CAJ | 학술논문

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.
목적 관찰항하후륜상병독(RRV)대BALB/c소서간장내조절성T(Treg)세포적억제작용시부여Treg세포차상두/시방상라선전록인자(Foxp3)기인계동자CpG도갑기화유관,탐토기재소서간장자신면역염증중적작용.방법 60지7～9주령BALB/c소서수궤분위3조(매조20지)복강주사공반형성단위(PFU)=106 RRV감염24h화48 h,정상대조조복강주사상동용적생리염수,Percoll비련속제도분리단핵세포,이용류식세포의분선소서간장CD4+ CD25+ Foxp3+ Treg세포.응용중아류산염수식후측서법(BSP)검측BALB/c소서비장조직중분선출적Treg세포DNA Foxp3기인계동자CpG도갑기화상태,Western blot검측Foxp3단백적표체.결과 Treg세포재정상대조조화RRV24、48 h점CD4+세포비례분별위(5.26±0.21)％、(4.30±0.46)％、(3.60±0.19)％,RRV24、48 h적비례여정상대조조세포비례차이유통계학의의(P＜0.05);대조조화RRV조24、48 hCpG도갑기화정도분별위(2.78±0.33)％、(26.70±1.87)％、(41.18±3.67)％,각시간점갑기화솔균교정상조명현승고(P＜0.01),Treg세포Foxp3단백표체야명현하강,여RRV감염적시간정정상관,차이균유통계학의의(P＜0.05).결론 RRV가이명현촉진BALB/c소서간장중Treg세포Foxp3기인계동자CpG도갑기화억제Treg세포적증식화정상적면역공능,저일과정가능삼여료소서자신면역성간염적발생.
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.