胞壁酰二肽-抗CD10免疫偶联物对急性淋巴细胞白血病患儿树突状细胞的影响

Effect of the novel immunomodulator composed of muramyl dipeptide and anti - CD10 monoclonal antibody on dendritic cells in children with acute leukemia

目的观察胞壁酰二肽与抗CD10单克隆抗体偶联物（MDP-Ab）对急性白血病患儿树突状细胞（DC）活性的影响。方法采用本课题组新合成的MDP-Ab作用于急性淋巴细胞性白血病患儿分离培养所得的DC.分为6组：对照组、未偶联的抗CD10抗体组、未偶联的MDP组、MDP-Ab组、脂多糖（LPS）组及MDP-Ab＋LPS组，培养8d，收集各组DC，检测细胞免疫表型、内吞作用及细胞上清液中白细胞介素12（IL-12）水平。采用活细胞荧光剂羧基荧光素二醋酸盐琥珀酰亚胺酯（CFSE）染色法检测各组作用的淋巴细胞增殖情况，酶联免疫吸附法检测上清液干扰素-γ（IFN-γ）水平。结果1．DC细胞免疫表型：MDP-Ab促进白血病患儿外周血DC表达人白细胞（位点）DR抗原（HLA-DR）、共刺激分子（CD80、CD86）及成熟分子（CD83）的阳性细胞均明显高于对照组、未偶联的抗CD10抗体组及未偶联的MDP组，但低于LPS组及MDP-Ab＋LPS组（F=629．62，P=0．000）。2．IL-12水平：MDP-Ab组、LIPS组及MDP-Ab＋LPS组均明显高于对照组、未偶联的抗CD10抗体组及未偶联的MDP组（F=857．87，P=0．000）。MDP-Ab组与LPS组、MDP-Ab＋LPS组相比，亦有显著性差异（P〈0．05）。3．DC吞噬功能：对照组（即未成熟DC）为（81．3±10．1）％，明显高于未偶联的抗CD10抗体组、未偶联的MDP组、MDP-Ab组、LPS及MDP-Ab＋LPS组（F=383．04，P=0．000）。4．混合淋巴细胞反应及IFN-γ水平：与对照组、未偶联的抗CD10抗体组及未偶联的MDP组相比，MDP-Ab组、LPS组及MDP-Ab＋LPS组CFSE阳性细胞及IFN-γ水平明显增高，以MDP-Ab＋LPS组为最高（F=393．36，P=0．000；F=2497．18，P=0．000）。结论新合成的免疫偶联物MDP-Ab能促进白血病患儿外周血DC成熟。

Objective To study the effect of a new immunomodulator composed of muramyl dipeptide（MDP） and anti - CD10 monoclonal antibody （ MDP - Ab ） on the dendritic cells （ DC ） of children with acute leukemia. Methods DC was adopted to divide the children with acute lymphoblastic leukemia into 6 groups,including the control group,unconjugated anti- CD10 alone,unconjugated MDP alone ,MDP- Ab alone ,lipopolysaccharide （LPS） alone and MDP- Ab ＋ LPS. The immunophenotypes, the endoeytosis interleukin- 12 （IL- 12） were detected. The stimulation index of autologous lymphocytes was assayed by adopting 5 - （ and 6） - carboxyfluorescein diacetate, succinimidyl ester（CFSE） -staining method. The supernatants of DC and autologous lymphocytes were used to detect the level of interferon - γ（ IFN - γ） by using enzyme - linked immunosorbent assay. Results （ 1 ） DC immunophenotype ： The expressions of human leukocyte antigen - DR （ HLA - DR ）, mature molecule （ CD83 ） and co - stimulatory molecules （ CD80 and CD86 ） were increased significantly upon DC triggered with MDP - Ab, compared with the control group, unconjugated anti -CD10 group, and unconjugated MDP group, but lower than those in LPS and combination of MDP - Ab with LPS （ F = 629.62, P = 0. 000 ）. （ 2 ） The level of IL - 12 ： a significant increase in IL - 12 level was detected in MDP - Ab group, LPS group, and combination of MDP - Ab with LPS group, compared with the control group, unconjugated anti -CD10 group, and unconjugated MDP group （F = 857.87 ,P =0. 000）. There were significant differences among the first three groups. （3） Endocytosis assay ： The uptake of DCs stimulated by unconjugated anti - CD10, unconjugated MDP, MDP -Ab immunoconjugate, LPS or combination was lower than that of immature DC in the control group which was （ 81.3 ± 10.1 ） %. （4） Mixed lymphocyte reaction and IFN -γ level ： DC, treated with MDP - Ab, LPS and combination, stimulated more CFSE positive cells and higher level of IFN -γ secretion than the control group and unconjugated anti - CD10 group, unconjugated MDP group. The most significance was observed in combination of MDP-Ab with LPS（F=393.36,P=0. 000;F=2 497.18,P=0.000）. Conclusion It is concluded that MDP-Ab could promote the proliferation and maturation of DC derived from blood of children with acute leukemia.