Bcg Vaccine With Inactivated Bcg On Immune Function And Anti-leukemia Effect

PartⅠEffect of Bacillus Calmette-Guerin and Non-viable Bacillus Calmette-Guerin on the Culture and Proliferation of Dendritic Cells Derived from Children Acute Leukemia Peripheral Blood in vitroObjective To investigate the effect of Bacillus Calmette-Guerin(BCG) on the culture and proliferation of dendritic cells(DCs) derived from leukemia children acute leukemia peripheral blood in vitro.To compare the BCG and non-viable BCG(NV-BCG).Methods The mononuclear cells were isolated from blood of Leukemia children by the method of Ficoll-Hypaque.The experiment was divided into six groups.①the control group(RPMI1640+1×10~6PBMNCS)②the BCG group(1×10~6PBMNCS+3×10~4cfuBCG)③theNV-BCGgroup(1×10~6PBMNCS+3×10~4cfuNV-BCG)④theGTIgroup(1×10~6PBMNC S+rhGM-CSF,rhTNF-α,rhIL-4)⑤theGTIBgroup(1×10~6PBMNCS+rhGM-CSF,rhTNF-α, rhIL-4+3×10~4cfuBCG)⑥the GTINB group(1×10~6PBMNCS+rhGM-CSF,rhTNF-α, rhIL-4+3×10~4cfu NV-BCG).The numbers of DC from different groups were counted and the phenotypes of DCs were determined by flow cytometry on the ninth day of culture,and some harvest cells were stained with Wright-Giemsa,then observed and photographed under the oil immersion objective.Results①The test groups all attained a certain amount of typical DCs;The number of cells of control group was(0.83±0.25)×10~5/L,the numbers of the test groups were (2.58±0.42)×10~5/L;(2.36±0.38)×10~5/L;(3.63±0.37)×10~5/L;(4.15±0.36)×10~5/L and(3.96±0.35)×10~5/L,they were higher than the control group(p＜0.01).There was no difference between the BCG and the NV-BCG group,but the BCG group was lower than the GTI group,GTIB group and GTINB group(p＜0.01),and there is no difference between the GTI and the GTIB group,GTINB group;there is also no difference between the GTIB and the GTINB group.②The rates of CD1a+ of the BCG group,the NV-BCG group,the GTIB group and the GTINB group were 24.76±7.51,22.98±7.32,36.12±5.16,38.06±4.20 and 37.96±4.12,There was no difference between the BCG group and the NV-BCG group,but was higher than the control group(p＜0.05) and was lower than the GTI group, the GTIB group and the GTINB group(p＜0.05).There was no difference between the GTI and the GTIB group,GTINB group(p＞0.05).There was no difference between the GTIB and GTINB group.The rates of HLA-DR+ of the BCG group,the NV-BCG group,the GTIB group and the GTINB group were 36.73±5.34,35.69±5.11,53.22±1.46,69.29±6.21 and 65.45±5.98,There was no difference between the BCG and the NV-BCG group,but was higher than the control group(p＜0.05) and was lower than the GTI group,the GTIB group and the GTINB group(p＜0.05).The GTI group was lower than the GTIB group,GTINB group(p＜0.05).There was no difference between GTIB and GTINB group(p＞0.05).The rates of CD83+ of the BCG group,the NV-BCG group,the GTIB group and the GTINB group were 30.22±7.63,29.80±7.51,48.11±8.14,62.83±7.46 and 60.59±7.50,There was no difference between the BCG and the NV-BCG group,but was higher than the control group(p＜0.05) and was lower than the GTI group,the GTIB group and the GTINB group(p＜0.05).The GTI group was lower than the GTIB group,GTINB group(p＜0.05).There was no difference between GTIB and GTINB group(p＞0.05).Conclusions1.BCG can promote the proliferation of DCs,but the power of the BCG is lower than that of the isolation of the rhGM-CSF,rhTNF-αand rhIL-4.2.BCG not only promotes the proliferation of DCs derived from children acute leucemia peripheral blood of leukemia patients in vitro,but also cooperates with rhGM-CSF,rhTNF-αand rhIL-4 in promoting the maturity of DCs.3.There is no difference between BCG and NV-BCG in promoting the proliferation and the maturIty of DCs. PartⅡEffect of Bacillus Calmette-Guerin and Non-viable Bacillus