IL-12单克隆抗体阻断DC上清诱导FBL-3细胞分化的实验研究
发布时间：2019-04-04 12:00
【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P0.05;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P0.05;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P0.05;致敏DC组、阳性对照组分别与阴性对照组、阻断试验组比较P0.05;阻断试验组与阴性对照组比较P0.05。 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。
[Abstract]:Objective: to establish a method for in vitro culture and amplification of dendritic cells (dendritic cell, DC) from C57BL/6 mice, and to prepare mouse erythroleukemia FBL-3 tumor cell antigen and sensitize DC; by repeated freeze-thaw method. To observe the effect of sensitized DC culture supernatant on inducing differentiation of mouse erythroleukemia cell line FBL-3, and to observe the effect of IL-12 monoclonal antibody blocking sensitized DC culture supernatant on inducing differentiation of mouse erythroleukemia cell FBL-3. Furthermore, the mechanism of differentiation of leukemic cells induced by DC culture supernatant was discussed, which provided a new way for differentiation therapy of leukemia. Methods: 1Bone marrow cells of C57BL/6 mice were induced by 1ng/ml IL-4 (interleukin, IL-4 and 10ng/ml granulocyte-macrophage colony stimulating factor (granulocyte-macrophage colony stimulating factor, GM-CSF). The morphological changes during the development of DC were observed by light and electron microscopy. Flow cytometry was used to detect the expression of CD80,CD86,H-2Kb and I-Ab on the surface of DC. (2) the FBL-3 cells induced by C57BL/6 were cultured and collected, and FBL-3 cells were frozen and thawed repeatedly to prepare tumor antigens. 3The prepared FBL-3 tumor antigen was co-incubated with cultured DC of C57BL/6 mice, and the sensitized DC;4 was used to detect the concentration of IL-12 in the supernatant of sensitized DC on the 8th day (cell concentration 2.0 脳 106/ml) by ELISA method. Five groups were divided into four groups: standard IL-12 group (positive control group, group A), culture supernatant group of sensitized DC (sensitized DC group, group B) on the 8th day, and culture supernatant group of sensitized DC with IL-12 monoclonal antibody on the 8th day (blocking experimental group). C group, RPMI-1640 group (negative control group, D group). After 72 hours of incubation with FBL-3 cells in each of the four groups, mature monocytes were counted by Rayleigh staining, the ultrastructure of mature cells was observed by transmission electron microscope, and the positive expression rate of CD14 molecules on the surface of the cells was detected by flow cytometry. Observe the differences among the four groups. Results: 1the bone marrow cells of C57BL/6 mice were co-cultured with IL-4 and GM-CSF, and the typical DC cells were obtained. The concentration of IL-12 in the supernatant of DC (cell concentration 2.0 脳 106/ml) was (699.77 卤16.67) pg/ml; on the 8th day of culture of sensitized DC by 2ELISA method. 3The percentage of mature monocytes counted by Rayleigh staining: the conversion rate of the negative control group was (3.06 卤1.41)%, and that of the blocking group was (17.11 卤1.25)% (P 0.05); The ratio of monocytes in sensitized DC group was (46.03 卤2.41)%, which was significantly higher than that in negative control group and blocking group (P < 0.05). The percentage of monocytes in the positive control group was (48.07 卤1.96)%, compared with that in the negative control group and the blocking group (P 0.05) and in the sensitized DC group (P 0.05). (4) the percentage of mature monocytes counted by transmission electron microscope (TEM) was the same as that of mature monocytes by Rayleigh staining, (5) the expression rate of CD14 in negative control group was (3.24 卤1.39)%, and the expression rate was (3.24 卤1.39)% in the negative control group. In the positive control group, sensitized DC group and blocking test group, some cells expressed CD14 molecule. The positive rates of CD14 were (49.39 卤1.88)%, (48.28 卤1.10)%, (18.02 卤0.92)%, respectively. The sensitized DC group and the positive control group were compared with P0.05; the sensitized DC group, the positive control group and the negative control group were respectively compared with the negative control group; the blocking test group and the negative control group were compared with P0.05. Conclusion: 1Mic bone marrow cells cultured with IL-4 combined with GM-CSF can amplify the DC cultured with mature DC, in a large amount in accordance with its own characteristics, and the sensitized DC can secrete a large amount of IL-12;. (3) sensitized DC culture supernatant could induce partial transformation of FBL-3 cells into monocytes, and the transformed monocytes could accord with their own characteristics, and 4IL-12 monoclonal antibody could block the induction and transformation of FBL-3 cells induced by sensitized DC supernatant. 5 there may be other substances in the supernatant of sensitized DC that can induce the differentiation of FBL-3.
