摘要
目的:对小鼠凋亡相关新基因PNAS-4(mPNAS-4)进行克隆,构建其真核表达载体,并在小鼠Lewis肺癌细胞系LL2中转染表达;探讨mPNAS-4基因转染LL2细胞过表达所诱导的体外肿瘤细胞凋亡情况。方法:用RT-PCR从小鼠肝脏组织中克隆mPNAS-4编码区cDNA,构建重组真核表达载体pcDNA3.1(+)-mPNAS-4;用脂质体将pcDNA3.1(+)-mPNAS-4转染小鼠Lewis肺癌LL2细胞;用RT-PCR检测转染细胞中mPNAS-4的过表达情况;通过MTT、流式细胞术(FCM)及DNALadder分别检测转染细胞的增殖与凋亡情况。结果:从小鼠肝脏组织中克隆到mPNAS-4全长cDNA并成功构建其真核表达载体pcDNA3.1(+)-mPNAS-4,转染小鼠Lewis肺癌LL2细胞可使其mRNA表达明显上调。mPNAS-4过表达能抑制LL2细胞增殖并诱导其凋亡。结论:mPNAS-4过表达对小鼠Lewis肺癌LL2细胞的生长有明显的抑制和诱导凋亡作用。
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 overpressed 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 Upofectamine 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 mFINA 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.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2007年第12期1140-1143,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家重点基础研究发展计划(973)资助项目(2005CB522506)
关键词
PNAS-4
基因转染
真核表达
凋亡
PNAS-4
gene transfection
eukaryotic expression
apoptosis
