Bcg Study On The Impact Of Children With Leukemia, Peripheral Blood Dendritic Cells

Objective To investigate the effect of Bacillus Calmette Guerin(BCG) on the culture and proliferation of dendritic cells(DC) derived from peripheral blood of Leukemia Children in vitro.Methods The mononuclear cells were isolated from peripheral blood of Leukemia Children with Ficoll-Hypaque method and cultured with RPMI-1640 in the control group.The test groups was divided into three subset groups,they were respectively BCG(include BCG only),GTI(include cytokines only),GTIB(include GTI and BCG).Then PBMCs were induced into DCs.The growth of DC was observed under a light microscope.We determined the phenotypes of DC by flow cytometry on the 8th day of culture,then observed and photographed them under a light microscope.Results 1.On the 8^th day of the culture,all the experimental groups attained some number of typical DC.The number of the BCG group((2.69±0.46)×10⁶/L),GTI group((3.83±0.42)×10⁶/L),GTIB group((4.22±0.28)×10⁶/L)was higher than that of the control(t value is respectively 3.92,7.90,11.40,P <0.05 ).The number of the BCG group cells was lower than that of the GTI group(t=3.17,P<0.05 ).There was no siginificant difference betweenGTI and GTIB(t=1.34,P>0.05).2.On the 8^th day of the culture,CD1a positive cell rate of the BCG,GTI and GTIB group was respectively(15.64±2.16)%,(30.18±2.49)%,(34.03±2.62)%,which was higher than that of the control(t value is respectively3.29,11.43,13.06,P<0.05).CD1a positive cell rate of the BCG was lower than that of the GTI group(t=6.31,P<0.01 ).There was no siginificant difference between GTI and GTIB group(t=1.84,P> 0.05).HLA-DR positive cell rate of the BCG,GTI and GTIB group was respectively(32.28±3.82)%,(43.12±5.43)%,(56.69±4.26)%,while CD83 positive cell rate of the BCG,GTI and GTIB group was (30.62±4.12)%,(45.64±3.26)%,(53.90±3.87)%.HLA-DR and CD83 positive cells rate of the BCG group was higher than that of the control(t value is respectively 5.81,5.29, P<0.01),but it was lower than those of the GTI group(t value is respectively 2.82,4.95,P<0.05).HLA-DR and CD83 positive cells rate of the GTI group were lower than those of the GTIB group(t value is respectively 3.40,2.82,P<0.05)Conclusion 1.Bacillus Calmette Guerin can induce production of the typical DCs derived from human peripheral blood of Leukemia Children in vitro,which shows Bacillus Calmette Guerin can promote the proliferation of DC,though the effect of Bacillus Calmette Guerin is worse than GTI.2.Bacillus Calmette Guerin can increase the rate of HLA-DR and CD83 positive cells, especially in the GTIB group.It is showed that Bacillus Calmette Guerin can co-operates with rhGM-CSF,rhTNF-αand rhIL-4 in promoting the maturity of DC derived from peripheral blood of Leukemia Children. Objective To investigate the effect of Bacillus Calmette Guerin on the anti-leukemia immune effect induced by dendritic cells derived from peripheral blood of Leukemia Children.Methods The mononuclear cells were isolated from peripheral blood of Leukemia Children with Ficoll-Hypaque method,which was distilled into the concentration of 10⁶/ml and separated into six groups:Group A(GTI) was cultured with PBMC+rhGM-CSF+rhTNF-α+ rhIL-4,Group B(BCG) was divided into three subset groups(B1,B2 and B3),which were cultured with PBMC +differet concentration of BCG(respectively 25ul,50ul,75ul).;Group C (GTI+BCG) was cultured with PBMC+rhGM-CSF+rhTNF-α+rhIL-4+ BeG,while Group D(control group)was cultured with PBMC+ RPMI-1640.Then PBMCs were induced intoDCs.The growth of DC was observed under a light microscope.We determined the phenotypes of DC by flow cytometry on the 8th day of culture,then mixed DC with HL-60 cells and T lymphocytes derived from paracmastic childs who suffered from acute myeloblastic leukemia to induce antigen specific cytotoxic T cells.Cytotoxicity assay was measured by MTT method.Results 1.The CD1a positive cell rate in groupB1,B2,B3 were respectively(14.98±3.24)%,(15.64±3.12)%,(15.89±4.06)%,and so there was no siginificant diference among the three subset groups.The CD1a positive cell rate in group A,B,C and D were(23.18±2.49)%,(15.60±3.39)%,(28.03±2.62)%,all of which were obviously different from the control group(t value is respectively 7.75,3.09,10.00,P< 0.05).2.The HLA-DR positive cell rate in groupB1,B2,B3 were respectively(31.43±4.32)%,(32.28±3.82)%,(32.03±4.53)%,and so there was no siginificant diference among the three subset groups.The HLA-DR positive cell rate in group A,B,C and D were (43.12±5.43)%,(32.10±4.20)%,(56.69±4.26)%,all of which were obviously different from the control group(t value is respectively 6.71,4.54,11.55,P<0.05).3.The CD83 positive cell rate in groupB1,B2,B3 were respectively(29.86±3.89)%,(30.62±4.12)%,(30.86±3.05)%,and so there was no siginificant difference among the three subset groups.The CD83 positive cell rate in group A,B,C and D were(45.64±3.26)%,(30.23 ±3.72)%,(54.60±4.16)%,all of which were obviously different from the control group(t value is respectively 11.50,4.25,11.68,P<0.05).4.The cytotxicity for killing of HL-60 cells in groupB1,B2,B3 were respectively (59.32±2.51)%,(60.51±2.25)%,(60.36±1.61)%,and so there was no siginificant difference among the three subset groups.The cytotxicity in group A,B,C and D were (51.32±1.77)%,(60.18±2.20)%,(70.09±0.65)%,all of which were obviously different from the control group(t value is respectively 7.96,12.62,29.69,P<0.01).The cytotxicity from group B was stronger than group A(t=5.44,P<0.01).The cytotxicity from group C increased most significantly,much higher than group A,B(t value is respectively 17.24,7.48,P<0.01,).Conclusion 1.BCG can improve the maturity of DC in peripheral blood of lukemia children.2.BCG can hence the anti-leukemia immune response mediated by DC of the leukemia children peripheral blood.3.The concentration of BCG has no effect on the maturity of DC and the anti-leukemia immune response mediated by DC in peripheral blood of lukemia children.