【作者】 赵梅；

【导师】 叶欣；

【作者基本信息】 泰山医学院 ， 老年医学， 2009， 硕士

【摘要】 目的:建立体外培养扩增C57BL/6小鼠树突状细胞(dendritic cell ,DC )的方法;应用反复冻融法制备小鼠红白血病FBL-3肿瘤细胞抗原并致敏DC;观察致敏DC培养上清对小鼠红白血病细胞FBL-3的诱导分化作用;观察IL-12单克隆抗体阻断致敏DC培养上清对小鼠红白血病细胞FBL-3的诱导分化作用;进而探讨DC培养上清诱导白血病细胞分化作用的机制,为白血病的诱导分化治疗提供一条新的途径。方法:①应用1ng/ml白介素-4(interleukin , IL-4)和10ng/ml粒细胞—巨噬细胞集落刺激因子(granulocyte-macrophage colony stimulating factor ,GM-CSF)联合诱导培养C57BL/6小鼠骨髓细胞,光镜和电镜观察DC发育过程中形态学变化;流式细胞术检测DC表面分子CD80、CD86、H-2Kb及I-Ab的表达情况;②常规培养并收集C57BL/6小鼠诱导的红白血病FBL-3细胞,反复冻融FBL-3细胞制备肿瘤抗原,收集备用;③制备的FBL-3肿瘤抗原与培养的C57BL/6小鼠DC共孵育,致敏DC;④用ELISA法检测致敏DC第8天培养上清中IL-12的浓度(细胞浓度2.0×106/ml);⑤分四组:标准IL-12组(阳性对照组,A组),致敏DC第8天的培养上清组(致敏DC组,B组),加入IL-12单克隆抗体的致敏DC第8天的培养上清组(阻断实验组,C组),RPMI-1640组(阴性对照组,D组)。四组分别与FBL-3细胞共孵育72小时后,用瑞氏染色计数成熟单核细胞、透射电镜观察成熟细胞的超微结构、流式细胞仪检测细胞表面CD14分子的阳性表达率,观察四组有何不同。结果:①用IL-4和GM-CSF联合培养C57BL/6小鼠骨髓细胞,可得到符合DC特征的典型细胞;②ELISA法检测致敏DC培养第8天DC(细胞浓度2.0×106/ml)上清中IL-12的浓度为:(699.77±16.67)pg/ml;③瑞氏染色计数成熟单核细胞的比率:阴性对照组转化率为(3.06±1.41)%,阻断实验组单核细胞比率为(17.11±1.25)%,两者比较P<0.05;致敏DC组单核细胞比率为(46.03±2.41)%,与阴性对照组和阻断实验组比较P均<0.05;阳性对照组单核细胞比率(48.07±1.96)%,与阴性对照组和阻断实验组比较P均<0.05,与致敏DC组比较P>0.05;④透射电镜计数成熟单核细胞的比率与瑞氏染色计数成熟单核细胞的比率基本相同;⑤流式细胞仪分析显示:阴性对照组细胞基本无CD14表达,表达率为(3.24±1.39)%;阳性对照组、致敏DC组、阻断试验组作用后均有部分细胞表达CD14分子,CD14阳性率分别为(49.39±1.88) %、(48.28±1.10) %、(18.02±0.92) %。致敏DC组和阳性对照组比较P>0.05;致敏DC组、阳性对照组分别与阴性对照组、阻断试验组比较P<0.05;阻断试验组与阴性对照组比较P<0.05。结论:①应用IL-4联合GM-CSF培养小鼠骨髓细胞,可大量扩增成熟DC,培养的DC符合其自身的特性;②致敏DC可分泌大量的IL-12;③致敏DC培养上清能诱导FBL-3细胞部分转化为单核细胞,转化后的单核细胞符合其自身的特性;④IL-12单克隆抗体能够阻断致敏DC培养上清对FBL-3细胞的诱导转化作用;⑤致敏DC培养上清中可能还存在其它物质对FBL-3有诱导分化作用。更多还原

【Abstract】 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.更多还原