BALB/c小鼠胸腺细胞发育模型的建立

Establishment of Model for Thymocytes Development of BALB/c

目的建立胸腺细胞体外研究模型,为T细胞早期发育及相关实验提供有效技术平台。方法无菌摘取14.5天龄胚鼠胸腺,胸腺器官体外培养,FACS检测不同培养时间点细胞存活状况和表型变化,计数每个胸腺小叶的细胞数变化。结果胸腺体外培养显示:经过6d的培养,胸腺细胞存活率均在88.0%以上,由双阴性发育至双阳性细胞,CD4+CD8+双阳性细胞由培养第0天的0上升到第6天的89.07%;细胞数量随胸腺发育(培养时间的增加)而增多,每个胸腺小叶胸腺细胞数量也由培养0d的0.75×104个上升到第6天的1.38×106个。而体内数据显示:胸腺细胞由胚龄14.5d的双阴性阶段发育到新生鼠的双阳性阶段,每个胸腺小叶细胞数量由14.5d的0.75×104个上升到新生鼠的2.56×106个,同时间段CD4+CD8+双阳性细胞比例由0上升到91.22%。体内、外胸腺细胞表型、数量变化趋势一致。结论体外培养状态下胸腺细胞数量和表型变化与体内自然变化趋势一致,在体外培养可以模拟体内胸腺细胞由双阴性到双阳性的发育过程,这将为T细胞发育的研究提供简易、有效的技术平台。

Purpose To set up the model for thymocytes development study in vitro. Methods Fetal mice thymus lobes were picked out and cultured on medium in complete 1640 with 20 % FBS. Phenotypes of thymocytes were analyzed by FACS and number of cells per lobe was counted. Results Data showed in vivo that thymocytes developed gradually from DN at day 14.5 to DP at newborn, the cell number of each lobe increased from 0.75 × 10^4 to 2.56 × 10^6, and the proportion of CD4^＋ CD8^＋ T cells increased from 0 to 91.22 % . The data in vitro showed that the thymocytes developed from DN to DP in 6 days. The livability of thymocytes was more than 88.0 % . The percentage of CD4^＋ CD8^＋ T cells in all thymocytes increased from 0 at 0 day to 89.07 % at the sixth day; the number of thymocytes increased from 0.75 × 10^4 at the beginning of culture to 1.38 × 10^6 at the sixth day. Conclusions Thymocytes development from DN to DP whether in pbenotype or in number in vitro were the same as that of in vivo, which showed that the model for thymocytes development is consistent with its development from DN to DP in vivo.