DRAM基因真核表达载体的构建及其表达对HepG_2细胞的作用

Construction of DRAM gene eukaryotic expression vector and effect of the expression on HepG_2 cells

目的:构建DRAM全长cDNA的真核表达载体pEGFP-DRAM,观察DRAM在人肝细胞HepG2中的作用。方法:应用RT-PCR技术扩增获得DRAM全长cNDA,克隆扩增产物,经酶切、DNA测序鉴定正确后,构成pEGFP-DRAM的真核表达重组质粒。用脂质体将重组质粒转染入HepG2细胞,在激光共聚焦显微镜(LSCM)下观察DRAM的表达情况,用流式细胞仪检测细胞凋亡、细胞周期和死亡细胞,MTS法检测细胞增殖能力,Westernblot鉴定细胞表达蛋白质的水平,并在透射电子显微镜下观察自噬体。结果:构建完成的真核表达重组质粒pEGFP-DRAM,经DNA测序证实与GenBank中的人DRAM cDNA序列一致。LSCM下可观察到转染DRAM的细胞中有绿色荧光蛋白的表达。流式细胞分析仪检测显示,转染DRAM的HepG2死亡细胞明显增高(P<0.05);G1期细胞百分数则明显减少(P<0.05);MTS法检测结果显示转染DRAM的HepG2细胞增殖力明显增高(P<0.05);Western blot检测到DRAM蛋白表达明显增高;转染pEGFP-N1的HepG2细胞超微结构基本正常,转染DRAM的一些细胞中可见自噬体。结论:本研究成功构建了表达DRAM的真核质粒,并在HepG2细胞中表达,DRAM在肿瘤细胞存在双向效应,既可诱导HepG2细胞发生自噬性死亡,又可促进HepG2细胞增殖。

Objective To construct damage-regulated autophagy modulator （DRAM）gene full-length cDNA eukaryotic expression vector pEGFP-DRAM and to assess effect of the expression on human HepG2 cells. Methods The full-length cDNA encoding DRAM gene was amplified by reverse transcription polymerase chain reaction （RT-PCR） and cloned to eukaryotic expression vector pEGFP-N1, the recombinant pEGFP-DRAM was identified by double digestion with restriction endonucleases and sequencing. The recombinant plasmid was transfected into HepG2 cells by lipofectamine method. The expression of DRAM mRNA in HepG2 cells was assayed by laser scanning confocal microscopy （LSCM）. Cell apoptosis,cell cycle and death cell were assessed by flow cytometry, cell proliferation capacity was determined by MTS assay, and autophagosome was detected by transmission electron microscopy. Results For the constructed eukaryocyte expression recombinant plasmid pEGFP-DRAM, DNA sequencing showed that the sequence was identical to that of human DRAM cDNA in GenBank. LSCM demonstrated that cells transfected with recombinant plasmid had expression of green fluorescent protein. Flow cytometry analysis showed that cell death was significantly higher in HepG2 ceils transfected with plasmid pEGFP-DRAM （P〈0.05）. Percentage of cells in G1 phase was significantly lower （P〈0.05）. MTS assay showed that the proliferation capacity of HepG2 cells transfected with plasmid pEGFP-DRAM was significantly higher （P〈0.05）. Ultrastructure of HepG2 cells transfected with pEGFP-N1 was essentially normal, whereas autophagosome could be seen in ceils transfected with pEGFP-DRAM. Conclusions Eukaryotic expression plasmid pEGFP-DRAM is successfully constructed, and could be expressed in HepG2 cells. DRAM has a two-sided effect in tumor cells. In HepG2 cells, DRAM could induce autophagic death, yet on the other side it also could promote cell proliferation.