IL-12單克隆抗體阻斷DC上清誘導(dǎo)FBL-3細(xì)胞分化的實(shí)驗(yàn)研究
發(fā)布時(shí)間：2019-04-04 12:00
【摘要】： 目的:建立體外培養(yǎng)擴(kuò)增C57BL/6小鼠樹突狀細(xì)胞(dendritic cell ,DC )的方法;應(yīng)用反復(fù)凍融法制備小鼠紅白血病FBL-3腫瘤細(xì)胞抗原并致敏DC;觀察致敏DC培養(yǎng)上清對小鼠紅白血病細(xì)胞FBL-3的誘導(dǎo)分化作用;觀察IL-12單克隆抗體阻斷致敏DC培養(yǎng)上清對小鼠紅白血病細(xì)胞FBL-3的誘導(dǎo)分化作用;進(jìn)而探討DC培養(yǎng)上清誘導(dǎo)白血病細(xì)胞分化作用的機(jī)制,為白血病的誘導(dǎo)分化治療提供一條新的途徑。 方法:①應(yīng)用1ng/ml白介素-4(interleukin , IL-4)和10ng/ml粒細(xì)胞—巨噬細(xì)胞集落刺激因子(granulocyte-macrophage colony stimulating factor ,GM-CSF)聯(lián)合誘導(dǎo)培養(yǎng)C57BL/6小鼠骨髓細(xì)胞,光鏡和電鏡觀察DC發(fā)育過程中形態(tài)學(xué)變化;流式細(xì)胞術(shù)檢測DC表面分子CD80、CD86、H-2Kb及I-Ab的表達(dá)情況;②常規(guī)培養(yǎng)并收集C57BL/6小鼠誘導(dǎo)的紅白血病FBL-3細(xì)胞,反復(fù)凍融FBL-3細(xì)胞制備腫瘤抗原,收集備用;③制備的FBL-3腫瘤抗原與培養(yǎng)的C57BL/6小鼠DC共孵育,致敏DC;④用ELISA法檢測致敏DC第8天培養(yǎng)上清中IL-12的濃度(細(xì)胞濃度2.0×106/ml);⑤分四組:標(biāo)準(zhǔn)IL-12組(陽性對照組,A組),致敏DC第8天的培養(yǎng)上清組(致敏DC組,B組),加入IL-12單克隆抗體的致敏DC第8天的培養(yǎng)上清組(阻斷實(shí)驗(yàn)組,C組),RPMI-1640組(陰性對照組,D組)。四組分別與FBL-3細(xì)胞共孵育72小時(shí)后,用瑞氏染色計(jì)數(shù)成熟單核細(xì)胞、透射電鏡觀察成熟細(xì)胞的超微結(jié)構(gòu)、流式細(xì)胞儀檢測細(xì)胞表面CD14分子的陽性表達(dá)率,觀察四組有何不同。 結(jié)果:①用IL-4和GM-CSF聯(lián)合培養(yǎng)C57BL/6小鼠骨髓細(xì)胞,可得到符合DC特征的典型細(xì)胞;②ELISA法檢測致敏DC培養(yǎng)第8天DC(細(xì)胞濃度2.0×106/ml)上清中IL-12的濃度為:(699.77±16.67)pg/ml;③瑞氏染色計(jì)數(shù)成熟單核細(xì)胞的比率:陰性對照組轉(zhuǎn)化率為(3.06±1.41)%,阻斷實(shí)驗(yàn)組單核細(xì)胞比率為(17.11±1.25)%,兩者比較P0.05;致敏DC組單核細(xì)胞比率為(46.03±2.41)%,與陰性對照組和阻斷實(shí)驗(yàn)組比較P均0.05;陽性對照組單核細(xì)胞比率(48.07±1.96)%,與陰性對照組和阻斷實(shí)驗(yàn)組比較P均0.05,與致敏DC組比較P0.05;④透射電鏡計(jì)數(shù)成熟單核細(xì)胞的比率與瑞氏染色計(jì)數(shù)成熟單核細(xì)胞的比率基本相同;⑤流式細(xì)胞儀分析顯示:陰性對照組細(xì)胞基本無CD14表達(dá),表達(dá)率為(3.24±1.39)%;陽性對照組、致敏DC組、阻斷試驗(yàn)組作用后均有部分細(xì)胞表達(dá)CD14分子,CD14陽性率分別為(49.39±1.88) %、(48.28±1.10) %、(18.02±0.92) %。致敏DC組和陽性對照組比較P0.05;致敏DC組、陽性對照組分別與陰性對照組、阻斷試驗(yàn)組比較P0.05;阻斷試驗(yàn)組與陰性對照組比較P0.05。 結(jié)論:①應(yīng)用IL-4聯(lián)合GM-CSF培養(yǎng)小鼠骨髓細(xì)胞,可大量擴(kuò)增成熟DC,培養(yǎng)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培養(yǎng)上清能誘導(dǎo)FBL-3細(xì)胞部分轉(zhuǎn)化為單核細(xì)胞,轉(zhuǎn)化后的單核細(xì)胞符合其自身的特性;④IL-12單克隆抗體能夠阻斷致敏DC培養(yǎng)上清對FBL-3細(xì)胞的誘導(dǎo)轉(zhuǎn)化作用;⑤致敏DC培養(yǎng)上清中可能還存在其它物質(zhì)對FBL-3有誘導(dǎo)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 鹵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 鹵1.41)%, and that of the blocking group was (17.11 鹵1.25)% (P 0.05); The ratio of monocytes in sensitized DC group was (46.03 鹵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 鹵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 鹵1.39)%, and the expression rate was (3.24 鹵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 鹵1.88)%, (48.28 鹵1.10)%, (18.02 鹵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.
【學(xué)位授予單位】：泰山醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392
本文編號：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 鹵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 鹵1.41)%, and that of the blocking group was (17.11 鹵1.25)% (P 0.05); The ratio of monocytes in sensitized DC group was (46.03 鹵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 鹵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 鹵1.39)%, and the expression rate was (3.24 鹵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 鹵1.88)%, (48.28 鹵1.10)%, (18.02 鹵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.
【學(xué)位授予單位】：泰山醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392
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