强制泛素化乙型肝炎核心抗原诱导树突状细胞自噬以增强特异性细胞毒性T淋巴细胞反应的作用  

Effect of ubiquitination hepatitis B virus core antigen on inducing dendritic cells autophagy to enhance specific cytotoxic T lymphocyte responses

作　　者：黄润 陈洁[1] 谭全会 马思远 陈小华[1] 余永胜[1] 臧国庆[1] 汤正好[1] Huang Run;Chen Jie;Tan Quanhui;Ma Siyuan;Chen Xiaohua;Yu Yongsheng;Zang Guoqing;Tang Zhenghao(Department of Infectious Diseases,Shanghai Jiao Tong University Affiliated Sixth People′s Hospital,Shanghai 200233,China)

机构地区：[1]上海交通大学附属第六人民医院感染病科,200233

出　　处：《中华传染病杂志》2021年第4期228-233,共6页Chinese Journal of Infectious Diseases

基　　金：国家自然青年科学基金项目(81702050)。

摘　　要：目的了解强制泛素化乙型肝炎核心抗原(ubiquitination of hepatitis B virus core antigen,Ub-HBcAg)对树突状细胞(dendritic cell,DC)自噬的影响,以及调节细胞自噬对DC抗原提呈功能和细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)反应的作用。方法构建Ub-HBcAg慢病毒载体(lentiviral vector,LV,即为LV-Ub-HBcAg组)、无泛素化的乙型肝炎核心抗原(hepatitis B virus core antigen,HBcAg)重组慢病毒载体(LV-HBcAg组)和空载质粒慢病毒载体(LV组),并分别转染至DC2.4细胞,设置空白对照组(NC组)。采用蛋白质印迹法检测自噬相关蛋白P62、微管相关蛋白1轻链3B(microtubule associated protein 1 light chain 3 beta,LC3B)、自噬相关蛋白5(autophagy related 5,ATG5)和自噬效应蛋白1(Beclin-1)的蛋白质相对表达量。流式细胞术检测DC表面共刺激分子CD86、CD80、主要组织相容性复合体(major histocompatibility complex,MHC)-Ⅱ的表达。CCK-8法检测T淋巴细胞增殖。非放射性的乳酸脱氢酶释放实验检测特异性CTL杀伤活性。统计学分析采用独立样本t检验。结果NC组自噬相关蛋白LC3B-Ⅱ/LC3B-Ⅰ、Beclin-1和ATG5蛋白质相对表达量分别为0.445±0.076、0.522±0.026、0.761±0.038,分别低于LV-Ub-HBcAg组的0.926±0.021、0.919±0.016、1.451±0.028,而NC组P62相对表达量为1.875±0.016,高于LV-Ub-HBcAg组的0.647±0.121,差异均有统计学意义(t=6.102、9.842、17.490、10.590,均P<0.01)。LV-Ub-HBcAg组DC的表面共刺激分子CD86(75.51%)、CD80(83.35%)和MHC-Ⅱ(66.66%)的表达也较高,NC组分别为8.03%、7.49%、0.04%。LV-Ub-HBcAg组特异性CTL杀伤率为(65.310±2.091)%,高于NC组的(14.400±0.497)%和LV-HBcAg组的(54.870±1.443)%,差异均有统计学意义(t=23.690、4.111,均P<0.05)。结论Ub-HBcAg可促进DC自噬,并上调DC表面共刺激分子的表达,有利于DC成熟活化,并在泛素化作用下能更有效地激活T淋巴细胞功能,产生强烈的特异性CTL反应。Objective To clarify the effect of ubiquitination hepatitis B virus core antigen(Ub-HBcAg)on dendritic cells(DC)autophagy,and to explore the mechanism of autophagy in enhancing DC antigen presentation and inducing hepatitis B virus-specific cytotoxic T lymphocyte(CTL)responses.Methods Ub-HBcAg lentiviral vector(LV-Ub-HBcAg),lentiviral vector-hepatitis B virus core antigen(LV-HBcAg)and no-load plasmid LV(LV)were constructed and packaged.DC2.4 cells were divided into LV-Ub-HBcAg group,LV-HBcAg group and LV group.The blank control group(NC group)was also set.The protein expression of autophagy-related protein P62,microtubule associated protein 1 light chain 3 beta(LC3B),autophagy related 5(ATG5)and Beclin-1 were detected by Western blotting.The expressions of co-stimulatory molecules such as CD86,CD80 and major histocompatibility complex(MHC)-Ⅱwere detected by flow cytometry.Cell counting kit-8(CCK-8)method was used to detect T lymphocytes proliferation.The non-radioactive lactic acid dehydrogenase(LDH)release method was applied to detect the killing ability of CTL.Statistical analysis was conducted by independent sample t test.Results The relative protein expressions of LC3B-Ⅱ/LC3B-Ⅰ,Beclin-1 and ATG5 in NC group were 0.445±0.076,0.522±0.026 and 0.761±0.038,respectively,which were all lower than those in LV-Ub-HBcAg group(0.926±0.021,0.919±0.016 and 1.451±0.028,respectively).The relative protein expression of P62 in the NC group was higher than that in LV-Ub-HBcAg group((1.875±0.016)vs(0.647±0.121)).The differences were all statistically significant(t=6.102,9.842,17.490 and 10.590,respectively,all P<0.01).The expressions of CD86(75.51%),CD80(83.35%),MHC-Ⅱ(66.66%)in the LV-Ub-HBcAg group were high,and those in the NC group were 8.03%,7.49%,0.04%,respectively.The specific CTL killing rate((65.310±2.091)%)of the LV-Ub-HBcAg group was significantly higher than both NC group((14.400±0.497)%)and LV-HBcAg group((54.870±1.443)%),and the differences were both statistically significant(t=23.690 and 4.111,r

关 键 词：肝炎病毒 乙型 自噬 泛素化乙型肝炎核心抗原 树突状细胞 T淋巴细胞 细胞毒性 

分 类 号：R512.62[医药卫生—内科学]