| Objective: The mononuclear cells were extracted from the patients, peripheral blood, and then were cultured as dendritic cells(DC) loaded with WT1 peptide. The cytokine-induced killer cells were induced in vitro, and the killing activity to WT1 positive K562 cells were detected after the DC and the CIK mixed culture, DC and CIK cell proliferation and immune film marked change were observed.Methods: DC cells culture: Peripheral blood with heparin anti-coagulation were collected from WT1 gene positive acute leukemia patients. The mononuclear cells were isolated by H- F(Hypaque- Fieoll) lymphocyte separation medium(relative density of 1.077±0.001), were washed three times by RPMI 1640 culture. Cell concentration was adjusted to 4×106 per ml, after four hours’ incubation, adherent cells were collected with the cell concentration to 1×106 per ml, then cells were cultured at 37℃, 5% CO2 with human granulocyte macrophage colony stimulating factor(rh- granulocyte- macroph- age, Colony- stimulating Factor, rh GM- CSF) 1000 U/ml, human recombinant interleukin 4(rh- interleukin- 4, rh IL –4) 1000 U/ml. According to the conventional cultivation, medium was half changed every two days, with cytokines added, then the cells were count. On the fifth day of DC cell cultivation joined WT1 peptide so that part of the DC cells became the WT1 antigen peptide-loaded group, the rest part became the non-WT1 antigen peptide-loaded group, on the sixth day DC cell cultivation, recombinant human tumor necrosis factor-a(rh-tumor necrosis factor- a, rh TNF-a) 1000 u/ml was added, to induce the DC to maturation.CIK cells cultivation: the homologous suspension cells appealed to, which were washed three times by RPMI 1640 broth. The cell concentration adjusted to 4×106 per ml. After four hours, incubation, suspension cells were collected with cell concentration to 1×106 per ml with RPMI 1640 medium which containing 10% fetal calf serum, then were cultured at 37 ℃, 5%CO2 with different cytokines on day 1 and day 2. The replacement of the new culture medium with rh IL-2 were added every two days, the cells were counted at the same time. On the seventh day, CIK cells and DC cells were collected, and living cells were counted by trypan blue exclusion method, with the DC cells concentration to 1×106 per ml and CIK cells concentration to 5×106 per ml in RPMI1640 liquid containing 10% FBS. The two kinds cells were mixed to culture, according to the ratio of the DC and CIK cells for l: 5. On the first day, 3th day, 7th day after co-cultivation, the mixture were collected, counted the biological activities were detected.Results:1 Cell proliferation between the WT1 antigen peptide-loaded group and non-WT1 antigen peptide-loaded group were compared.After incubation the adherent mononuclear cells by GM-CSF and IL-4-induced cells into DC, the training process significant changes in cell morphology, cell volume also increased. In the first two days of cultivation, the cells became larger, the surface cytoplasmic appeared projections with irregular morphology. With time extending, cell surface protrusions increased longer and clustered into a group of growth(see Fig.1).Suspension mononuclear cells were induced by stimulation of various cytokines, a part of lymphoid cells was amplified into CIK cells and gradually form a cell pellet; the cell body was smaller than DC, initially scattered in uniform distribution, small size, bright and round(See Fig.2). In the first four days of cultivation, the cells began to show an irregular shape, and showed clusters growth(see Fig.3). After the sixth day of cultivation, the cells proliferated significantly, clumped into colonies growing state, and suspended in culture medium(see Fig.4). As the incubation time went by, the cells clumped and cell colonies increased.Co-culture DC and CIK cells into DC-CIK cells on the seventh day of cultivation, different groups were compared with their changes of immune phenotype and biological activity. On the first day of cell co-culture, viable cell were counted by trypan blue exclusion method. The proliferation of multiple DC-CIK cells of WT1 antigen peptide-loaded group was(10.01±0.71), and the DC-CIK cells of non WT1 antigen peptide-loaded group was(10.20±0.60). On the last day of cell co-culture, DC-CIK cells of WT1 antigen peptide-loaded group was(17.16±0.93) times the original, while the DC-CIK cells of non-WT1 antigen peptide-loaded