摘要
目的 观察恒河猴轮状病毒（RRV）对BALB/c小鼠肝脏内调节性T（Treg）细胞的抑制作用是否与Treg细胞叉状头/翅膀状螺旋转录因子（Foxp3）基因启动子CpG岛甲基化有关,探讨其在小鼠肝脏自身免疫炎症中的作用.方法 60只7～9周龄BALB/c小鼠随机分为3组（每组20只）腹腔注射空斑形成单位（PFU）=106 RRV感染24h和48 h,正常对照组腹腔注射相同容积生理盐水,Percoll非连续梯度分离单核细胞,利用流式细胞仪分选小鼠肝脏CD4＋ CD25＋ Foxp3＋ Treg细胞.应用重亚硫酸盐修饰后测序法（BSP）检测BALB/c小鼠脾脏组织中分选出的Treg细胞DNA Foxp3基因启动子CpG岛甲基化状态,Western blot检测Foxp3蛋白的表达.结果 Treg细胞在正常对照组和RRV24、48 h占CD4＋细胞比例分别为（5.26±0.21）％、（4.30±0.46）％、（3.60±0.19）％,RRV24、48 h的比例与正常对照组细胞比例差异有统计学意义（P＜0.05）;对照组和RRV组24、48 hCpG岛甲基化程度分别为（2.78±0.33）％、（26.70±1.87）％、（41.18±3.67）％,各时间点甲基化率均较正常组明显升高（P＜0.01）,Treg细胞Foxp3蛋白表达也明显下降,与RRV感染的时间呈正相关,差异均有统计学意义（P＜0.05）.结论 RRV可以明显促进BALB/c小鼠肝脏中Treg细胞Foxp3基因启动子CpG岛甲基化抑制Treg细胞的增殖和正常的免疫功能,这一过程可能参与了小鼠自身免疫性肝炎的发生.
Objective To investingate whether T （Treg） cells forkhead/winged helix transcription factor P3 （Foxp3） gene promoter CpG island methylation was participated in the Rhesus rotavirus （RRV） inhibiting regulatory Treg cells in BALB/c mice liver,to explore its role in autoimmune liver inflammation.Methods A total of 60 7-9-week-old BALB/c mice were divided randomly into 3 groups （20 each）：injected intraperitoneally PFU =106 RRV infection 24 h and 48 h,and normal control group injected the same volume of saline.Discontinuous Percoll gradient separation of monocytes,Using flow cytometry sorting liver CD4 ＋ CD25 ＋ Foxp3 ＋ Treg cells.Application bisulfite sequencing method modified after sulphate （BSP） to detect BALB/c mouse spleen tissue carve DNA Foxp3 Treg cells selected gene promoter CpG island methylation status,Western blotting assay,spectrophotometer assay respectively.Results In the control group and after injection RRV 24 h or 48 h,accounted for the proportion of CD4 ＋ cells was （5.26 ± 0.21） ％,（4.30 ± 0.46） ％,（3.60 ± 0.19） ％ respectively,and the difference were statistically significance （P＜ 0.05）.Foxp3 gene CpG island methylation were （2.78 ± 0.33）％,（26.70 ± 1.87） ％,（41.18 ± 3.67）％ respectively and the difference were statistically significance （P ＜ 0.01）.Expression of Foxp3 Treg cells also decreased,with RRV infection time was positively correlated,and the differences were statistically significant（P ＜ 0.05）.Conclusion RRV significantly promote the Treg cells Foxp3 gene promoter CpG island methylation in BALB/c mice liver,and Inhibit the proliferation of Treg cells and normal immune function.That may involved in autoimmune hepatitis in mice.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2015年第6期1320-1322,共3页
Chinese Journal of Experimental Surgery
关键词
调节性T细胞
叉状头/翅膀状螺旋转录因子
甲基化
自身免疫性肝炎
Regulatory T Cells
Forkhead/winged helix transcription factor P3 gene
CpG island
Methylation
Autoimmune hepatitis
