中华风湿病学杂志
中華風濕病學雜誌
중화풍습병학잡지
CHINESE JOURNAL OF RHEUMATOLOGY
2010年
11期
732-737
,共6页
王国兵%李成荣%杨军%温鹏强%贾实磊
王國兵%李成榮%楊軍%溫鵬彊%賈實磊
왕국병%리성영%양군%온붕강%가실뢰
黏膜皮肤淋巴结综合征%T淋巴细胞,辅助诱导%甲基化%SOCS1%SOCS3
黏膜皮膚淋巴結綜閤徵%T淋巴細胞,輔助誘導%甲基化%SOCS1%SOCS3
점막피부림파결종합정%T림파세포,보조유도%갑기화%SOCS1%SOCS3
Mucocutaneous lymph node syndrome%T-lymphocytes,helper-inducer%Methylation%SOCS1%SOCS3
目的 探讨SOCS1和SOCS3低甲基化对川崎病Th1/Th2细胞失衡的影响.方法 急性期川崎病患儿36例,健康同年龄对照组16名.酶联免疫吸附试验(ELISA)检测血浆白细胞介素(IL)-6蛋白浓度;实时荧光定量聚合酶链反应(PCR)检测CD4+T细胞SOCS1、SOCS3、T-bet、干扰素(IFN)-γ、GATA3、IL-4基因mRNA表达;流式细胞术检测外周血Th1/Th2细胞比例和CD4+T细胞磷酸化STAT3(pSTAT3)蛋白平均荧光强度(MFI);甲基化特异性定量PCR(MethySYBR PCR)检测CD4+T细胞SOCSl基因外显子2、SOCS3基因5'端非翻译区(5'-UTR)3个可能的STAT3结合位点CpG岛甲基化水平.采用t检验进行统计分析.结果 ①急性期川崎病患儿血浆IL-6浓度[分别为(51.8±16.3)pg/ml和(8.6±2.0)pg/ml]、CD4+T细胞pSTAT3 MH水平[分别为(52±14)和(10±4)]显著上调(P＜0.05).其中川崎病合并冠状动脉损伤组(川崎病-CAL+)IL-6和pSTAT3 MFI水平均明显高于无冠状动脉损伤组[IL-6为(87.2±27.4)pg/ml与(36.2±12.8)pg/ml,P＜0.05;pSTAT3 MFI为(75±15)和(42±11),P＜0.05].②急性期川崎病患儿Th1、Th2细胞比例及相关因子(T-bet、IFN-γ、GATA3和IL-4)表达明显增高(P＜0.05),Th1/Th2比值低于健康对照组(P＜0.05).其中川崎病-CAL+组Th1、Th2细胞比例及相关因子表达水平高于川崎病-GAL-组(P＜0.05),而Th1/Th2比值则略低于后者(P＜0.05).③急性期川崎病患儿CD4+T细胞SOCS1和SOCS3 mRNA水平显著高于同年龄对照组(P＜0.05),其中川崎病-CAL+组 SOCS1和SOCS3 mRNA表达低于川崎病-CAL-组(P＜0.05);健康对照组SOCS1基因外显子2、SOCS3基因5'-UTR区第3个STAT3结合位点的CpG岛完全去甲基化,急性期川崎病患儿呈低甲基化状态(P＜0.05),其中川崎病-CAL+组去甲基化水平明显低于川崎病-CAL组(P＜0.05);各组SOCS3基因5'-UTR区第1、2个STAT3结合位点CpG岛均处于完全去甲基化状态(P＞0.05).结论 SOCS1和SOCS3基因低甲基化所致表达相对不足可能是川崎病Th1/Th2细胞失衡的因素之一.
