人血管内皮细胞生长因子受体2小干扰RNA的设计及其表达载体的构建

doi:10.3877/cma.j.issn.1673-5250.2007.03.103

中华妇幼临床医学杂志(电子版) ›› 2007, Vol. 03 ›› Issue (03) : 129 -f3. doi: 10.3877/cma.j.issn.1673-5250.2007.03.103

论著

人血管内皮细胞生长因子受体2小干扰RNA的设计及其表达载体的构建

郭海霞, 薛红漫, 黎阳, 孟哲, 陈环, 李文益

中山大学附属第二医院儿科(广州，510120)

出版日期:2007-06-01
基金资助:
广东省科技计划项目(2006B36301003)

Design of human vascular endothelial growth factor receptor 2 siRNA and construction of its expression vector

Hai-xia GUO, Hong-man XUE, Yang LI, Zhe MENG, Huan CHEN, Wen-yi LI

The Department of Pediatrics, the Second Affiliated Hospital, Sun Yat-Sen University, Guangzhou, 510120, China

Published:2007-06-01

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目的

寻找并探讨干扰效果强的人血管内皮细胞生长因子受体2(vascular endothelialgrowth factor receptor 2，VEGFR-2)小干扰RNA (small interference RNA, siRNA)，以抑制白血病细胞。

方法

设计人VEGFR-2 siRNA，并转染HL60细胞，MTT法筛选最有效siRNA；合成VEGFR-2shRNA，并构建VEGFR-2 shRNA表达载体；比较VEGFR-2 shRNA表达载体转染HL60细胞后干扰的效果。

结果

筛选出最有效siRNA; VEGFR-2 shRNA正确正向克隆入pENTR TM/U6，成功构建VEGFR-2 shRNA表达载体；以其将VEGFR-2 shRNA瞬时转染入HL60细胞后，细胞生长抑制率为84.9%，P<0.01;VEGFR-2蛋白表达明显减少。

结论

通过必要和有效的筛选机制，才能找到抑制效果相对高的siRNA靶序列；VEGFR-2 shRNA表达载体可在细胞内简单、快速地克隆shRNA，VEGFR-2的表达抑制率高。

关键词: 小干扰RNA, 血管内皮细胞生长因子受体2(VEGFR-2), HL60细胞, 表达载体

Objective

To search and investigate human vascular endothelial growth factor receptor 2 (VEGFR-2) small interference RNA (siRNA) with strong interfering effect to suppress leukemia cell.

Methods

Designed human VEGFR-2 siRNA and screened the most efficient with MTT method after transfecting HL60 cells ; synthesized shRNA and constructed VEGFR-2 shRNA expression vector ; compared the interfering effects after transfecting HL60 cell with VEGFR-2 shRNA expression vector.

Results

The most efficient siRNA was screened ; VEGFR-2 shRNA was cloned correctly into pENTR TM/U6 and constructed VEGFR-2 shRNA expression vector successfully; transfecting HL60 cell transiently with it, cell inhibitory rate was as high as 84. 9%, P<0. 01; expression of VEGFR-2 protein decreased significantly.

Conclusion

SiRNA target sequence with relatively good inhibitory effect should follow necessary and effective screening principals. VEGFR-2 shRNA expression vector can clone shRNA simply and quickly in cells and has high inhibitory rate of VEGFR-2 expression.

Key words: small interference RNA (siRNA), vascular endothelial growth factor receptor 2(VEGFR-2), HL60, expression vector

图1　VEGFR-2 shRNA表达载体对HL60 VEGFR-2表达影响的Western blot分析

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Design of human vascular endothelial growth factor receptor 2 siRNA and construction of its expression vector

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