Ad.RGD-ING4-PTEN对MEG01人白血病细胞的抑制作用

doi:10.13523/j.cb.20140302

中国生物工程杂志

2014, Vol. 34

Issue (3): 9-17 DOI: 10.13523/j.cb.20140302

研究报告

Ad.RGD-ING4-PTEN对MEG01人白血病细胞的抑制作用

黄晨, 王家融, 杨吉成, 盛伟华, 缪竞诚

苏州大学医学部苏州 215123

The Inhibition of Ad. RGD-ING4-PTEN on MEG01 Human Leukemia Cell

HUANG Chen, WANG Jia-rong, YANG Ji-cheng, SHENG Wei-hua, MIAO Jing-cheng

Medical College of Soochow University, Cell and Molecular Biology Institute, Suzhou 215123, China

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摘要： 目的：构建RGD修饰的 ING4和PTEN 双基因共表达重组腺病毒载体（Ad.RGD-ING4-PTEN），研究其对人白血病细胞MEG01的抑制作用。方法：采用AdEasyTM腺病毒重组系统，在本科室已成功构建的pAdTrack-CMV-ING4-polyA-promoter、pAdTrack-CMV-polyA-promoter腺病毒转移质粒的基础上，在多克隆酶切位点的Not I、Xho I间插入PTEN片段，得到pAdTrack-CMV-ING4-polyA-promoter-PTEN重组转移载体，与RGD-4C修饰的腺病毒骨架质粒pAdEasy-1（RGD）同源重组后，经包装和扩增获得ING4和PTEN双基因共表达重组腺病毒Ad.RGD-ING4-PTEN。将Ad.RGD-ING4-PTEN体外感染人白血病细胞MEG01，检测腺病毒对MEG01细胞的感染效率，Western blot法检测外源基因 ING4和PTEN 在MEG01细胞中的表达，CCK8法检测外源基因的导入对MEG01细胞的生长抑制作用，流式细胞仪检测外源基因导入对MEG01细胞的凋亡和周期的影响。结果：鉴定结果显示，成功构建了Ad.RGD-ING4-PTEN双基因共表达腺病毒，其能有效地感染MEG01细胞。Western blot检测到 ING4和PTEN 在MEG01细胞中的表达； ING4和PTEN 基因能明显抑制MEG01细胞的生长，诱导其凋亡并产生G2/M期阻滞作用，且双基因联合作用较单基因作用更明显。结论：成功构建了RGD-4C修饰的ING4和PTEN双基因共表达腺病毒载体Ad.RGD-ING4-PTEN。收获的腺病毒能有效感染人白血病MEG01细胞。Ad.RGD-ING4-PTEN具有抑制MEG01细胞的生长、诱导其凋亡及G2/M期阻滞的作用。

关键词： 腺病毒; ING4; PTEN; 白血病

Abstract: Objective: To construct a recombinant adenoviral vector modified by RGD-4C co-expressing inhibitor of growth 4 ( ING4 ) and phosphatase and tensin homolog deleted on chromosome ten (PTEN) and to study its inhibitory effects on human leukemia cell line MEG01. Methods:The recombinant adenovirus Ad.RGD-ING4-PTEN was obtained by gene recombination and in vitro packaging technique. Firstly, between the restriction enzyme cutting sites NotⅠand XhoⅠ, the PTEN fragment were inserted respectively, at the foundation of pAdTrack-CMV-ING4-polyA-promoter and pAdTrack-CMV-PolyA-promoter. Secondly, the transfered vector linearized by Pme I digestion and the backbone vector modified by RGD-4C were further co-transformed into the bacteria BJ5183 for homologous recombination. Lastly, the QBI-293A cell was used for packaging and amplification. The human leukemia cell line MEG01 was infected with Ad.RGD-ING4-PTEN. The infection efficiency of adenovirus to MEG01 was detected by flow cytometry. The expression of ING4 and PTEN was detected by Western blot. Cell growth inhibition was detected by CCK-8 assay. The cell apoptosis and the cell cycle were detected by flow cytometry. Results: The results of identification test show that the successful build of Ad.RGD-ING4-PTEN,the adenovirus which can express the double gene of ING4 and PTEN.It can infect MEG01 cell line effectively. The expression of ING4 and PTEN in MEG01 is obvious; ING4 and PTEN gene can significantly inhibit the growth of MEG01, induce its apoptosis and produce cycle arrest, and the fuction of double gene combination is more obvious than Single gene. Conclusion:Adenoviral vector modified by RGD-4C co-expressing ING4 and PTEN were successfully constructed. RGD-4C can significantly improve the efficiency of the infection to human leukemia cell line MEG01. Ad.RGD-ING4-PTEN can inhibit the cell growth of MEG01 and induce its apoptosis and block the cell cycle.

Key words: Adenovirus ING4 PTEN Leukemia

收稿日期: 2013-12-24 出版日期: 2014-03-25

ZTFLH:

Q813

基金资助: 国家自然科学基金资助项目（81001016）

通讯作者: 缪竞诚 E-mail: jcmiao@163.com

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引用本文:

黄晨, 王家融, 杨吉成, 盛伟华, 缪竞诚. Ad.RGD-ING4-PTEN对MEG01人白血病细胞的抑制作用[J]. 中国生物工程杂志, 2014, 34(3): 9-17.

HUANG Chen, WANG Jia-rong, YANG Ji-cheng, SHENG Wei-hua, MIAO Jing-cheng. The Inhibition of Ad. RGD-ING4-PTEN on MEG01 Human Leukemia Cell. China Biotechnology, 2014, 34(3): 9-17.

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https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140302 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I3/9

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