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为了借助FTOC体系探讨Flt3L对小鼠胸腺树突状细胞分化发育的影响。摘取15～16d龄胎鼠胸腺进行体外器官培养(胎鼠胸腺器官培养-FTOC),根据所使用培养基的不同实验分为两组:对照组(基础培养基)和Flt3L组(培养基中含有细胞因子Flt3L),在体外进行FTOC常规培养,12d后分别收集两实验组的胸腺细胞,流式细胞仪检测细胞表面分子CD4、CD8、CD11c、Ia等的表达,通过光学显微镜观察细胞形态。骨髓来源的c-kit+造血干细胞通过悬滴培养方法种植入2-脱氧鸟苷处理过的胸腺,随后将胸腺放置于组织器官培养皿中所使用的培养基为基础培养基或加入细胞因子Flt3L的培养基,进行为期12d的FTOC常规培养。12d后收集不同条件下FTOC培养的胸腺细胞,通过流式细胞仪对胸腺细胞的表型进行分析;将在不同条件下FTOC培养获得的胸腺细胞进行MACS分选,从而获得胸腺树突状细胞(CD11c+DC),再与异源的CD4+T细胞进行混合淋巴细胞反应,通过MTT法检测T细胞的增殖情况。结果:在正常FTOC体系中,流式细胞仪检测结果和细胞形态学结果显示:Flt3L组胸腺DC有明显的增加,且FTOC联合悬滴培养体系中Flt3L组胸腺DC的生成率明显高于对照组;MTT检测结果也显示:没有CpG2006刺激时,胸腺DC刺激T细胞增殖的能力比较弱,但添加CpG2006刺激后,胸腺DC趋向于成熟表型,刺激T细胞增殖的能力有所增强。提示,Flt3L在小鼠胸腺DC的分化发育中发挥重要的调节作用,明显促进小鼠骨髓来源的c-kit+造血干细胞向胸腺DC的分化。
In order to investigate the effect of Flt3L on the differentiation and development of mouse thymus dendritic cells by FTOC system. In vitro organ culture (fetal thymus organ culture-FTOC) was performed in 15-16 days old fetus thymus and divided into two groups according to different experiments of the medium used: control group (basal medium) and Flt3L group Containing cytokine Flt3L) were cultured in vitro by FTOC. After 12 days, the thymocytes of two experimental groups were collected. The expression of CD4, CD8, CD11c and Ia were detected by flow cytometry. The morphology of the cells was observed by optical microscope. Bone marrow-derived c-kit + hematopoietic stem cells were inoculated into 2-deoxyguanosine-treated thymus by hanging drop culture, and then the thymus was placed in a tissue culture medium as a basal medium or a cytokine Flt3L Medium, for a period of 12d conventional FTOC culture. After 12 days, the thymocytes cultured in FTOC were collected under different conditions, and the phenotypes of thymocytes were analyzed by flow cytometry. The thymocytes obtained from FTOC culture under different conditions were subjected to MACS sorting to obtain thymus dendritic cells CD11c + DC), and then mixed lymphocyte reaction with allogeneic CD4 + T cells. The proliferation of T cells was detected by MTT assay. Results: In normal FTOC system, the results of flow cytometry and morphological examination showed that the thymus DC in Flt3L group increased significantly, and the generation rate of thymus DC in Flt3L group was significantly higher than that in control group The results of MTT assay also showed that the ability of thymus DC to stimulate T cell proliferation was weak in the absence of CpG2006 stimulation, but thymus DC tended to mature phenotype after stimulation with CpG2006, and the ability of stimulating T cell proliferation was enhanced. These results suggest that Flt3L plays an important regulatory role in the differentiation and development of mouse thymus DCs and significantly promotes the differentiation of mouse bone marrow-derived c-kit + hematopoietic stem cells into thymocytes.