目的 探討SOCS1和SOCS3低甲基化對川崎病Th1/Th2細胞失衡的影響.方法 急性期川崎病患兒36例,健康同年齡對照組16名.酶聯免疫吸附試驗(ELISA)檢測血漿白細胞介素(IL)-6蛋白濃度;實時熒光定量聚閤酶鏈反應(PCR)檢測CD4+T細胞SOCS1、SOCS3、T-bet、榦擾素(IFN)-γ、GATA3、IL-4基因mRNA錶達;流式細胞術檢測外週血Th1/Th2細胞比例和CD4+T細胞燐痠化STAT3(pSTAT3)蛋白平均熒光彊度(MFI);甲基化特異性定量PCR(MethySYBR PCR)檢測CD4+T細胞SOCSl基因外顯子2、SOCS3基因5'耑非翻譯區(5'-UTR)3箇可能的STAT3結閤位點CpG島甲基化水平.採用t檢驗進行統計分析.結果 ①急性期川崎病患兒血漿IL-6濃度[分彆為(51.8±16.3)pg/ml和(8.6±2.0)pg/ml]、CD4+T細胞pSTAT3 MH水平[分彆為(52±14)和(10±4)]顯著上調(P＜0.05).其中川崎病閤併冠狀動脈損傷組(川崎病-CAL+)IL-6和pSTAT3 MFI水平均明顯高于無冠狀動脈損傷組[IL-6為(87.2±27.4)pg/ml與(36.2±12.8)pg/ml,P＜0.05;pSTAT3 MFI為(75±15)和(42±11),P＜0.05].②急性期川崎病患兒Th1、Th2細胞比例及相關因子(T-bet、IFN-γ、GATA3和IL-4)錶達明顯增高(P＜0.05),Th1/Th2比值低于健康對照組(P＜0.05).其中川崎病-CAL+組Th1、Th2細胞比例及相關因子錶達水平高于川崎病-GAL-組(P＜0.05),而Th1/Th2比值則略低于後者(P＜0.05).③急性期川崎病患兒CD4+T細胞SOCS1和SOCS3 mRNA水平顯著高于同年齡對照組(P＜0.05),其中川崎病-CAL+組 SOCS1和SOCS3 mRNA錶達低于川崎病-CAL-組(P＜0.05);健康對照組SOCS1基因外顯子2、SOCS3基因5'-UTR區第3箇STAT3結閤位點的CpG島完全去甲基化,急性期川崎病患兒呈低甲基化狀態(P＜0.05),其中川崎病-CAL+組去甲基化水平明顯低于川崎病-CAL組(P＜0.05);各組SOCS3基因5'-UTR區第1、2箇STAT3結閤位點CpG島均處于完全去甲基化狀態(P＞0.05).結論 SOCS1和SOCS3基因低甲基化所緻錶達相對不足可能是川崎病Th1/Th2細胞失衡的因素之一.
목적 탐토SOCS1화SOCS3저갑기화대천기병Th1/Th2세포실형적영향.방법 급성기천기병환인36례,건강동년령대조조16명.매련면역흡부시험(ELISA)검측혈장백세포개소(IL)-6단백농도;실시형광정량취합매련반응(PCR)검측CD4+T세포SOCS1、SOCS3、T-bet、간우소(IFN)-γ、GATA3、IL-4기인mRNA표체;류식세포술검측외주혈Th1/Th2세포비례화CD4+T세포린산화STAT3(pSTAT3)단백평균형광강도(MFI);갑기화특이성정량PCR(MethySYBR PCR)검측CD4+T세포SOCSl기인외현자2、SOCS3기인5'단비번역구(5'-UTR)3개가능적STAT3결합위점CpG도갑기화수평.채용t검험진행통계분석.결과 ①급성기천기병환인혈장IL-6농도[분별위(51.8±16.3)pg/ml화(8.6±2.0)pg/ml]、CD4+T세포pSTAT3 MH수평[분별위(52±14)화(10±4)]현저상조(P＜0.05).기중천기병합병관상동맥손상조(천기병-CAL+)IL-6화pSTAT3 MFI수평균명현고우무관상동맥손상조[IL-6위(87.2±27.4)pg/ml여(36.2±12.8)pg/ml,P＜0.05;pSTAT3 MFI위(75±15)화(42±11),P＜0.05].②급성기천기병환인Th1、Th2세포비례급상관인자(T-bet、IFN-γ、GATA3화IL-4)표체명현증고(P＜0.05),Th1/Th2비치저우건강대조조(P＜0.05).기중천기병-CAL+조Th1、Th2세포비례급상관인자표체수평고우천기병-GAL-조(P＜0.05),이Th1/Th2비치칙략저우후자(P＜0.05).③급성기천기병환인CD4+T세포SOCS1화SOCS3 mRNA수평현저고우동년령대조조(P＜0.05),기중천기병-CAL+조 SOCS1화SOCS3 mRNA표체저우천기병-CAL-조(P＜0.05);건강대조조SOCS1기인외현자2、SOCS3기인5'-UTR구제3개STAT3결합위점적CpG도완전거갑기화,급성기천기병환인정저갑기화상태(P＜0.05),기중천기병-CAL+조거갑기화수평명현저우천기병-CAL조(P＜0.05);각조SOCS3기인5'-UTR구제1、2개STAT3결합위점CpG도균처우완전거갑기화상태(P＞0.05).결론 SOCS1화SOCS3기인저갑기화소치표체상대불족가능시천기병Th1/Th2세포실형적인소지일.
