Effects Of Lentinan On The Proliferation And Surface Markers Of γδT Cells

Objective To establish a T cell culture system induced by cytokine differentiation in healthy adult peripheral blood,and to study the effects of lentinan of different concentrations(0 μg/ ml,5 μg/ ml,10 μg/ ml,25 μg/ ml,50 μg/ ml,75 μg/ ml)on the proliferation and surface marker expression of human T cells in vitro.Methods Peripheral blood mononuclear cells(PBMC)were isolated from 3 healthy adult peripheral blood samples under aseptic conditions,and human T cells were amplified in vitro with zoledronic acid(Zol)and interleukin-2(IL-2).In 10 days later,gamma delta T cells can be divided into 6 groups,respectively in different concentration of Lentinan(0 μg/ml,5 μg/ml,10 μg/ml,25 μg/ml and 50 μg/ml,75μg/ml),marked as blank group,the LNT-I group,the LNT-II group,LNT-group,the LNT III-IV group,the LNT-Ⅴgroup.After 72 h induction culture,the following studies were conducted :(1)MTT method was used to detect the effect of lentinan at different concentrations on the proliferation rate of T cells;(2)Flow cytometry was used to detect the effect of lentinan of different concentrations on the purity of T cells and the expression of CD3,CD44 and CD107 a.Results(1)On the 10 th day of culture in vitro,lentinan of different concentration was added for 72 hours.The cell proliferation rates of LNT-I group,LNT-II group,LNT-III group,LNT-IV group and LNT-V group were(23.35±1.69)%,(31.79±1.46)%,(4.58±1.11)%,(-9.22±1.70)%,(-13.65±2.98)%,respectively.Comparison between the experimental group and control group and experimental group,the difference was statistically significant(P < 0.05).Lentinan at 5,10,25μg/ml could significantly promote the proliferation of gamma-delta T cells 72 hours later,while Lentinan at 50,75 μg/ml inhibited the proliferation of gamma-delta T cells.Lentinan at 10 μg/ml had the highest effect on the proliferation rate of gamma-delta Tcells,which was the best concentration of cell growth promoting liquid.Most of the gamma-delta T cells in LNT-IV group and LNT-V group had died.LNT-III group was set as the final experimental group.(2)After 72 hours of co-culture with lentinan of different concentrations,(1)the expression of cell marker Anti-TCR-gamma Delta in the blank group,LNT-I group,LNT-II group and LNT-III group was(49.05±3.02)%,(50.33±4.81)%,(58.34±3.58)%,(69.90±4.55)%.The expression of cell surface marker Anti-TCR-gamma Delta in the 5μg/ml lentinan group was different from that in the blank control group,but there was no statistical difference(P>0.05).The expression of gamma-delta T in 10 and 25 μg/ml experimental groups was higher than that in blank group and 5 μg/ml lentinan group,and the difference was statistically significant(p < 0.05).(2)The cell surface marker CD3 of blank group,LNT-I group,LNT-II group and LNT-III group were(84.59±3.78)%,(93.38±2.32)%,(96.33±1.13)%,(94.58±2.59)%.The cell surface marker CD3 of lentinan group was higher than that of blank control group(p < 0.05),but there was no significant difference among the experimental groups(P > 0.05).(3)The cell surface marker CD44 of blank group,LNT-I group,LNT-II group and LNT-III group were(33.53±3.10)%,(44.78±2.48)%,(48.37±3.05)%,(54.68±3.20)%.the experimental group surface marker expression of CD44 was better than control group,the difference was statistically significant(p < 0.05),of which 5,10 μg/ml lentinan group cell surface expression of CD44 below 25 μg/ml lentinan group,The difference was statistically significant(P < 0.05),while there was no statistically significant difference between the 5 and 10 μg/ ml lentinan group(P>0.05).(4)The cell surface markers CD107 a in blank group,LNT-I group,LNT-II group and LNT-III group were(19.54±4.09)%,(19.45±3.28)%,(15.63±2.26)%,(13.31±2.25)%,respectively.Although the expression of CD107 a was different between the experimental groups and between the experimental group and the blank control group,the difference was not statistically significant(p > 0.05).Conclusion(1)Lentinan can promote the proliferation of gamma-delta T cells invitro in a certain concentration range,but high concentration of Lentinan can inhibit the proliferation of cells.The optimum concentration of lentinan for inducing gamma-delta T cells was 10 ug/ml.(2)Lentinan at 10 and 25 μg/ml could promote the expression of anti-TCR-gamma-delta on the surface of gamma-delta T cells;Lentinan could promote the expression of CD3 on the surface of gamma-delta T cells,but not related to the concentration of Lentinan;Lentinan could promote the expression of CD44 on the surface of gamma-delta T cells,especially at 25μg/ml;Lentinan had no significant effect on the expression of CD107 a on the surface of gamma-delta T cells.