| Objective: We establish the method of culturing dendritic cells from C57BL/6 mouse bone marrow in vitro. We make use of repeated freeze-thaw method to prepare FBL-3 leukemic cell tumor antigen and utilize the antigen to sensitize DC. We investigate that the interleukin-12 in sensitized DC supernatant can induce differentiation of mouse erythroleukemia cell FBL-3,and observe that the IL-12 monoclonal antibody block the induction of differentiation. Further we explore the mechanism involved in the differentiation of FBL-3 cells induced by DC supernatant. At the same time, this research will offer a new method of immunotherapy for leucocythemia.Methods:①With interleukin-4 and GM-CSF, the cells from mouse bone marrow were cultured. The morphological changes of DCs were observed under light microscope and electron microscope after culture. The expressions of CD80,CD86,H-2Kd and I-Ad were tested by flow-cytometric analysis;②Conventional culture and to collect FBL-3 cells, to make use of repeated freeze-thaw methed to prepare FBL-3 leukemic cell tumor antigen and to collect and reserve it for future use;③To cultural the FBL-3 cells tumor antigen together with DCs for five days, so we can sensitize the DC cells;④To detect the concentration of interleukin-12 in the sensitized DCs supernatant with the enzyme-linked immunospecific assay (cell density 2.0×106/ml);⑤Divide into four groups: standard IL-12 group (positive control group, A group); The prepared sensitized DC supernatant of the 8th day (sensitized DC group, B group); The prepared sensitized DC supernatant of the 8th day with the added IL-12 monoclonal antibody (Blocked experimental group, C group); RPMI-1640 group(negative control group, D group). FBL-3 cells were incubated separately with the four groups for 72 hours.Then we use the Wright's staining methed to record the mature monocyte cell population, to use the transmission electron microscope to observe the ultramicrostructure , to use the flow cytometry to detect the positive expression rate of the surface molecular CD14. Then we observe the differences of the four groups.Result:①After culturing mouse bone marrow cells with interleukin -4 combined GM-CSF, we can get the cells with typical DC characteristics.②The density of interleukin-12 in sensitize DC (cell density 2.0×106/ml)cultivate supernatant at the eighth day is (699.77±16.67)pg/ml.③Count the ratio of transformed to mature monocytes by Wright's stain: the ratio of negative control group is (3.06±1.41)% ,the ratio of blocked experimental group is (17.11±1.25)%, and the result of comparison between the two groups is P<0.05; The ratio of sensitized DC group is (46.03±2.41)%, comparison with the negative control group and the blocked experimental group the results are P<0.05; The ratio of positive control group is(48.07±1.96)%, Comparison with the negative control group and the blocked experimental group the results are P<0.05.④The ratio of transformed to mature monocytes between transmission electron microscope and Wright's stain is basically consistent.⑤Flow cytometry analysis showed: In the negative control group, there is rare the CD14 molecule expressing ,and the CD14 positive rate is (3.24±1.39)%; At the same time , the CD14 positive rate of the positive control group, the sensitized DC group and the blocked experimental group is respectively(49.39±1.88) %,(48.28±1.10) %,and (18.02±0.92)%. According to the results of ANOVA statistical analysis: there is no differences (P>0.05) between the sensitized DC group and the positive control group, but there are significant differences (P<0.05= in the sensitized DC group and the positive control group separately comparison with the negative control group and the blocked experimental group; At the same time, there are significant differences (P<0.05) between the negative control group and the blocked experimental group.Conclusion:①Cultivation of mouse bone marrow cells by interleukin-4 associated with GM-CSF could produce many mature DCs in keeping with its character.②Sensitized DCs can secrete a lot of IL-12.③After being induced by the supernatant of the sensitized DC, the FBL-3 cells can partly differentiate into mature monocytes, and the mature monocytes differentiated are consistent with the personal characteristics.④IL-12 monoclonal antibody can block the differentiation of the sensitization DC culture supernatant.⑤There are some other substances which can make FBL-3 differentiation,in sensitized DC culture supernatant.
|
PDF Full Text Request