利用线粒体膜电位测定筛选参与细胞凋亡的人类新基因

细胞凋亡(apoptosis)属于细胞程序化死亡(programmed cell death),是细胞内涉及到许多生化反应的复杂过程.建立了基于细胞水平的凋亡筛选模型,用于筛选人类基因组中功能未知的序列,以发现与细胞凋亡相关的新基因.通过构建人类未知基因的表达文库,并将未知基因表达载体瞬时转染HeLa细胞,用阳离子染料JC-1标记HeLa细胞线粒体内膜并检测线粒体跨膜电位,用流式细胞术进行阳性结果的验证.经过对未知基因表达文库内600个新基因的筛选,得到7个线粒体跨膜电位下降相关新基因(CHMP6、CGI-38、hCAP-H2、NUDT16L1、ARMC1、PHF17和FLJ21103),经实验验证,其中3个基因(CHMP6、CGI-38和hCAP-H2)与细胞凋亡相关.结果表明,所建立的基于细胞的凋亡筛选模型稳定高效,3个细胞凋亡相关基因将被进行深入研究.

Screening and Validation of Human Novel Genes Associated With Cell Apoptosis

With the success of human genome project, a large number of predicted genes were sequenced, requiring functional assays for their characterization in a high-throughput manner. To identify novel human genes associated with cell apoptosis, a high-throughput assay was established. Candidate sequences were amplified and cloned in pcDNA3.1/myc-His(-)B, then were transfected into HeLa cells respectively. The expression vector encoding BAX was used as the positive control, which was wildly-known to effectively induce programmed cell death. JC-1 staining was utilized to assess the mitochondrial membrane potential, which could collapse in the early stage of apoptosis. 600 human novel genes were screened and seven positive genes (CHMP6, CGI-38, hCAP-H2, NUDT16L1, ARMC1, PHF17, and FLJ21103) were found out. A subsequent validation by flow cytometry revealed that three of the seven genes (CHMP6, CGI-38, hCAP-H2) were with functions related to cell apoptosis. In HeLa cells transfected with the above three expression vectors, the proportion of single annexin-V-positive cells was evidently increased (8.01%, 6.88%, 5.01%) compared with PCDB-transfected cells (3.43%). Bioinformatics analysis of the three positive genes reveals that their functions are known little and the relationships between the three genes and cell apoptosis have not previously been reported. These results therefore indicate that a rapid and effective screening system has been studied. Further studies will perform on the 3 genes associated with cell apoptosis.