Yuan Zhu, Yan Fei, Zhao Xin-Yu, Deng Hong-Xin, Li Jiong
School of Life Science, Sichuan University, Chengdu 610064, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2007 Dec;23(12):1140-3.
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.
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.
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.
Overexpression of mPNAS-4 may have apoptotic effects and therefore inhibit the proliferation of LL2 cells.
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。
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。
成功构建了重组真核表达载体pcDNA3.1(+)-mPNAS - 4。用pcDNA3.1(+)-mPNAS - 4转染的LL2细胞中，mPNAS - 4在mRNA水平的表达显著上调。LL2细胞的增殖受到抑制。
mPNAS - 4的过表达可能具有凋亡作用，从而抑制LL2细胞的增殖。
