CTLA-4 siRNA对慢性乙型肝炎患者外周血Th1/Th2细胞因子的影响

中国临床药理学与治疗学 ›› 2011, Vol. 16 ›› Issue (5): 553-557.

CTLA-4 siRNA对慢性乙型肝炎患者外周血Th1/Th2细胞因子的影响

余永胜, 吴昊, 汤正好, 臧国庆

上海交通大学附属第六人民医院感染病科,上海 200233

收稿日期:2011-03-22 修回日期:2011-04-09 发布日期:2011-07-08
作者简介:余永胜,男,主任医师,硕导,研究方向:乙型病毒性肝炎的免疫机制。Tel:021-64369181-8675 E-mail:yuyongsheng@medmail.com臧国庆,男,主任医师,博导,研究方向:病毒性肝炎临床及发病机制。E-mail:zangguoqin@126.com

Effect of silencing CTLA4 with siRNA on the Th1/Th2 cytokines in peripheral blood of chronic hepatitis B patient

YU Yong-sheng, WU Hao, TANG Zheng-hao, ZANG Guo-qing

Department of Infectious Diseases, Shanghai Sixth People’s Hospital, Shanghai Jiao Tong University, Shanghai 200233, China

Received:2011-03-22 Revised:2011-04-09 Published:2011-07-08

摘要/Abstract

摘要： 目的: 观察化学合成的小干扰RNA(small interfering RNA,siRNA)对慢性乙型肝炎(chronic hepatitis B,CHB)患者外周血淋巴细胞细胞毒T淋巴细胞相关抗原4(cytotoxic T-lymphocyte antigen 4,CTLA-4)表达的抑制作用及干扰后对细胞因子IFN-γ、IL-2和IL-4分泌的影响,探讨CTLA-4对慢性乙型肝炎的T细胞免疫调节作用。方法: 检测CHB患者外周血淋巴细胞CTLA-4,观察其与HBV-DNA的相关性(P＜0.05);根据人淋巴细胞CTLA-4的基因序列,设计合成CTLA-4 siRNA及阴性对照siRNA(siRNA-co),电穿孔法转染CHB患者外周血淋巴细胞,采用实时荧光定量PCR(real-time Q-PCR)方法检测淋巴细胞CTLA-4 mRNA的表达, 采用Western Blotting检测淋巴细胞CTLA-4蛋白表达,采用酶联免疫吸附试验(enzyme-linked immuno sorbent assay,ELISA)检测IFN-γ、IL-2和IL-4分泌。结果: CHB患者外周血淋巴细胞CTLA-4表达量与血清HBV-DNA载量有关;CTLA-4 siRNA转染CHB患者外周血淋巴细胞后,CTLA-4 mRNA及CTLA-4蛋白表达均受到抑制,IFN-γ、IL-2分泌增加,与阴性对照相比差异具有统计学意义(P＜0.01)。而IL-4分泌没有变化,与阴性对照相比差异没有统计学意义(P＞0.05)。结论: CHB患者外周血淋巴细胞CTLA-4表达一定程度抑制免疫反应,利于HBV-DNA的复制;利用siRNA在mRNA水平抑制CHB患者外周血淋巴细胞CTLA-4的表达,能够诱导Th1型细胞因子IL-2、IFN-γ分泌增加,对Th2型细胞因子IL-4无影响。说明抑制CTLA4有助于慢性乙型肝炎患者T细胞免疫的增强。