Objective To investigate the effect of SOCS1 and SOCS3 hypomethylation on homeostasis of Th1/Th2 in Kawasaki disease(KD). Methods Thirty-six children with KD and sixteen age-matched healthy children consented to participate in this study. Protein concentration of IL-6 in plasma was measured by ELISA. Transcriptional levels of SOCS1, SOCS3, T-bet, IFN-γ, GATA3 and IL-4 were assessed by realtime PCR. The proportion of Th1 and Th2 cells, and mean fluorescence intensity(MFI)for phosphorylated STAT3(pSTAT3)protein in CD4+ T cells was analyzed by flow cytometry. A quantitative methylation specific PCR based on SYBR Green was used to evaluate methylation status of CpG islands in SOCSl exon2, and three potential binding sites for STAT3 in 5'-untraslated region(5'-UTR)of SOCS3 in CD4+T cells. Comparisons between groups were performed with t-test. Results ①Compared with healthy volunteers, plasma IL-6 concentration[(51.8±16.3)pg/ml vs(8.6±2.0)pg/ml, respectively]and MFI for pSTAT3[(52±14)vs(10±4), respectively]in CD4+ T cells were elevated significantly during acute phase of KD(P＜0.05), and the two items in KD patients with coronary artery lesion(KD-CAL+)were found to be higher than those in KD patients without coronary artery lesion(KD-CAL-)[IL-6:(87.2±27.4)pg/ml vs(36.2±12.8)pg/ml, P＜0.05; pSTAT3 MFI:(75±15)vs(42±11), P＜0.05]. ② The proportions of Th1 and Th2 cells and transcription levels of Th-associating factors(T-bet, IFN-γ, GATA3 and IL-4)in CD4+ T cells increased significantly in acute KD(P＜0.05), while the rate of Thl div Th2 in KD patients was found to be lower than that in normal controls(P＜0.05). In addition, the proportions of Th1 and Th2 cells and expressions levels of Th-associating factors in KD-CAL+ group were higher than those in KD-CAL-group, as well as the rate of Thl div Th2 cells in KD -CAL+ group were lower than that in KD-CAL- group(P＜0.05). ③ The mRNA levels of SOCSl and SOCS3 in CD4+ T cells increased significantly during acute phase of KD(P＜0.05), while the two items in KDCAL+ group were lower than those in KD-CAL- group(P＜0.05). Furthermore, CpG islands in SOCSl exon2 and the third potential binding site for STAT3 in SOCS3 5'-UTR were hypomethylated in acute KD, while those in healthy volunteers were fully demethylated(P＜0.05). Demethylation levels of the two items mentioned above in the KD-CAL+ group were lower than those in the KD-CAL-group(P＜0.05). CpG islands in the other two binding sites for STAT3 in SOCS3 5'-UTR were fully demethylated among all the groups(P＞0.05).Conclusion Relative insufficiency of SOCS1 and SOCS3 expression caused by hypomethylation may be one contributing factor for the imbalance of Th1/Th2 in KD.
