Effect Of Bovine Lactoferricin On The Proliferation, Differentiation And Gene Expression Level Of Jurkat Cell

Bovine Lactoferricin(LfcinB) is a kind of active peptides in milk, and has cytotoxic effect for a wide variety of tumor cells, but has no effect on normal cellls. LfcinB was choosed to affect Jurkat cells in this experiment, by determinting its influence on Jurkat cells proliferation and differentiation, and the expression level of miRNA19b and miRNA223to research the effect of LfcinB induction Jurkat cell differentiation and proliferation inhibition, which provide theoretical and experimental basis for developing LfcinB into a creature type of antitumor drugs. MethodsThe LfcinB concentrations were set as follows:LfcinB was dissolved in RMPI-1640and the LfcinB concentrations were set as follows:100、200、300、400μg/mL.Using the above concentrations of LfcinB dealing with Jurkat cells, and blank control group was set up without adding LfcinB stimulating logarithmic phase cells, experimental groups medium was added rapamycin (RAPA) only. MTT was used to determine the effect on multiplication capacity of Jurkat cells after different concentrations of LfcinB cultivation with Jurkat cells24h,48h and72h;The cell cycle distribution and cell surface differentiation antigen CDllb expression level were analysised by flow cytometry after different concentrations of LfcinB cultivation with Jurkat cells for24h and48h; Morphological changes of Jurkat cell nucleus effected by LfcinB were analysed through flurescenceinverted microscope after treated24h; NBT was used to measure the effect of LfcinB on Jurkat cell differentiation level; The expression level of miRNA19b and miRNA223was determined through RT-PCR after treated36h by different concentrations of LfcinB. Results(1) MTT results showed that LfcinB could inhibit Jurkat cell proliferation by dose dependence and time dependence;(2) LfcinB could induce Jurkat cell cycle stagnation at G0/G1phase, and improved the expression level of cell surface differentiation antigen CDllb with dose and time dependent. Those indicated that LfcinB could induce Jurkat cell differentiation and inhibit proliferation which maybe through interfereing with the cell cycle process to block cell cycle at G0/G1phase;(3) Fluorescent inverted microscope observations results showed, that the.blank group cells appeared morphology complete, cells nucleus shading evenly and Fluorescence was diffuse, but the experimental groups cells were chromatin condensation and nucleus are cracking, and appeared apoptotic body and cell apoptosis phenomenon;(4) NBT reduction results showed that LfcinB could significantly improved LfcinB cells OD value, by dose dependence and time dependence.This OD value reflected cells reducing capacity, also reflected cells differentiated degree indirectly. So the above results suggested LfcinB could induce Jurkat cell differentiation;(5) RT-PCR results suggested miRNA19b expression level decreased, and miRNA223expression level rise, with dose dependent. Conclutions(1)100～400μg/mL LfcinB could inhibit Jurkat proliferation and induce apoptosis significantly, and its proliferation inhibition ratio and induction apoptosis effect rise as LfcinB concentration increasing.300μg/mL is the best concentration for LfcinB inhibiting Jurkat proliferation.Inducing cell apoptosis may be one of the pathways for LfcinB inhibiting cell proliferation.(2)100～400μg/mL LfcinB could induce Jurkat cell cycle stagnation at G0/G1, suggesting that LfcinB mayby block DNA synthesis by interfering with the cell cycle process to inhibit cell proliferation.(3) LfcinB could significantly improved LfcinB cells OD value, suggesting LfcinB could induce Jurkat cell differentiation, and speculated that LfcinB maybe inhibit Jurkat proliferation by inducing cell differentiation.(4)100～400μg/mL LfcinB could improved miRNA223expression level, and reduce miRNA19b expression level significantly, suggesting LfcinB could induce Jurkat cell differentiation and inhibit proliferation by regulating differentiation and proliferation microRNA.(5) It coexisted that LfcinB induced Jurkat cell differentiation and inhibited proliferation, maybe LfcinB could anti leukemia by inducing differentiation and inhibiting proliferation.