关键词: 慢性乙型肝炎, 细胞毒性T淋巴细胞相关抗原4, siRNA, Th1/Th2

Abstract: AIM: To explore the inhibition of expression for cytotoxic T-lymphocyte antigen 4(CTLA-4) in peripheral blood lymphocytes in patients with CHB by chemically synthesized small interfering RNA (siRNA) and the secretion of IFN-γ, IL-2, IL-4, then to analyze the T cellular immune activation by the interference of CTLA-4 gene.METHODS: CTLA-4 of peripheral blood lymphocytes in patients with CHB was detected to observe the correlation with the HBV-DNA; according to human CTAL-4 gene sequence, CTAL-4 siRNA and the negative control (siRNA-co) were designed, synthesized, and then they were transfected into peripheral blood lymphocytes separated from CHB patients by electroporation. The expression of CTLA-4 mRNA was detected by real-time Q-PCR, the CTAL-4 protein was measured by Western Blot and the secretion of IFN-γ,IL-2 and IL-4 were assayed by enzyme-linked immunosorbent assay (ELISA).RESULTS: CTLA-4 of peripheral blood lymphocytes in patients with CHB was correlated to HBV-DNA (P＜0.05); after CTLA-4 were transfected into peripheral blood lymphocytes separated from CHB patients, the expression of CTLA-4 mRNA, CTAL-4 protein were suppressed and the production of IFN-γ, IL-2 were enhanced, compared with the negative control group (P＜0.01), while IL-4 concentrations did not show any significant differences (P＞0.05).CONCLUSION: CTLA-4 of peripheral blood lymphocytes in patients with CHB might contribute to the persistent infection of HBV. Inhibiting the expression of CTLA-4 mRNA in peripheral blood lymphocytes in patients with CHB could up-regulate the secretion of Th1-type cytokines IFN-γ, IL-2 except Th2-type cytokines IL-4. It suggested that inhibiting CTAL-4 is helpful to activating T cellular immunity of patients with CHB.

Key words: CHB, CTLA-4, siRNA, Th1/Th2

中图分类号:

R542.96

余永胜, 吴昊, 汤正好, 臧国庆. CTLA-4 siRNA对慢性乙型肝炎患者外周血Th1/Th2细胞因子的影响[J]. 中国临床药理学与治疗学, 2011, 16(5): 553-557.

YU Yong-sheng, WU Hao, TANG Zheng-hao, ZANG Guo-qing. Effect of silencing CTLA4 with siRNA on the Th1/Th2 cytokines in peripheral blood of chronic hepatitis B patient[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2011, 16(5): 553-557.

参考文献

[1] Bocher WO, Galun E, Marcus H, et al. Reduced hepatitis B virus surface antigen-specific Th1 helper cell frequency of chronic HBV carriers is associated with a failure to produce antigen-specific antibodies in the trimera mouse [J]. Hepatology, 2000, 31(2): 480-487.
[2] Priimiagi LS, Tefanova VT, Tallo TG, et al. Th1-cytokines in chronic hepatitis B and C[J]. Voprosy Virusologii, 2002, 47(3): 23-27.
[3] June CH, Ledbetter JA, Linsley PS, et al. Role of the CD₂₈ receptor in T-cell activation[J]. Immunol Today, 1990, 11(6): 211-216.
[4] Sharpe AH, Freeman GJ. The B7-CD₂₈ superfamily[J]. Nat Rev Immunol, 2002, 2(2): 1l6-126.
[5] Alegre ML, Frauwirth KA, Thompson CB. T-cell regulation by CD₂₈ and CTLA-4[J]. Nat Rev Immunol, 2001, 1(3): 220-228.
[6] Reuben JM, Lee BN, Li C, et al. Biologic and immunomodulatory events after CTLA-4 blockade with ticilimumab in patients with advanced malignant melanoma[J]. Cancer, 2006, 106(11): 2437-2444.
[7] Annunziato F, Cosmi L, Liotta F, et al. Phenotype, localization, and mechanism of suppression of CD₄⁺CD₂₅⁺ human thymocytes[J]. J Exp Med, 2002, 196(30): 379-383.
[8] Nasta F, Corinti S, Bonura A, et al. CTLA-4 regulates allergen response by modulating GATA-3 protein level per cell [J]. Immunology, 2007, 121(1): 62-70.
[9] Das G, Augustine MM, Das J, et al. An important regulatory role for CD₄⁺CD₈ αα T cells in the intestional epithelial layer in the prevention of inflammatory bowel disease[J]. Proc Natl Acad Sci USA, 2003, 100(9): 5324-5329.
[10] Zhou C, Peng G, Jin X, et al. Vaccination with a fusion DNA vaccine encoding hepatitis B surface antigen fused to the extracellular domain of CTLA4 enhances HBV-specific immune responses in mice: implication of its potential use as a therapeutic vaccine[J]. Clin Immunol, 2010, 137(2): 190-198.
[11] Schurich A, Khanna P, Lopes AR, et al. Role of the co-inhibitory receptor CTLA-4 on apoptosis-prone CD₈ T cells in persistent HBV infection [J]. Hepatology, 2011; 25(5):1494-1503.