Calmette-Guerin on the Immunifaction of Dendritic Cells Derived from Children Acute Leucemia Peripheral BloodObjective To investigate the effect of Bacillus Calmette-Guerin on the immunifaction of Dendritic Cells Derived from Children Leucemia Peripheral Blood.To compare the BCG and non-viable BCG(NV-BCG).Methods The mononuclear cells were isolated from blood of Leukemia children by the method of Ficoll-Hypaque.the experiment was divided into five groups①the control group(the GTI group)(1×10~6/mlPBMNC+rhGM-CSF100ng/ml,rhIL-450ng/ml,rhTNF-α10ng/ml)②the BCG group(1×10~6/mlPBMNC+3×10~4cfuBCG)③the NV-BCG group (1×10~6/mlPBMN+3×10~4cfuNV-BCG)④the GTIB Group(rhGM-CSF,rhTNF-α,rhIL-4 +3×10~4cfuBCG+1×10~6PBMNCS)⑤the GTINB group(rhGM-CSF,rhTNF-α,rhIL-4+ 3×10~4cfu NV-BCG+1×10~6PBMNCS) The growth of DCs was observed under a invert microscope everyday and collected on the 9th day.The DCs of different groups were mixed with T cells which just separated from another acute lymphoblastic leucemia peripheral blood,then they were cultured with rhIL-2+RPMI-1640 for 96 hours,then the numbers of different groups were counted and the contents of IL-12,IFN-γwere tested by ELISA.The DCs of different groups and T cells(Cultured with rhIL-2,PHA50u/ml+ RPMI-1640 for 7 days) and HL-60 cells were Cultured for 72 hours.Cytotoxicity assay was measured by MTT method.Results①Mixed lymphocyte reaction:(1)the stimulation index of T cell When the number of stimulation cells was 1×10~3,the stimulation index of T cell of the GTIB group and GTIB group were 2.56±0.27;2.54±0.25,There was no difference between them,but was higher than GTI group,the BCG group and the NV-BCG group (p＜0.05).Also with the increasing of DCs,the stimulation index of T cell was rising,and was significantly higher than the GTI group,the BCG and the NV-BCG group have no difference which had the same number of DCs.(2) The contains of IL-12,IFN-γWhen the number of stimulation cells was 1×10~4,the contains of IL-12 of the GTIB group and the GTINB group were(79,70±13.55),(77.83±12.51) pg/ml There was no difference between them,but was higher than GTI group,the BCG group and the NV-BCG group (p＜0.05);There was no difference between the BCG and the NV-BCG group.the contains of IFN-γof the GTIB group and the GTINB group were(127.47±22.51), (118.25±20.32) pg/ml,There was no difference between them,but was higher than GTI group,the BCG group and the NV-BCG group(p＜0.05);There was no difference between the BCG and the NV-BCG group.②killing activity on HL-60 cells:Cytotoxicity assay shew that killing activity on HL-60 cells of T cells in BCG group,the NV-BCG group,the GTIB group and the GTINB group was more higher than the GTI group(P＜0.05),the activity of the GTIB group and the GTINB group was the most strong,there was no difference between them,and there was also no difference between the BCG group and the NV-BCG group.Conclusions1.The DCs induced by BCG can particularly enhance the proliferation and secretion of T cells,with the increasing numbers of DCs the power of the proliferation is stronger and the cytotoxicity on HL-60 cells of T cells is higher than the GTI group,reinforce its antigen presenting capability,accordingly enhance the anti-leukemic immune effect of DCs which derived from children acute leucemia peripheral blood.2.It is more notable when BCG combined with rhGM-CSF+rhTNF-α+rhIL-4,to promote the proliferation,secretion of T cells and the activity of killing HL-60 cells3.There is no difference between BCG and NV-BCG in enhancing the proliferation and secretion of T cells and killing the HL-60 cells.4.DCs can be induced by the children acute lymphoblastic leucemia peripheral blood,the CTL cells can secret some IL-12,IFN-γand kill the HL-60 cells well With the help of DCs.