D-半乳糖诱导小鼠免疫衰老的体外研究

Investigation of D-galactose Induced Cellular and Organic Immunosenescence

目的建立和评价小鼠体外细胞及器官水平D-半乳糖致免疫衰老体系,探讨D-半乳糖对于免疫衰老的作用机制。方法建立细胞水平的D-Gal作用体系:设置不同浓度的D-Gal处理体外培养的小鼠胸腺和脾脏淋巴细胞,3H-TdR掺入实验检测增殖活性;建立器官水平的D-Gal作用体系:设置D-Gal注射处理组和对照组进行小鼠胸腺器官培养,检测方法为HE染色法观察组织切片和PI染色流式细胞术检测细胞周期和凋亡。结果D-Gal对于小鼠胸腺和脾脏淋巴细胞的增殖都有明显抑制作用,并且呈剂量相关效应;胸腺器官培养15 d后,HE染色结果显示:对照组的胸腺皮髓质分界清晰,细胞形态清晰,状态良好。而D-Gal组胸腺皮髓质分界混乱,细胞形态异常;D-Gal处理后,进行器官培养的胸腺3、7、11、15 d的亚二倍体峰的百分率分别为9.19±0.41、21.65±2.68、28.59±0.93、46.36±2.20,较对照组差异显著。结论D-Gal显著抑制了T淋巴细胞的增殖活性,可以诱导细胞免疫功能的衰退,诱导细胞水平的免疫衰老;切片结果表明,胸腺器官培养15 d后,D-Gal组胸腺发生组织退化。另外,胸腺器官培养中,对照组和D-Gal组的细胞凋亡率差异显著,D-Gal组的凋亡率随时间的增加而显著上升,证实D-Gal可在胸腺内诱导细胞的凋亡,破坏胸腺微环境,在体外器官水平诱导免疫衰老。

Objective To establish and evaluate D - galactose induced cellular and organic system and investigate the mechanism on D- galactose induced immunosenescence. Methods We developed the cellular system of D - Gal induced immunosenescence by ConA - induced proliferation of thymocytes and splenocytes and detected with 3H - TdR in- corporations; we developed the organic system of D - Gal induced immunosenescence by young thymus organ culture system and detected with HE staining and PI flow cytometry analysis. Results D - Gal had significantly decreased thymocytes and splenocytes proliferation; After 15 d organ culture, HE staining showed that control group thymus status was good and had no cell apoptosis phenomenon .In contrast, D- Gal treated group thymus cortex and medulla cell became sparse and abnormal; After 3 d, 7 d, 11 d, 15 d organ culture, D- Gal treated group apoptosis percentage respectively was9.19±0.41,21.65 ±2.68,28.59±0.93,46.36±2.20（ P 〈0.05）; Concusions D-Gal decreased T cell proliferation and could induce cellular immunosenescence; D- Gal increased apoptosis level and could induce organic immunosenescence; we estanlished effectively D- Gal induced immunosenescence system in vitro.