Flt3L对小鼠胸腺树突状细胞在FTOC体系中分化发育的作用  

The effects of cytocine Flt3L treatment on the development of mouse thymic dendritic cells by using fetal thymic organ culture system

作　　者：许云云[1] 李毅平[1] 李芳[2] 朱雪明[1] 汪健[1] 王泳[3] 张学光[3] 

机构地区：[1]苏州大学附属儿童医院儿科研究所,苏州215005 [2]苏州大学医学部人体解剖与组织胚胎系,苏州215005 [3]苏州大学医学生物技术研究所,苏州215005

出　　处：《现代免疫学》2011年第6期450-455,共6页Current Immunology

基　　金：国家自然科学基金资助项目(30400395);国家教育部留学回国人员科研基金资助项目(K5120506);苏州市科技计划项目(SYS201042)

摘　　要：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。To investigate the effect of Flt3L on the differentiation of thymic dendritic cells by using the fetal thymic orgsn culture（FTOC） system,thymic lobes of mice were aseptically removed from 15-or 16-day-old mouse fetuses and cultured by the way of FTOC system in vitro,in which two groups were divided according to the culture medium used： the control group（Basal medium used）,and experimental group（cytocine Flt3L added）.After 12 days,thymocytes in the cultured lobes were collected for FACS analysis to detect the expression of CD4,CD8,CD11c,and its morphology was observed under light microscopy.Then,by way of hanging drop culture,the c-kit＋ progenitor stem cells（HPCs） derived from bone marrow were added to 2-deoxyguanosine（2-dGuo） treated thymic lobes,and cultured in FTOC with or without Flt3L.After 12 days,thymocytes in the lobes cultured under different conditions were collected for FACS analysis.The thymic dendritic cells（TDCs） among the collected thymocytes were sorted by MACS and used as the stimulator for allogeneic T cells,whose proliferation were detected later by MTT method.As indicated by FACS and morphologies analysis,the amount of TDCs incresaed obviously after been cultivated with Flt3L by using the FTOC system,and the production rate of TDCs in the Flt3L group was significantly higher than that by using FTOC and hanging drop culture system.MTT assay also showed that in CpG2006 untreated group,the stimulation of TDCs on T cells＇ proliferation was relatively weaker compared to CpG2006 treated group,indicating that T cell proliferation had been enhanced on account of the matured TDCs.It is evident that Flt3L played an important role in the regulation of differentiation and development of mouse thymic dendritic cells,and obviously improved the differentiation of TDCs from mouse c-kit＋HPCs.

关 键 词：FTOC FLT3L 胸腺树突状细胞 c-kit+造血干细胞 

分 类 号：R392.1[医药卫生—免疫学]