【关键词】 cd4+ cd25+ foxp3+ t细胞 cd4+ cd25- foxp3+ t细胞 衰老 小鼠
gao qi, ma tian, zhang xia, jiang yang, zhang lining
(institute of immunology, school of medicine, shandong university, jinan 250012, china)
to explore the relationship between t cells and aging. methods 15 mice were divided into three groups: the 2month (young), the 6 month (middleaged) and the 15month (aged) groups. splenocyte suspensions were prepared in sterile conditions. surface marks were stained with the pecy5conjugated cd4 antibody and the fitcconjugated cd25 antibody. after being washed with pbs, cells were treated with stiletto fluid and fixation fluid and stained with the peconjugated foxp3 antibody. the proportion of cd4+ regulatory t cells was analyzed by flow cytometry. results the proportion of cd4+ t cells and cd4+ cd25+ /cd4+ t cells in splenocytes had no significant differences among the three groups. but the proportion of cd4+ foxp3+ t cells was significantly elevated with an increase of age. the proportion of cd4+ foxp3+ t cells was(16.83±0.44)% in aged mice, which was obviously higher than young and middleaged mice(p<0.01), and the proportion of cd25+ foxp3+ regulatory t cells gradually decreased. the proportion of cd25+ foxp3+ t cells in the aged group or in middleaged mice was lower than that in young mice(p<0.05). however, the proportion of cd25- foxp3+ regulatory t cells continuously elevated with an increase of age. its proportion in aged mice was significantly higher than that in middleaged mice(p<0.01), and that in middleaged mice was higher than that in young mice(p<0.01). in addition, the ratio of cd4+ cd25+ foxp3+ t to cd4+ cd25- foxp3+ t cells showed a decreasing tendency with an increase of age. conclusion the proportion of cd4+ cd25+ foxp3+ t cells shows an decreasing tendency with an increase of age, however, the proportion of cd4+ cd25- foxp3+ t cells shows an increasing tendency. these suggest that the two regulatory subsets play different roles in aging.
key words: cd4+ cd25+ foxp3+ t cell ; cd4+ cd25- foxp3+ t cell; aging; flow cytometry
