[新型凋亡相关小鼠基因mPNAS-4真核表达载体的构建、表达及其体外抗肿瘤活性]

[Construction and expression of eukaryotic expression vector of a novel apoptosis-related mouse gene, mPNAS-4 and its antitumor activity in vitro].

作者信息

Yuan Zhu, Yan Fei, Zhao Xin-Yu, Deng Hong-Xin, Li Jiong

机构信息

School of Life Science, Sichuan University, Chengdu 610064, China.

出版信息

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2007 Dec;23(12):1140-3.

PMID:18062887

Abstract

AIM

To construct eukaryotic vector of a novel apoptosis-related gene from mouse (mPNAS-4) and overexpress it in mouse Lewis lung carcinoma(LL2) cell line. To explore the apoptosis of the tumor cells induced by overexpressed mPNAS-4 in LL2 cells via transfection.

METHODS

RT-PCR was applied to amplify the encoding region of mPNAS-4 gene from the mouse liver. The cDNA was cloned into the eukaryotic expression vector pcDNA3.1(+) and the resulting recombinant expression plasmid pcDNA3.1(+)-mPNAS-4 was then transfected into mouse Lewis lung carcinoma (LL2) cells through Lipofectamine 2000. Overexpression of mPNAS-4 in the transfected cells and proliferation of the transfected cells was detected by RT-PCR and MTT assay,respectively, while the apoptosis was analysed by FCM and DNA ladder.

RESULTS

The recombinant eukaryotic expression vector pcDNA3.1(+)-mPNAS-4 was successfully constructed.The expression of mPNAS-4 at the mRNA level in LL2 cells transfected with pcDNA3.1(+)-mPNAS-4 was up-regulated significantly. The proliferation of LL2 cells was inhibited.

CONCLUSION

Overexpression of mPNAS-4 may have apoptotic effects and therefore inhibit the proliferation of LL2 cells.

摘要

目的

构建小鼠新型凋亡相关基因（mPNAS - 4）的真核表达载体，并使其在小鼠Lewis肺癌（LL2）细胞系中过表达。通过转染探讨过表达的mPNAS - 4诱导LL2细胞中肿瘤细胞凋亡的情况。

方法

应用逆转录 - 聚合酶链反应（RT - PCR）从小鼠肝脏中扩增mPNAS - 4基因的编码区。将该cDNA克隆到真核表达载体pcDNA3.1(+)中，然后通过脂质体2000将所得重组表达质粒pcDNA3.1(+)-mPNAS - 4转染到小鼠Lewis肺癌（LL2）细胞中。分别通过RT - PCR和MTT法检测转染细胞中mPNAS - 4的过表达情况和转染细胞的增殖情况，同时通过流式细胞术（FCM）和DNA梯带分析细胞凋亡情况。