胞壁酰二肽对儿童急性白血病骨髓树突状细胞功能的影响

Effect of Muramyl Dipeptide on the Immunifaction of Dendritic Cells Derived from Bone Marrow in Children with Acute Leukemia

摘要: 目的：探讨胞壁酰二肽(MDP)对儿童急性白血病骨髓树突状细胞(DC)功能的影响。方法：应用Ficoll-Hypaque法分离急性白血病患儿骨髓单个核细胞，对照组：用细胞因子诱导培养DC；实验组实验组分为5个亚组，1组：与对照组等量的MNC+ MDP (10 ug/l)，2~5组分别对应为：与对照组等量的MNC + 与对照组等量的细胞因子 + 不同浓度的MDP (10² ug/l，10² ug/l，10³ ug/l，10⁴ ug/l)；分别诱导培养DC，第9天收获各组DC，与同种异体外周血T淋巴细胞混合反应96 h，观察淋巴细胞的增殖情况，ELISA方法检测上清液中IL-12、IFN-γ的含量；经HL-60抗原致敏后的DC与T淋巴细胞混合培养6 d后，加入HL-60细胞共同孵育12 h，LDH释放改良法检测各组细胞毒T淋巴细胞(CTL)的杀伤活性。结果：1) 混合淋巴细胞反应显示：① T细胞扩增指数：固定刺激细胞数，不同组比较，对照组与1组、2组比较均无显著性差异(p均 > 0.05)；3组、4组、5组T细胞扩增指数明显高于对照组，亦明显高于1组(p均 < 0.01)。当刺激细胞数量为1 × 10³至1 × 10⁴，同组间T细胞扩增指数逐渐增加(p均 < 0.01)，1 × 10⁴与5 × 10⁴之间T细胞扩增指数的增加无统计学意义(p > 0.05)。② T细胞分泌细胞因子：当刺激细胞数为5 × 10⁴时，对照组与1组、2组IL-12含量的比较均无显著性差异(p > 0.05)；3组、4组、5组明显高于对照组，亦明显高于1组(p均 < 0.01)。对照组与1组、2组IFN-γ含量的比较均无显著性差异(p > 0.05)；3组、4组、5组明显高于对照组，亦明显高于1组(p均 < 0.01)。2) 杀瘤活性结果显示：实验各组杀伤HL-60细胞活性均较对照组显著增强(p均 < 0.01)；当MDP浓度由10² ug/l递增至10⁴ ug/l，2组、3组、4组、5组组间比较，杀伤活性逐渐增加(p均 < 0.01)，以5组杀伤HL-60细胞活性最强。结论：① 当MDP诱导后的DC数量 ≥ 1 × 10³时，即能促进T细胞的增殖，且有MDP浓度依赖性。② 经MDP诱导后的DC具有促进T细胞分泌的作用，与细胞因子联合作用更强。③ MDP能从儿童急性白血病骨髓中诱导出有较强的抗原刺激能力的DC，且有MDP浓度依赖性。

Abstract: Objective: To investigate the effect of Muramyl Dipeptide (MDP) on the function of dendritic cells (DC) derived from bone marrow in children with acute leukemia. Methods: The mononuclear cells were isolated from bone marrow in children with acute leukemia by the method of Ficoll-Hypaque and cultured with cytokines in the control group. The test groups contained five subgroups: used MDP (10² ug/l) alone (Group 1), cytokines combined with MDP of different concentration, which was put in 3rd day (Group 2, 10 ug/l; Group 3, 10² ug/l; Group 4, 10³ ug/l; Group 5, 10⁴ ug/l). The DCs were collected in the 9th day. The DCs of different groups were mixed with allogeneic peripheral blood T cells for 96 hours, the numbers of T cells in different groups were counted and the contents of interleukin (IL)-12, interferon-γ (IFN-γ) were tested by enzyme-linked immunosorbent assay (ELISA). The DCs of different groups and T cells (cultured with rhIL-2 + 10%FCS of RPMI1640 for 6 days) and HL-60 cells were cultured for 12 hours, cytotoxicity assay were measured by LDH release. Results: 1) Mixed lymphocyte reaction ① The stimulation index of T cell: When the number of stimulation cells was fixed, the proliferation of allogeneic lymphocytes could be strongly stimulated. There was no difference among the control group, group 1 and group 2; but the group3, group 4 and group 5 were higher than the control group and group 1 (p < 0.01). With the increasing of the number of stimulation cells from 1 × 10³ to 1 × 10⁴, the stimulation index of T cells was rising, and the group3, group 4 and group 5 were significantly higher than the control group and group 1, but there was no difference among the control group, group 1 and group 2. ② The contains of IL-12, IFN-γ: When the number of stimulation cells was 5 × 10⁴, there was no difference among the contains of IL-12 of the control group, group 1 and group 2; the group 3, group 4 and group 5 were higher than the control group and group 1 (p < 0.01). There was no difference among the contains of IFN-γ of the control group, group 1 and group 2; the group 3, group 4 and group 5 were higher than the control group and group 1 (p < 0.01). 2) Cytotoxicity assay showed that killing activity on HL-60 cells of T cells in test groups was higher than that of the control group, and in test groups, with the increasing of the concentration of MDP from 10² ug/l to 10⁴ ug/l, the killing activity on HL-60 cells was dramatically enhanced (p < 0.01), among which the killing activity of the group 5 was the strongest. Conclusion: ① The proliferation of T cells could be promoted when the number of DCs induced with MDP is ≥ 1 × 10³, and its function has MDP concentration dependence. ② The DCs induced with MDP could promote T cells secretion, and has no difference between cytokines, but the combined application effect is more obvious. ③ MDP could induce functional DCs to be generated from bone marrow in children with acute leukemia, and its function has MDP concentration dependence.