β-胡萝卜素对病毒转化细胞的影响

目的 探讨β-胡萝卜素 (β- C)对病毒转化细胞的抑制作用 .方法 体外培养人腺病毒转化的 2 93细胞和 EB病毒转化的 Raji细胞 ,培养时添加 0 ,10 ,5 0和 10 0 μmol· L- 1的β- C作用 2 4,48和 72 h后 ,进行细胞计数 ,测定细胞生长抑制率 ;EL ISA-结晶紫方法测定 A值 ,计算细胞存活量 ;采用快速 CPE(细胞病变 )方法测定复制缺陷型腺病毒在 2 93细胞上的病毒滴度 .结果 10～ 10 0μmol· L- 1 的β- C对 2 93细胞和 Raji细胞均有抑制作用 ,细胞存活量减少 ,抑制率分别是15 .6 % ,10 .1% (10 μm ol· L- 1 ) ;19.7% ,2 2 .4% (5 0 μmol· L- 1 )和 33.6 % ,2 9.3% (10 0μmol· L- 1 ) ,P<0 .0 5 .快速CPE结果显示加入 β- C后 ,复制缺陷型腺病毒在其辅助细胞 -2 93细胞上的复制增殖能力明显下降 ,病毒滴度降低 ,空斑形成单位 (× 10 9pfu· L- 1 )分别为 98± 41(10μmol· L- 1 ) ,75± 2 9(5 0 μmol· L- 1 )和 6 3± 31(10 0 μmol· L- 1 ) ,与对照组 185±2 5 (0 μmol· L- 1 )相比 ,P<0 .0 5 .结论 β- C对病毒转化的2 93细胞和 Raji细胞的生长增殖具有明显的抑制作用 ,对 2 93细胞内整合的腺病毒早期基因

EBV转化的人乳头瘤病毒感染者B淋巴母细胞系的建立及应用

建立EB病毒(EBV)转化的B淋巴母细胞系(LCL),应用此细胞系作为抗原递呈细胞筛选抗原特异性的T细胞克隆。收集人乳头瘤病毒(HPV)感染者外周血20份,分离外周血单个核细胞(PBMC),利用Pan T细胞试剂盒去除PBMC中的T细胞,获得non-T淋巴细胞,EBV病毒转化non-T淋巴细胞。应用转化的LCL细胞作为抗原递呈细胞,ELISPOT筛选可能的HPV抗原特异性的T细胞克隆。成功建立18个LCLs,建株成功率为90%。转化成功的LCL细胞体积增大,聚集成团状或簇状。建株失败的两份标本,用于转化的non-T淋巴细胞数少于2×106。应用LCL细胞作为抗原递呈细胞筛选出64个HPV抗原特异性的T细胞克隆。成功建立LCLs,此细胞系细胞可作为体外研究HPV特异性免疫反应的刺激细胞。

阿司匹林诱导EB病毒转化的人B淋巴细胞凋亡

目的探讨阿司匹林诱导EB病毒转化的人B淋巴细胞凋亡及其机制。方法 EB病毒转化的人B淋巴细胞经一定浓度的阿司匹林处理后,首先通过MTT法分析细胞的增殖情况;然后用光学显微镜和电子显微镜、PI染色和流式细胞术分析以及琼脂糖凝胶电泳等方法对细胞凋亡进行评价;最后,通过免疫印记法检测凋亡相关蛋白、mTOR信号通路和PU.1-Bim信号通路蛋白表达情况。结果阿司匹林能抑制EB病毒转化的人B淋巴细胞增殖。形态学和超微结构观察发现,阿司匹林处理的细胞固缩变小、数目减少、细胞核畸形、染色质边缘化和细胞质空泡化。阿司匹林处理导致细胞膜通透性增加、细胞存活率明显下降和细胞DNA的弥散现象。免疫印迹分析显示,阿司匹林处理抑制了细胞的mTOR信号和转录因子PU.1的表达水平,激活了细胞凋亡相关蛋白caspase-3、PARP和Bim。结论阿司匹林可能通过抑制EB病毒转化的人B淋巴细胞增殖和诱导细胞凋亡表现一定的抗淋巴瘤效应,mTOR信号途径和PU.1-Bim轴可能参与了阿司匹林抗肿瘤作用机制。

