以重组人类CD40配体为主的细胞因子体外诱导急性髓系白血病完全缓解患者及健康人外周血单个核细胞来源的树突状细胞形态及表型分析

Morphological and phenotypic analysis of monocyte-derived dendritic cells with rhCD40L in acute myeloid leukemia with complete remission and the healthy persons in vitro

目的以健康人AB血清与以重组人类CD40配体（rhCD40L）为主的细胞因子体外诱导急性髓系白血病完全缓解（AML—CR）患者及健康人外周血的单个核细胞，并分析其形态及表型差异。方法以健康人AB血清为基础的培养体系中加入GM-CSF、rhIL-4、rhCD40L等细胞因子，诱导单个核细胞分化形成树突状细胞（DC）。瑞特-吉姆萨染色观察形态；流式细胞术鉴定表型；M3T法检测DC对淋巴细胞抗原刺激能力；ELISA法检测DC培养上清IL-12的分泌。结果培养7d后的细胞具有典型的MoDC形态，并上调表达MoDC特征性表面分子CD83及共刺激分子CD40CD80CD86，AML-CR组与健康人组相比差异无统计学意义（均P〉0．05），但加rhCD40L组较未加rhCD40L组间差异有统计学意义（均P〈0．05）。培养后的MoDC可较强地刺激同种自体淋巴细胞增殖，AML．CR组与健康人组间刺激淋巴细胞增殖作用的差异无统计学意义（P〉0．05），但加rhCD40L组较未加rhCD40L组间差异均有统计学意义（均P〈0．05）。培养的MoDC自培养第5天始即有IL-12的分泌，AML-CR组与健康人组相比IL-12分泌水平差异无统计学意义（P〉0．05），加rhCD40L组与未加rhCD40L组间差异有统计学意义（P〈0．05）。结论以rhCD40L为主的细胞因子体外可诱导AML-CR患者及健康人外周血DC生成，两者在形态及表型上无明显差异，DC可刺激白血病特异毒性T细胞增殖，尤以由rhCn40L诱导的DC作用明显。

Objective To induce monocyte-derived dendritic cells （MoDC） from acute myeloid leukemia （AML） and healthy persons by rhCD40L in normal human AB serum system in vitro and to identify the morphology and phenotype of MoDC. Methods Peripheral blood mononuclear cells （PBMNC） of AML and healthy persons were cultured in RPMI 1640 media including human AB serum, GM-CSF, rhIL-4 and rhCD40L, respectively. MoDC were identified by morphological features, surface antigen expression and the ability to stimulate T cells. Results After cultured for 7 days, MoDC displayed typical morphology with elongated dendritic process, and upregulation of the costimulatory molecules CD40 CD40 CD86 and CD83. The morphology and expression of costimulatory molecules were not significantly different between AML and healthy persons （P 〉 0.05）, but were significantly different between rhCD40L group and without rhCD40L group （P 〈 0.05）. MoDC had the ability to activate T cells, and there were no statistical differences between AML and healthy persons （P 〉 0.05）, but were significant differences between rhCD40L group and without rhCD40L group （P〈 0.05）. MoDC started to secrete IL-12 on day 5, and there was no statistical differences between AML and healthy persons （P 〉 0.05）, and had differences between rhCD40L group and without rhCD40L group （P 〈 0.05）. Conclusion MoDC can be cultured from the peripheral blood of AML and healthy persons. There were no significant differences in morphology and phenotype. Monocyted-derived DC can be used as an alternative to generate leukemia-specific cytotoxic T cells, especially in the presence of rhCD40L.