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目的优化多细胞因子联合诱导分离树突状细胞(DC)工艺,提高小鼠骨髓来源树突状细胞(BMDC)体外诱导分离效率。方法采用重组小鼠粒细胞-巨噬细胞集落刺激因子(rmGM-CSF)、重组小鼠白细胞介素4(rmIL-4)、脂多糖(LPS)、重组小鼠肿瘤坏死因子α(rmTNF-α)和诱导时间为考察因素,未成熟树突状细胞(imDC)和成熟树突状细胞(mDC)获得量为考察指标,采用Box-Behnken设计试验,响应面法分析并验证试验结果,并结合光学显微镜、电子显微镜、流式细胞术分析BMDC形态、表面标志物等指标。结果响应面优化诱导imDC最佳诱导工艺rmGM-CSF为46 ng/mL、rmIL-4为24 ng/mL,诱导时间为6 d;imDC获取量为(4.58±0.28)×10~6个,相对偏差为4.00%。诱导mDC最佳工艺LPS为1.4μg/m L、rmTNF-α为30 ng/m L,诱导时间为1 d;m DC获取量为(4.21±0.15)×10~6个,相对偏差为3.80%。体外诱导5~7 d,可获得足量的具有典型树突状的DC,流式细胞术检测发现imDC较高表达CD11c(68.62%±2.3%),低表达CD86(37.95%±1.8%);mDC高表达CD11c(82.05%±1.6%)和CD86(90.34%±1.4%)。结论单因素实验与响应面优化联合优化多细胞因子体外快速、高效诱导扩增DC,为进一步研究提供了工具。
OBJECTIVE: To optimize the process of dendritic cells (DC) induced by multi-cytokines and to improve the efficiency of in vitro isolation of mouse bone marrow-derived dendritic cells (BMDC). Methods Recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF), recombinant mouse interleukin-4 (IL-4), lipopolysaccharide (LPS) and recombinant mouse tumor necrosis factor- ) And induction time were investigated, the immature dendritic cells (imDC) and mature dendritic cells (mDC) access to the amount of indicators for the study, the use of Box-Behnken design test, response surface analysis and validation test results, and combined Optical microscope, electron microscope, flow cytometry analysis of BMDC morphology, surface markers and other indicators. Results The response surface optimization of imDC optimal induction process was 46 ng / mL for rmGM-CSF, 24 ng / mL for rmIL-4 and 6 d for imDC. The acquisition of imDC was (4.58 ± 0.28) × 10 ~ 6, Deviation is 4.00%. The optimum conditions for inducing mDC were as follows: LPS was 1.4μg / m L, rmTNF-α was 30 ng / m L, induction time was 1 d, m DC acquisition was (4.21 ± 0.15) × 10 ~ 6 with relative deviation of 3.80% . A large number of DCs with typical dendritic morphology were obtained after induction for 5-7 d in vitro. Flow cytometry showed that imDC showed high expression of CD11c (68.62% ± 2.3%) and low expression of CD86 (37.95% ± 1.8%). mDC overexpressed CD11c (82.05% ± 1.6%) and CD86 (90.34% ± 1.4%). Conclusions Single factor experiments and response surface optimization combined with optimization of multicellular factors to induce DCs expansion rapidly and efficiently in vitro provide the tools for further research.