调节性t细胞(regulatory t cell, treg)是能抑制其他免疫细胞活化、增殖的一群细胞,在维持自身内环境的稳定、防止自身免疫性疾病的发生、抑制移植排斥反应等疾病中发挥重要的保护作用。目前对其特性了解最多的是cd4+ cd25+ foxp3+调节性t细胞,它们数量和功能的降低与移植排斥反应和多种自身免疫性疾病的发生有关,其数量的升高成为肿瘤免疫耐受的重要机制,然而,对于 cd4+ cd25+ foxp3+调节性t细胞与衰老的关系的报道较少。另外,机体内尚有cd4+ cd25- foxp3+调节性t细胞的存在,它们与疾病的关系,特别是与衰老的关系尚不清楚。本研究分析cd4+ cd25+ foxp3+ 和cd4+ cd25- foxp3+两群调节性t细胞在不同鼠龄小鼠的变化规律,为研究不同调节性t细胞亚群与衰老的关系提供试验依据。
1 材料与方法
1.1 材料
1.1.1 动物 15只健康雄性c57bl/6小鼠购于山东大学实验动物中心,其中鼠龄2个月(青年)、6个月(中年)和15个月小鼠(老年)各5只,均在清洁级环境下喂养。
1.1.2 试剂 pecy5标记的抗小鼠cd4(pecy5cd4)抗体、fitc 标记的抗小鼠cd25(fitc cd25)抗体购于bd bioscience公司,pe标记的抗小鼠foxp3(pefoxp3)抗体、核内染色用打孔液和固定液均购于ebioscience公司, mccoy′s 5a培养基购于sigma公司,胎牛血清购于上海微科生化试剂有限公司。
1.2 方法
1.2.1 小鼠脾细胞悬液的制备 取不同鼠龄c57bl/6小鼠脾脏,用200目铜网研磨制备单个脾细胞悬液,离心弃上清后pbs洗1遍,然后加入5ml红细胞裂解液室温作用5min,pbs调整细胞浓度为1×107/ml。
1.2.2 流式细胞染色及检测 取1×106个脾细胞,分别加入pecy5cd4、fitccd25抗体,4℃下避光孵育30min,进行表面染色;pbs洗涤2次,加入1ml打孔液,混匀,4℃下避光作用2h;用2ml固定液洗涤细胞2次并固定细胞,然后加入pefoxp3抗体,混匀,4℃下避光孵育30min,进行核内染色;用固定液洗涤细胞2次,最后加600μl pbs(含0.1%bsa)重悬细胞,用beckman cytomics(tm) fc 500流式细胞仪进行检测。
1.2.3 统计学处理 所有数据用spss 11.5统计学软件进行分析。计量数据以±s表示,各项指标比较采用t检验,p<0.05为差异有统计学意义。
2 结 果
2.1 小鼠脾细胞中cd4+ foxp3+ t细胞的数量变化 见图1a、b和图2a、b。由图1a、b可见,流式细胞术的检测结果显示,2个月、6个月和15个月小鼠脾脏中cd4+ t细胞的数量差异无统计学意义(2个月:26.58±0.89; 6个月:24.12±0.50;15个月:27.3±0.30;p>0.05),3组间cd4+ cd25+ t/cd4+ t细胞数量也无明显改变(2个月:8.28±0.06;6个月:7.05±0.55;15个月:7.67±0.27;p>0.05)。由 a: facs analysis of splenocytes for the proportion of cd4+ and cd4+ cd25+ t cells; b: statistic results of the proportion of cd4+ and cd4+ cd25+ t cells图2a、b可见,随小鼠年龄的增加cd4+ foxp3+ t/cd4+ t的数量明显升高,15个月小鼠cd4+ foxp3+ t/cd4+ t的数量较2个月和6个月小鼠明显升高(2个月:16.83±0.44;6个月:11.5±0.25;15个月:12.30±0.64;p<0.01)。 图2 cd4+ 调节性t细胞在不同年龄小鼠脾细胞中的数量
2.2 小鼠脾细胞中cd4+ cd25+ foxp3+ t细胞的数量随年龄增加而降低 见图2a、c。进一步分析cd4+ foxp3+ t细胞中cd4+ cd25+ foxp3+ t细胞的数量发现,6个月小鼠脾细胞中cd4+ cd25+ foxp3+ t细胞数量较2个月小鼠明显降低[(6个月:(5.92±0.47)%;2个月:(7.70±0.07)%;p<0.05)],15个月小鼠与6个月小鼠相比较差异无统计学意义(p>0.05);上述结果说明随小鼠年龄的增长,cd4+ cd25+ foxp3+ t/cd4+ t细胞的数量明显降低。
2.3 cd4+ cd25- foxp3+ t细胞的数量随年龄增加明显升高 见图2a、d。cd4+ t细胞中存在一群cd25- foxp3+ t细胞,这群细胞在小鼠2个月龄时数量为(3.65±0.19)%,6个月为(6.32±0.57)%,后者较前者明显升高(p<0.01);15个月小鼠cd25- foxp3+ t细胞的数量进一步升高,达到(11.0±1.37)%,与6个月小鼠相比较明显升高(p<0.01)。以上结果说明,数量升高的cd4+ foxp3+ t细胞中主要是cd25- foxp3+ t的细胞,随着年龄的增长cd4+ cd25- foxp3+ t/cd4+ t细胞的数量呈上升趋势。
2.4 cd4+ cd25+ foxp3+ t细胞与cd4+ cd25- foxp3+ t细胞的比值下降 见图3。根据上述结果可以看出,小鼠脾细胞中存在两群cd4+ 调节性t细胞,cd4+ cd25+ foxp3+ t细胞的数量随年龄增加而降低,cd4+ cd25- foxp3+ t的数量随年龄增加明显升高,计算这两个细胞亚群的比值结果显示呈现下降的趋势(2个月:1.97±0.07 ;6个月:0.94±0.10;15个月:0.73±0.06;p<0.001),6个月和15个月小鼠较2个月小鼠明显降低。
