| ObjectiveTo explore the positive effects of interleukin21combined with interleukin6on anti-leukemic activity though acting on regulatory T cells and cytotoxic activity of cytokine induced killer cells derived from umbilical cord blood.Methods1. Mononuclear cells were separated from fresh umbilical cord blood of healthy gravida and cultured with respective cytokines to establish CIK cell culture system.2. CD3+CD56+CIK cell sorting was operated by flow cytometry.3. Cytotoxic activity of CD3+CD56+CIK cells against to K562and HL-60cells in the presence of IL-21and IL-6was detected by CCK-8assay.4. Proliferation of cells in each group was was measured by CCK-8assay.5. Cytotoxic activity of cells from the CIK culture system cell against K562and HL-60cells in the presence of IL-21and IL-6was detected by LDH release assay.6. Immunophenotype of cells from the system was detected by flow cytometry.Results1. The cytotoxic activity of CD3+CD56+CIK cells against K562and HL-60cells in IL-21+IL-6group at the ratio of20:1was the highest one at (59.63±2.13)%,(45.72±3.85)%respectively, which was significantly higher than any other groups (P<0.01), while cytotoxic activity between in IL-21group [(48.37±3.06)%.(35.49±2.72)%] and in IL-6group [(47.09±1.25)%,(33.99±1.83)%] was similar. However, cytotoxic activity in all the experimental groups was higher than that in the control group which was at [(40.68±1.91)%,(23.77■3.01)%].2. IL-21and IL-6could enhance proliferative activity of cells from the CIK culture system separately or in conjuction, and the proliferative activity could reflected by the absorbance values detected by CCK-8assay in each group. The result showed that no difference existed in IL-21group and IL-6group about proliferative activity, howerer proliferative activity in IL-21+IL-6was much stronger than that in any other group.3. The cytotoxic activity of cells from the CIK cell culture system against K562 and HL-60.cells in control group at the ratio of20:1was (29.31±0.58)%,(20.14±1.27)%respectively, which was significantly lower than the experimental groups (P<0.01), while cytotoxic activity in IL-21+IL-6group [(63.79±0.91)%,(41.53±1.43)%] was higher than IL-21group [(52.99±1.26)%,(33.04±1.68)%] and IL-6group [(53.05±1.52)%,(32.18±3.83)%](p<0.01), but no difference existed between IL-21group and IL-6group.4. On the seventh day of cell culture system, compared with control group, proportion of CD3+CD56+CIK cells in IL-21group was increased from (7.30±1.20)%to (12.52±1.45)%, and increased to (11.01±1.04)%in IL-6group, and increased to (20.8±2.33)%in IL-21+IL-6group; however, expression of Treg cells in IL-21group was decreased from (26.18±1.03)%to (10.95±1.49)%, and decreased to (17.91±1.95)%in IL-6group, and proportion was decreased to (5.25±0.54)%in IL-21+IL-6group; on the fourteenth day, proportion of CD3+CD56+CIK cells in IL-21and in IL-6group was2times than the control group, and the proportion was increased to2.5times in IL-21+IL-6group compared with control group. While the expression of Treg cells in IL-21group was decreased from (9.24±0.42)%to (1.48±0.06)%, and decreased to (3.96±0.11)%IL-6group, and expression of Treg cells was decreased to (0.83±0.08)%in IL-21+IL-6group.ConclusionIL-21and IL-6could enhance the proliferative and cytotoxic activity of cells from CIK cell culture system derived from umbilical cord blood, meanwhile the significant reduction in proportion of Treg cells and increase in proportion of CD3+CD56+CIK cells indicate their enhancement to function of CIK cells in different aspects, moreover the enhanced cytotoxic activity of CD3+CD56+CIK against to leukemic cell line showed that IL-21and IL-6could act directly on effector cells in the culture system. And all the effect was stronger when IL-21and IL-6were used in conjuction. Thus IL-21and IL-6could strengthen anti-leukemic effect th rough directly acting on CIK cells and indirectly decreasing Treg cell expression. |
PDF Full Text Request