响应面优化多细胞因子联合诱导分离树突状细胞的工艺

Response surface methodology for optimizing the technology of induction and isolation of dendritic cells by multiple cytokines

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 promote the induction and separation efficiency of bone marrow-derived dendritic cells（ BMDCs） in vitro through optimizing the inducing and isolating process by multiple cytokines. Methods The factors to be optimized in single factor tests included recombinant mouse granulocyte macrophage-colony stimulating factor（ rm GM-CSF）,recombinant mouse interleukine 4（ rm IL-4）, lipopolysaccharide（ LPS）, recombinant mouse tumor necrosis factor α（ rm TNF-α） and inducing time. The numbers of immature dendritic cells（ im DCs） and mature dendritic cells（ m DCs） were investigated as the indicators. Box-Behnken experimental design-response surface methodology was used to analyze and verify the data. Morphological changes were observed using the inverted microscopy and the transmission electron microscopy.Surface molecules including CD11 c and CD86 were detected using the flow cytometry. Results The optimum inducing conditions for im DCs were obtained as follows： rm GM-CSF was 46 ng/m L,rm IL-4 was 24 ng/m L,inducing time was 6 days,and the number of im DCs was（ 4. 58 ± 0. 28） × 10^6 cells,and the relative deviation was 4. 00 %. The optimum inducing conditions for m DCs were as follows： LPS was 1. 4 μg/m L,rm TNF-α was 30 ng/m L,inducing time was 1 day,and the number of m DCs was（ 4. 21 ± 0. 15） ×10^6 cells,and the relative deviation was 3. 80%. Sufficient typical im DCs and m DCs were obtained within 5-7 days of induction in vitro. Also,flow cytometry showed that the amplified im DCs had a high expression of CD11c（ 68. 62% ± 2. 3%） and a low expression of CD86（ 37. 95% ± 1. 8%）,and the m DCs had high expressions of both CD86（ 90. 34% ± 1. 4%） and CD11c（ 82. 05% ± 1. 6%）. Conclusion The combination of single factor tests and Box-Behnken design-response surface methodology could optimize the inducing and isolating method for DCs in vitro by multiple cytokines rapidly and efficiently,which provided basic experiment materials for further studies.