【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392
本文编号：2453788
[Abstract]:Objective: to establish a method for in vitro culture and amplification of dendritic cells (dendritic cell, DC) from C57BL/6 mice, and to prepare mouse erythroleukemia FBL-3 tumor cell antigen and sensitize DC; by repeated freeze-thaw method. To observe the effect of sensitized DC culture supernatant on inducing differentiation of mouse erythroleukemia cell line FBL-3, and to observe the effect of IL-12 monoclonal antibody blocking sensitized DC culture supernatant on inducing differentiation of mouse erythroleukemia cell FBL-3. Furthermore, the mechanism of differentiation of leukemic cells induced by DC culture supernatant was discussed, which provided a new way for differentiation therapy of leukemia. Methods: 1Bone marrow cells of C57BL/6 mice were induced by 1ng/ml IL-4 (interleukin, IL-4 and 10ng/ml granulocyte-macrophage colony stimulating factor (granulocyte-macrophage colony stimulating factor, GM-CSF). The morphological changes during the development of DC were observed by light and electron microscopy. Flow cytometry was used to detect the expression of CD80,CD86,H-2Kb and I-Ab on the surface of DC. (2) the FBL-3 cells induced by C57BL/6 were cultured and collected, and FBL-3 cells were frozen and thawed repeatedly to prepare tumor antigens. 3The prepared FBL-3 tumor antigen was co-incubated with cultured DC of C57BL/6 mice, and the sensitized DC;4 was used to detect the concentration of IL-12 in the supernatant of sensitized DC on the 8th day (cell concentration 2.0 脳 106/ml) by ELISA method. Five groups were divided into four groups: standard IL-12 group (positive control group, group A), culture supernatant group of sensitized DC (sensitized DC group, group B) on the 8th day, and culture supernatant group of sensitized DC with IL-12 monoclonal antibody on the 8th day (blocking experimental group). C group, RPMI-1640 group (negative control group, D group). After 72 hours of incubation with FBL-3 cells in each of the four groups, mature monocytes were counted by Rayleigh staining, the ultrastructure of mature cells was observed by transmission electron microscope, and the positive expression rate of CD14 molecules on the surface of the cells was detected by flow cytometry. Observe the differences among the four groups. Results: 1the bone marrow cells of C57BL/6 mice were co-cultured with IL-4 and GM-CSF, and the typical DC cells were obtained. The concentration of IL-12 in the supernatant of DC (cell concentration 2.0 脳 106/ml) was (699.77 卤16.67) pg/ml; on the 8th day of culture of sensitized DC by 2ELISA method. 3The percentage of mature monocytes counted by Rayleigh staining: the conversion rate of the negative control group was (3.06 卤1.41)%, and that of the blocking group was (17.11 卤1.25)% (P 0.05); The ratio of monocytes in sensitized DC group was (46.03 卤2.41)%, which was significantly higher than that in negative control group and blocking group (P < 0.05). The percentage of monocytes in the positive control group was (48.07 卤1.96)%, compared with that in the negative control group and the blocking group (P 0.05) and in the sensitized DC group (P 0.05). (4) the percentage of mature monocytes counted by transmission electron microscope (TEM) was the same as that of mature monocytes by Rayleigh staining, (5) the expression rate of CD14 in negative control group was (3.24 卤1.39)%, and the expression rate was (3.24 卤1.39)% in the negative control group. In the positive control group, sensitized DC group and blocking test group, some cells expressed CD14 molecule. The positive rates of CD14 were (49.39 卤1.88)%, (48.28 卤1.10)%, (18.02 卤0.92)%, respectively. The sensitized DC group and the positive control group were compared with P0.05; the sensitized DC group, the positive control group and the negative control group were respectively compared with the negative control group; the blocking test group and the negative control group were compared with P0.05. Conclusion: 1Mic bone marrow cells cultured with IL-4 combined with GM-CSF can amplify the DC cultured with mature DC, in a large amount in accordance with its own characteristics, and the sensitized DC can secrete a large amount of IL-12;. (3) sensitized DC culture supernatant could induce partial transformation of FBL-3 cells into monocytes, and the transformed monocytes could accord with their own characteristics, and 4IL-12 monoclonal antibody could block the induction and transformation of FBL-3 cells induced by sensitized DC supernatant. 5 there may be other substances in the supernatant of sensitized DC that can induce the differentiation of FBL-3.
【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392
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