ABSTRACT
Objective To construct the human IL-24 recombined adenovirus(Ad-IL24)and to observe the effect of Ad-IL24 on ex vivo culture of HL-60 cells.Methods pAdTrack-CMV-IL24 was constructed by PCR with pcDNA3.0-IL24 recombined plasmid as a template,enzyme digestion and ligation.The pAdTrack-CMV-IL24 lineared by Pme Ⅰ was co-transformed into BJ5183 with pAdEasy-1.The pAdEasy-1-pTrack-CMV-IL24 recombined adenovirus vector was lineared with Pac Ⅰ and then transfected in to QBI-293A cells.The Ad-IL24 was obtained and used to infect HL-60 ceils,and the effect on HL-60 cells was tested by LSCM,Mrrr,FCM,ELISA and immunohistochemistry staining.Results The pAdEasy-1-pTrack-CMV-IL24 vector was constructed and the Ad-IL24 was successfully obtained.The effect of inhibiting growth of HL-60 cells and inducing cell apoptosis by IL-24 was proved by LSCM,MTT and FCM.IL-24 down-regulated the expression of anti-apoptosis factor Bcl-2 and increased the secretion of IFN-γ,TNF-α of HL-60 cells.Conclusion Ad-IL24 can inhibit the growth of HL-60 cells and induce cell apoptosis through down-regulating expression of anti-apoptosis factor and increasing the secretion of IFN-γ,TNF-α of HL-60 cells.The human Ad-IL24 may provide a basic study for the future target therapy to tumors.
ABSTRACT
Objective:To construct the cells which can express LIF protein steadily and study the effect of rhLIF and IL-24 on HL-60 cells.Methods:ECV-304 cells were infected by eukaryotic expression plasmid pcDNA3-LIF,then the positive cells were selected by G418.The positive cells were collected and their culture supernatant was stored for further use.Expression of LIF mRNA was detected by RT-PCR.The pAdeasy-1-pAdTrack-CMV-IL-24 was extracted from DH5a.The recombined adenovirus vector was lineared with PacI and transfected into 293 cells.The IL-24 recombined adenovirus(Ad-IL-24) was obtained and used to infect HL-60 cells.At the same time,the culture supernatant containing rhLIF was added into the HL-60 cells which was identified as positive expression of IL-24 by RT-PCR.Synergistic effect of the cytokines on HL-60 cells was tested by LSCM, FCM and immunohistochemistry stain assay.Results:The cells expressing LIF protein steadily were constructed successfully, the high titer of the recombined adenovirus(Ad-IL-24) was obtained. Expression of IL-24 in infected HL-60 cells was identified by RT-PCR.The apoptosis of HL-60 cells induced by rhLIF and IL-24 was proved by LSCM ,FCM and immunohistochemistry stain assay. They had synergistic effect.Conclusion:rhLIF and IL-24 can inhibit growth of HL-60 cells and induce apoptosis of the cells.They have synergistic effect.