋白质免疫印迹分析半胱天冬酶-3的活性。

结果

成功构建了shRNA真核表达载体。转染48小时后，P1和P2的转染率分别约为44.1%和47.2%。bcl-2的蛋白和mRNA水平分别为0.435和0.476，显著下调（F = 1672.4，P < 0.05）。HLEC凋亡率分别为42.3%和45.4%，显著提高（F = 1756.2，P < 0.05）。半胱天冬酶-3的活性显著增强。