[1]	张蕾, 肖兴鹏, 丁煌. 沉默ZKSCAN3基因表达加重脂多糖诱导的小鼠肺损伤[J]. 中国临床药理学与治疗学, 2023, 28(2): 164-170.
[2]	胡玉平，周海云，黄巧玲，姚轶敏. 丹皮酚对FSL-1与IL-4共刺激诱导树突状细胞成熟及细胞因子表达的影响[J]. 中国临床药理学与治疗学, 2019, 24(1): 14-19.
[3]	袁刚，胡爱荣，胡耀仁，曾传莉，朱德东，石小军. 恩替卡韦和阿德福韦酯治疗代偿期乙肝肝硬化临床疗效及远期预后[J]. 中国临床药理学与治疗学, 2018, 23(2): 170-174.
[4]	罗胜勇，贾德武，李小羿，戴向荣. 抗血小板溶栓素对大鼠脑缺血再灌注损伤保护作用机制研究[J]. 中国临床药理学与治疗学, 2018, 23(10): 1109-1115.
[5]	陈勇华，曹群奋，洪琼怿，王树民. 替诺福韦酯治疗对慢性乙型肝炎患者血清FGF-23、β2-MG、Cys-C及RBP水平的影响[J]. 中国临床药理学与治疗学, 2017, 22(7): 799-804.
[6]	陈星，吴为玲，陈姗姗，叶丽娅，梅双双，王静，何钰，柴泽英. 基于肿瘤相关巨噬细胞的宫颈癌免疫学病理机制研究[J]. 中国临床药理学与治疗学, 2017, 22(12): 1394-1399.
[7]	王萍，叶登美，窦德宇，董群. 白芍总苷对EAT小鼠Th1/Th2型细胞因子表达的影响[J]. 中国临床药理学与治疗学, 2016, 21(8): 894-898.
[8]	沈斌，蒋利亚，张小平，邓敏. 药物利用评价法和药物利用评估法在慢性乙型肝炎药物治疗中的应用研究[J]. 中国临床药理学与治疗学, 2016, 21(12): 1419-1424.
[9]	张毅，余永胜，汤正好，陈小华，王鹏，江红，奚敏，臧国庆. 恩替卡韦联合苦参素改善HBeAg阳性慢性乙型肝炎患者Th1/Th2失平衡临床研究[J]. 中国临床药理学与治疗学, 2016, 21(10): 1168-1172.
[10]	莫红缨, 江永南, 黎毅敏, 赖乐, 肖正伦. 巨噬细胞移动抑制因子siRNA对肺泡上皮细胞Toll样受体及其下游信号转导通路的影响[J]. 中国临床药理学与治疗学, 2013, 18(4): 382-387.
[11]	李桂秋, 陈淑兰, 崔兰英, 赵金英, 罗文涛, 路娟. HepG2.2.15细胞中siRNA与拉米夫定抗乙型肝炎病毒复制作用的比较[J]. 中国临床药理学与治疗学, 2010, 15(7): 727-731.
[12]	江永南, 莫红缨, 陈建海. 脂质微泡介导转染MIF siRNA对小鼠急性肺损伤的保护作用[J]. 中国临床药理学与治疗学, 2010, 15(7): 737-741.
[13]	彭官清, 张长, 刘雪峰. 拉米夫定停药后复发病例的临床特征[J]. 中国临床药理学与治疗学, 2010, 15(10): 1166-1169.
[14]	朱跃科, 段钟平, 王宝恩, 单晶, 陈煜, 韩大康, 赵军. 水飞蓟宾磷脂酰胆碱复合物治疗慢性乙型肝炎的疗效观察[J]. 中国临床药理学与治疗学, 2009, 14(12): 1392-1394.
[15]	金亮, 王宇, 朱爱华, 刘景晶. 融合蛋白HSP65-6 ×P277 在NOD 鼠中降糖作用的机制研究[J]. 中国临床药理学与治疗学, 2009, 14(10): 1142-1150.