hTERT基因电转染人γδT细胞建立可诱导的永生化细胞系

Establishment of Inducible Immortalized Human γδT Cell Lines with hTERT Genes by Electrotransfection

利用电击将外源基因hTERT与pTet-on调控质粒共转染人γδT细胞,建立可诱导的永生化人γδT细胞系.分离人PBMC,体外刺激增殖后经磁珠分选纯化,然后对其进行外源hTERT基因和pTet-on调控质粒的共同电转染,并加入强力霉素诱导目的基因表达.电转染程序T-23和T-20的效率分别为37.5%±0.9860%和30.5%±0.5590%.经鉴定,外源hTERT基因能够整合人γδT细胞基因组中并在诱导后表达.程序T-23的转染效率更高,强力霉素的有效诱导浓度是600ng/mL,志愿者本人的血清更利于电转后细胞的存活.

Inducible immortalized human γδT cell lines with exogenous hTERT genes and pTet-on plasmids were established by electrotransfection.Human PBMC were isolated from healthy donors and amplified in vitro.After being amplified for 7 days,human γδT cells were purified by using magnetic beads.The human γδT cells with a higher purity were eletrotransfected with exogenous hTERT genes and pTet-on plasmids.Identifications of these human γδT cells were performed after being treated with Doxycycline for 24 hours.In positive control groups,there were 37.5%±0.9860%(program T-23)and 30.5%±0.5590%(program T-20) human γδT cells expressing GFP when cultured for 4.5 hours after electrotransfection.The results of identifications with PCR and RT-PCR indicated that there were hTERT genes in the genome DNA,and there were transcripts of hTERT genes in RNA of human γδT cells.Program T-23 was the first choice to get a higher efficiency of electrotransfection when compared with program T-20.To induce the expression of hTERT genes effectively,the concentration of Doxycycline should be 600ng/mL.Using the donor's serum to culture γδT cells may help them to survive after electrotransfection.These data suggest that human γδT cells can be electrotransfected with exogenous hTERT genes and the hTERT genes integrated into the genome can be transcribed after being induced by Doxycycline.