3 讨 论
调节性t细胞是一群能抑制免疫功能的负调控细胞,其中cd4+ cd25+ 调节性t细胞,cd4+ cd25+ t细胞在小鼠中占外周cd4+ t细胞的数量大约为5%~10%,是一种具有抑制作用的t细胞[1]。cd4+ cd25+ t细胞主要从胸腺发育分化形成,foxp3 (forkhead/winged helix transcription factor 3 ,foxp3) 称*状头/翅膀状螺旋转录因子,在调控treg细胞的发育上起重要的作用。有资料显示,用载体向天然的cd4+ t细胞导入foxp3可以实现cd4+ t细胞向cd4+ cd25+ t细胞的转化,而缺乏foxp3的cd4+ cd25+ t细胞没有抑制功能[2]。fontenot等用foxp3突变小鼠发现了cd4+cd25+调节性t细胞发育缺陷而cd4+ cd25- t细胞则不受影响,异位表达foxp3的细胞对cd4+ cd25- t细胞具有抑制作用,以上均说明foxp3是cd4+ cd25+ t细胞发育和功能维持的重要调节因子[23]。分段目前认为cd4+ t细胞随年龄的增长其对抗原的应答能力减弱[4]。小鼠老化会伴随胸腺微环境发生很大的变化,主要表现为胸腺的退化,研究认为胸腺退化是t细胞功能减弱的主要原因[46],其后果可能会导致感染性疾病的发病率升高。胸腺是cd4+ cd25+ t细胞产生的中心,随着小鼠年龄的增长胸腺退化可能会导致cd4+ cd25+ t细胞产生减少,从而使cd4+ cd25+ foxp3+ t细胞的数量在年老小鼠外周免疫器官中降低。本研究区分了表达foxp3的细胞亚群,结果显示cd4+ t细胞群体中,cd4+ cd25+ foxp3+ t细胞到了小鼠6个月的时候数量明显减少,6个月之前这群细胞变化不很明显;另外还存在一群cd4+ cd25- foxp3+ t细胞,它们的数量随着年龄的增长呈现明显上升的趋势:年老小鼠体内出现了相当数量的cd25- foxp3+ t细胞,而cd4+ cd25+ foxp3+ t细胞反而降低。cd4+ cd25- foxp3+ t细胞与cd4+ cd25+ foxp3+ t细胞功能上的差别尚不清楚。cd4+ cd25- foxp3+ t细胞的发现提示,小鼠老化后其免疫功能的改变是否与cd4+ cd25- foxp3+ t细胞有关?这群cd25-调节性t细胞是否也具有抑制效应性t细胞的功能?这为进一步研究老化所导致的免疫功能低下提供了新的思路。
【参考文献】
[1] wing k, ekmark a, karlsson h, et al. characterization of human cd25+ cd4+ t cells in thymus, cord and adult blood[j]. immunology, 2002, 106(2):190199.
[2] karube k,ohshima k. expression of foxp3, a key molecule in cd4+ cd25+ regulatory t cells, in adult tcell leukaemia/lymphoma cells. br j haematol, 2004, 126(1):8184.
[3] fontenot j d, gavin m a, rudensky a y. foxp3 programs the development and function of cd4+ cd25+ regulatory t cells[j]. nat immunol, 2003, 4(4):330336.
[4] miller r a. the aging immune system: primer and prospectus[j]. science, 1996, 273(5271):7074.
[5] hirokawa k, utsuyama m, kasai m, et al. understanding the mechanism of the agechange of thymic function to promote t cell differentiation[j]. immunol lett, 1994, 40(3):269277.
[6] haynes l, eaton s m, swain s l. effect of age on naive cd4 responses: impact on effector generation and memory development. springer semin[j]. immunopathology, 2002, 24(1):5360.
中国论文网(www.lunwen.net.cn)免费学术期刊论文发表,目录,论文查重入口,本科毕业论文怎么写,职称论文范文,论文摘要,论文文献资料,毕业论文格式,论文检测降重服务。