per2对睡眠剥夺前后脾淋巴细胞功能影响的初步研究

Role of per2 in regulating splenic lymphocyte function under normal or stress conditions induced by sleep deprivation

目的探讨生物钟基因per2对72 h睡眠剥夺(SD)前后脾淋巴细胞功能的影响。方法将C57小鼠随机分为野生型小鼠对照组(WT)和野生型小鼠睡眠剥夺组(WT-SD);per2基因敲除小鼠(per2-/-)随机分为per2基因敲除小鼠对照组(per2-/-)和per2基因敲除小鼠睡眠剥夺组(per2-/--SD)。SD组小鼠使用睡眠剥夺仪进行连续睡眠剥夺72 h。小鼠麻醉后摘脾,使用淋巴细胞分离液分离获得淋巴细胞悬液,利用流式细胞术检测各组别CD4^(+)T、CD8^(+)T、CD3^(+)T以及B220^(+)B比例变化,并通过脂多糖(LPS)刺激3 d体外培养进行流式染色检测上述各淋巴细胞的比例变化,同时使用CCK-8法检测各组小鼠脾B细胞的增殖情况。结果与WT组小鼠相比,per2-/-组小鼠脾CD4^(+)T、CD8^(+)T以及B220^(+)B细胞比例无明显改变;在进一步的睡眠剥夺实验中发现,per2基因敲除影响T细胞,尤其是CD8^(+)T的组成变化。在LPS刺激实验中,与WT组相比,SD组小鼠脾淋巴细胞增殖更加明显,且与WT-SD组相比,per2-/--SD组B220^(+)B细胞比例增加。同样睡眠剥夺应激条件下,小鼠脾淋巴细胞增殖无明显变化,但与WT-SD组相比,per2-/--SD组脾B细胞降低同时对应T细胞比例升高。结论per2对脾淋巴细胞的组成影响较小,但在一定程度上参与调节睡眠剥夺后B淋巴细胞对LPS的增殖反应。

Objective To investigate the role of the circadian clock gene per2 in regulating splenic lymphocyte function under normal or stress condition induced by 72 h sleep deprivation(SD).Methods C57 mice were randomly divided into two groups:wild type group(WT)and wild type group subjected to deprivation sleep group(WT-SD).per2 gene knockout(per2-/-)mice were randomly divided into two groups:per2-/-group and per2-/-subjected to sleep deprivation(per2-/--SD)group.The mice of SD groups were subjected to 72 hours of sleep deprivation.The percentages of splenic CD4^(+)T,CD8^(+)T,CD3^(+)T and B220^(+)B cells in each group were detected by flow cytometry.The spleen cells were stimulated by LPS for three days,and the percentages of CD4^(+)T,CD8^(+)T,CD3^(+)T and B220^(+)B cells were detected by flow cytometry.CCK-8 kit was used to detect the proliferation of B cells.Results Compared with the cells in the spleen of WT mice,the percentages of CD4^(+)T,CD8^(+)T and B220^(+)B cells in the spleen of per2-/-mice did not change significantly.Moreover,per2 deficiency affected the CD8^(+)T spleen lymphocyte composition in the mice subjected to sleep deprivation.In the LPS stimulation experiment,higher proliferation of the spleen cells was observed in SD-/-mice than in WT mice.After sleep deprivation stress,the proportion of B220^(+)B lymphocytes in per2-/-group was higher than in WT group.Under the same sleep deprivation stress,there was no significant change in the proliferation of splenic lymphocytes in mice,but compared with WT-SD group,the number of B lymphocytes in per2-/-SD group decreased and the proportion of T lymphocytes increased.Conclusion Per2 has little effect on the composition of splenic lymphocytes,but to some extent,it is involved in the regulation of the proliferative response of B lymphocytes to LPS after sleep deprivation.