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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.
The hTERT and pTet-on regulatory plasmids were co-transfected into human γδT cells by electric shock to establish inducible immortalized human γδT cell lines.People were isolated from human PBMCs and stimulated with magnetic beads for proliferation in vitro and then subjected to magnetic particle sorting Co-transfection of exogenous hTERT gene and pTet-on regulatory plasmid, and the addition of doxycycline to induce the target gene expression.The efficiency of electroporation T-23 and T-20 programs were 37.5% ± 0.9860% and 30.5% ± 0.5590% .It has been identified that the exogenous hTERT gene can be integrated into the genome of human γδT cells and expressed after induction.The transfection efficiency of program T-23 is higher, the effective induction concentration of doxycycline is 600ng / mL, Serum is more conducive to cell survival after electroporation.