[Packaging of lentivirus carrying gene hßc and overexpression of gene hßc in NB4 cells].

ABSTRACT

This study was aimed to overexpress gene hßc in NB4 cells via the method of lentivirus-mediated gene transfer, to observe the differentiation behaviour change of hßc over-expressing NB4 cells treated with IL-3 or GM-CSF, to explore the relationship between hßc gene and the differentiation behaviour of NB4 cells. The targeted hßc gene was amplified by PCR from the cloned vector carrying ORF of hßc. The PCR product containing PmeI and BstBI site introduced by primer was digested, and then cloned into lentivirus vector pRRLSIN.cPPT.PGK/IRES/GFP.WPRE to construct a lentiviral vector carrying hßc, named pLV-hßc. And the pLV-hßc plasmid was confirmed by restriction and sequencing. The recombinant lentivirus was produced by co-transfecting three plasmids into 293T packing cells. After transfection, the lentiviral supernatant was collected to transfect NB4 cells. GFP expression was examined by fluorescent microscope and the expression of hßc gene was detected by Western blot. Then, the NB4 cells over-expressing hßc were treated with IL-3 (10 ng/ml), GM-CSF (10 ng/ml), ATRA (1 µmol/L) respectively, and the CD11b expression, morphology and differentiation behaviour changes of every groups were observed by flow cytometry and microscopy, while NB4 cells transfected with blank lentivirus (NB4-blank cells) were used as controls. The results showed that the recombinant lentivirus vector carrying hßc gene could efficiently transfect NB4 cells and made NB4 cells to stably over-express hßc gene. The expression of CD11b was up-regulated in NB4-hßc cells treated with of IL-3 or GM-CSF, but it was not as obvious as the effect of ATRA, and no morphological change was observed in NB4 hßc cells treated with the IL-3 or GM-CSF. It is concluded that IL-3 or GM-CSF can induce NB4 cells over-expressing hßc to differentiate to neutrophils, but can not make them fully matured.