乙型肝炎患者外周血CD4＋CD25＋调节性T细胞4个分子表达的研究

Expression of phenotypic molecules on CD4＋CD25＋ regμlatory T cells in peripheral blood of patients with chronic hepatitis B

目的 研究慢性乙型肝炎（chronic hepatitis B,CHB）患者和乙型肝炎病毒携带者（asymptomatic HBV carriers,ASC）外周血CD4＋CD25＋调节性T细胞（CD4＋CD25＋ regulatory T cells,Tregs）4个表型分子的表达情况,并分析其可能的作用.方法 收集50例CHB患者、28例ASC和24例健康献血者的外周静脉血,密度梯度离心法分离外周血单个核细胞,应用MACS免疫磁珠和AutoMACS分离系统分选CD4＋CD25＋ Tregs,提取CD4＋CD25＋ Tregs总RNA,逆转录成cDNA后,采用SYBR Green I荧光标记技术进行定量PCR检测待测分子的表达水平.结果 荧光定量PCR结果显示,4个待测分子中,CCR5、CTLA-4F和Foxp3的表达水平在各研究组之间差异均无统计学意义（均P ＞0.05）;GITR在CHB组外周血CD4＋CD25＋ Tregs中的表达水平明显高于健康对照组,差异均有统计学意义（均P＜0.05）,而GITR的表达水平在CHB组和ASC组之间及ASC组和健康对照组之间差异均无统计学意义（均P ＞0.05）.结论 CHB组患者外周血CD4＋CD25＋ Tregs的GITR表达水平显著增高,提示GITR在HBV感染过程中可能对外周血CD4＋CD25＋ Tregs的功能起着重要的调控作用.

Objective To analyze the expression of phenotypic molecules on CD4＋CD25＋ regulatory T cells （Tregs） in pe- ripheral blood of patients with chronic hepatitis B and asymptomatic HBV carriers. Methods Fifty patients with chronic hepatitis B （CHB）, 28 asymptomatic HBV carriers （ASC） and 24 healthy blood donators were enrolled in this study. Peripheral blood mononuclear ceils （PBMC） were isolated from blood samples by FicolI-Hypaque density gradient centrifugation. CD4＋CD25＋ Tregs were isolated by using CD4＋CD25＋ regulatory T Cell Isolation Kit, and autoMACS Separator（Miltenyi Biotec Inc）, and the to- tal RNA of isolated CD4＋CD25＋ Tregs was extracted. After reverse transcription, the expressions of CCR5, CTLA-4, Foxp3 and GITR were detected by quantitative Real-Time PCR using Applied Biosystems 7500 Real-Time PCR technology. Results The results of Real-Time PCR demonstrated that the expression of CCR5, CTLA-4 and Foxp3 showed no significant difference in cir- culating CD4＋CD25＋ Tregs among different groups. CHB patients presented a higher expression of GITR than that in normal controls （P〈0.05）, but there was no significant difference in GITR expression between CHB and ASC groups, and between ASC and normal control groups. Conclusion The expression of GITR in circulating CD4＋CD25＋ Tregs is up-regulated, indicating that GITR may be involved in the functional regulation of CD4＋CD25＋ Tregs after HBV infection.