Macrophage secretory products induce an inflammatory phenotype in hepatocytes

AIM:To investigate the influence of macrophages on hepatocyte phenotype and function.METHODS:Macrophages were differentiated from THP-1 monocytes via phorbol myristate acetate stimulation and the effects of monocyte or macrophageconditioned medium on HepG2 mRNA and protein expression determined.The in vivo relevance of these findings was confirmed using liver biopsies from 147 patients with hepatitis C virus(HCV)infection.RESULTS:Conditioned media from macrophages,but not monocytes,induced a transient morphological change in hepatocytes associated with upregulation of vimentin(7.8±2.5-fold,P=0.045)and transforming growth factor(TGF)-β1(2.6±0.2-fold,P<0.001)and downregulation of epithelial cadherin(1.7±0.02-fold,P=0.017)mRNA expression.Microarray analysis revealed significant upregulation of lipocalin-2(17-fold,P <0.001)and pathways associated with inflammation,and substantial downregulation of pathways related to hepatocyte function.In patients with chronic HCV,realtime polymerase chain reaction and immunohistochemistry confirmed an increase in lipocalin-2 mRNA(F0 1.0 ±0.3,F1 2.2±0.2,F2 3.0±9.3,F3/4 4.0±0.8,P= 0.003)and protein expression(F1 1.0±0.5,F2 1.3± 0.4,F3/4 3.6±0.4,P=0.014)with increasing liver injury.High performance liquid chromatography-tandem mass spectrometry analysis identified elevated levels of matrix metalloproteinase(MMP)-9 in macrophageconditioned medium,and a chemical inhibitor of MMP-9 attenuated the change in morphology and mRNA expression of TGF-β1(2.9±0.2 vs 1.04±0.1,P<0.001) in macrophage-conditioned media treated HepG2 cells.In patients with chronic HCV infection,hepatic mRNA expression of CD163(F0 1.0±0.2,F1/2 2.8±0.3,F3/4 5.3±1.0,P=0.001)and MMP-9(F0 1.0±0.4,F1/2 2.8±0.3,F3/4 4.1±0.8,P=0.011)was significantly associated with increasing stage of fibrosis.CONCLUSION:Secreted macrophage products alter the phenotype and function of hepatocytes,with increased expression of inflammatory mediators,suggesting that hepatocytes actively participate in liver injury.

Hepatitis C virus and ethanol alter antigen presentation in liver cells

Alcoholic patients have a high incidence of hepatitis C virus （HCV） infection. Alcohol consumption enhances the severity of the HCV disease course and worsens the outcome of chronic hepatitis C. The accumulation of virally infected cells in the liver is related to the HCV- induced inability of the immune system to recognize infected cells and to develop the immune responses. This review covers the effects of HCV proteins and ethanol on major histocompatibility complex （MHC） class Ⅰ- and class Ⅱ-restricted antigen presentation. Here, we discuss the liver which functions as an immune privilege organ; factors, which affect cleavage and loading of antigenic peptides onto MHC class I and class ~I in hepatocytes and dendritic cells, and the modulating effects of ethanol and HCV on antigen presentation by liver cells. Altered antigen presentation in the liver limits the ability ＇of the immune system to clear HCV and infected cells and contributes to disease progression. HCV by itself affects dendritic cell function, switching their cytokine profile to the suppressive phenotype of interleukin-10 （IL-10） and transforming growth factor beta （TGFβ） predominance, preventing cell maturation and allostimulation capacity. The synergistic action of ethanol with HCV results in the suppression of MHC class Ⅱ-restricted antigen presentation. In addition, ethanol metabolism and HCV proteins reduce proteasome function and interferon signaling, thereby suppressing the generation of peptides for MHC class I -restricted antigen presentation. Collectively, ethanol exposure further impairs antigen presentation in HCV-infected liver cells, which may provide a partial explanation for exacerbations and the poor outcome of HCV infection in alcoholics.