group was(17.40±0.94). Proliferation of multiple comparative analysis of the same group of cells increases over time were statistically significant(Table 1). The WT1 antigen peptide-loaded DC cells and the non-WT1 antigen peptide-loaded DC cells both could assistant CIK cells proliferation rapidly, and there was little different between tow groups, and the data showed that whether the tumor antigen peptide loaded or not did not have significant affect on DC-CIKs, proliferation.2 Comparison of cell molecular marker phenotype between the WT1 antigen peptide-loaded group and non-WT1 antigen peptiede-loaded groupCells were collected on the 1st day, 7th day and 14 th day to detect molecular marker phenotype by flow cytometry. The results showed that CD3 +CD8 + cells were(30.34±3.14)% before cultivation, CD3 + CD56 + cells were(5.49±0.45)%; DC-CIK cells of WT1 antigen peptide-loaded group for the 7th day and 14 th, the immune phenotype test results were as follows: CD3 + CD8 + cells were(40.25±2.66)%,(76.48±4.08)%, CD3 + CD56 + cells were(9.48±1.06)%,(29.11±4.27)%; and DC-CIK cells of non-WT1 antigen peptide-loaded group results were as follows: CD3 + CD8 + cells were(39.61±2.51)%,(75.61±6.27)%, CD3 + CD56 + cells were(9.44±1.10)%,(29.11±4.91)%(Table 2). The data showed that DC-CIK cells of CD3+CD8+ and CD3+CD56+ were significantly increased, and there was no significant differents between the two DC-CIK groups(P>0.05), and the data showed that whether the tumor antigen peptide loaded or not did not have significant affect on DC-CIKs, molecular marker phenotype.3 Comparison of cell cytotoxic activity between the WT1 antigen peptide-loaded group and non-WT1 antigen peptide-loaded group.DC-CIK cells were collected on the 14 th day of cultivation to detect the killing activity. DC-CIK cells were collected from the WT1 antigen peptide-loaded group as effector cells, and the target cells were K562 cells of WT1 gene positive and HL60 cells of WT1 gene negtive, the cytotoxic activity of the effector to target ratio within the range of 5:1 to 20:1, K562 cell killing activity results:(38.95±2.03)%,(59.48±3.97)%,(69.19±1.52)%, simultaneous detection efficiency in the corresponding target ratio of HL60 cells with killing results were(39.18±1.96)%,(60.44±2.79)%,(69.56±1.45)%(see table 3), the differents between the two groups were significant(P<0.05).DC-CIK cells were collected from the non-WT1 antigen peptide-loaded group as effector cells, and the target cells were K562 cells of WT1 gene positive and HL60 cells of WT1 gene negtive, the cytotoxic activity of the effector to target ratio within the range of 5:1 to 20:1, K562 cell killing activity results:(38.95±2.03)%,(59.48±3.97)%,(69.19±1.52)%, simultaneous detection efficiency in the corresponding target ratio of HL60 cells with killing results were(39.95±1.45)%,(60.88±1.30)%,(70.08±1.07)%(see table 4), and there was no significant differents between the two groups(P>0.05).The data showed that DC-CIK of two groups both had killing effects to HL60 cells of WT1 gene negtive, and there was no statistical significance. DC-CIK of two groups both had killing effects to K562 cells of WT1 gene positive, and the WT1 antigen peptide-loaded group was stronger than non-WT1 antigen peptide-loaded group, the differences between the two groups have statistical significance, showed that WT1 antigen peptide-loaded group,s specific cytotoxicity on tumor cells.Conclusions:1 Applicate peripheral blood mononuclear cells of acute leukemia patients could develop DC-CIK cells by standard culture method, and had killing activity.2 WT1 antigen peptide-loaded group and non-WT1 antigen peptide-loaded group after co-cultured, DC-CIK cells of CD3+CD8+ and CD3+CD56+ double-positive proportion was significantly increased, there was no significant difference.3 DC-CIK cells originated in WT1 antigen peptide-loaded group and non-WT1 antigen peptide-loaded group after co-cultured,both had killing effects to HL60 cells of WT1 gene negtive, and there was no statistical significance.4 DC-CIK of two groups both had killing effects to K562 cells of WT1 gene positive, and the WT1 antigen peptide-loaded group was stronger than non-WT1 antigen peptide-loaded group, the differences between the two groups had statistical significance, showed that WT1 antigen peptide-loaded group,s specific cytotoxicity on tumor cells. |
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