免疫机制新进展：Toll蛋白样受体的模式识别

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Science：引发男性高发肝癌风险的关键蛋白被发现

2007-7-8 22:55:21　信息来源： 生物谷

www.bioon.com/biology/cancer/304051.shtml

最近，研究人员报告，男性比女性产生更多的与肝癌相关的蛋白，这可以解释为什么男性的肝癌发病风险高于女性。身体产生一种称为IL-6的蛋白，她可以对肝脏造成损害，IL-6可以增加能够引起肝癌的肝脏慢性炎症。
    加州大学圣迭戈分校研究小组发现女性不仅产生较少的蛋白质，雌激素可以制衡蛋白的产生量。慢性肝病，过量饮酒，乙型或丙型肝炎病毒感染或癌症家族史都可以引起肝癌，男性患肝癌的风险是女性的3到5倍。
    在他们的研究中，他们首先给小鼠致癌剂，与五分之一的雌性小鼠产生肝癌相比，所有的雄性小鼠都产生了肝癌。他们发现雄性IL-6水平要比雌性高，当给不能产生IL-6的小鼠致癌剂时，仅仅十分之一的雄性鼠患傻上癌症。他们给第三组雄性小鼠雌激素，然后给予致癌剂，在这种情况下，肝癌的发生率与正常雌性小鼠相同。仔细分析干细胞发现，雌激素有助于降低IL-6的产生。加州大学圣迭戈分校临床医学系的Willscott Naugle在准备好的发言中说“有些器官，如乳房，就明显受到性别的影响，而别的器官，如肝，就不会。因此肝的炎症可以被雌激素明显抑制是非常有趣的，它产生了这样一种可能性，通常不受性别影响的器官可能由同一种机制控制。比如膀胱癌，男性比女性多发，这种差异可能源于高的IL-6水平和男性膀胱的炎症。”
    刊登在七月号科学杂志上的文章建议，减少男性IL-6水平或者让其使用类似雌激素的化合物，可能会有助于降低肝癌的发病率。

 

 

Fig. 2. Lower incidence of HCC tumors and longer survival of IL-6^–/– mice. (A)Livers of 8-month-old DEN-treated mice. Multiple HCCs are seen only in WT male liver. (B) Incidence of HCC (> 0.5 mm) in WT male (n = 14), WT female (n = 13), IL-6^–/– male (n = 14), and IL-6^–/– female (n = 15) mice 8 months after DEN (25 mg/kg) injection. Asterisks indicate significant (P < 0.05; Student's t test) differences relative to WT male mice. (C) Survival curves of WT and IL-6^–/– mice injected with DEN (25 mg/kg) at 15 days of age (P = 0.0006; log-rank test for significance). [View Larger Version of this Image (139K JPEG file)]

 

 

原文出处：

Science 6 July 2007:   Vol. 317. no. 5834, pp. 121 - 124
DOI: 10.1126/science.1140485

Gender Disparity in Liver Cancer Due to Sex Differences in MyD88-Dependent IL-6 Production

Willscott E. Naugler, Toshiharu Sakurai, Sunhwa Kim, Shin Maeda, KyoungHyun Kim, Ahmed M. Elsharkawy, and Michael Karin
The greater production of an inflammatory cytokine in male mice explains their higher susceptibility to liver cancer.
Abstract »|   Full Text »|   PDF »|   Supporting Online Material »|  

作者简介：

Michael Karin

Affiliation: UCSD SOM

Professor, Department of Pharmacology, School of Medicine

Biography

B.Sc. Biology, 1975, Tel Aviv University, Tel Aviv, Israel
Ph.D. Molecular Biology, 1979, University of California, Los Angeles

Dr. Michael Karin is currently a Professor of Pharmacology at the School of Medicine, University of California, San Diego, where has been on the faculty since 1987. He has served as a member of the Signal Research Division of Celgene since 1992. Dr. Karin also serves as a member of the National Advisory Council for Environmental Health Sciences and has been an American Cancer Society Research Professor since 1999. He is a leading world authority on signal transduction pathways that regulate gene expression in response to extracellular stimuli. Key achievements include definition of cis elements that mediate gene induction by hormones, cytokines and stress, identification and characterization of the transcription factors that recognize these elements and the protein kinase cascades that regulate their activities. Dr. Karin received his Ph.D. in Molecular Biology from UCLA and completed his postdoctoral training at the Fox Chase Institute for Cancer Research (Dr. Beatrice Mintz) and the Departments of Medicine and Biochemistry at the University of California, San Francisco (Dr. John Baxter). He has published over 200 scientific articles and is an inventor on over 14 different patents or pending patent applications. Recently Dr. Karin was ranked first worldwide by the Institute of Scientific Information (ISI) in a recent listing of most-cited molecular biology and genetic research papers published in prestigious journals.

Research Summary

Dr. Karin's research interests focus on five areas of study. 1) Regulation of transcription in mammalian cells by steroid hormones, growth factors, and adverse environmental conditions and during cellular differentiation. Biochemical and genetic approaches are utilized to isolate transacting regulatory proteins, which mediate responses to developmental, hormonal and environmental signals, by binding to specific DNA sequences. Current efforts are to understand the regulation of gene transcription by growth factors, cytokines and polypeptide hormones and cell type specific gene expression. 2) Response of the human genome to stress. The molecular basis for the UV response, the mammalian counterpart of the bacterial SOS response is being studied by various molecular genetics techniques. 3) Protein kinase cascades and their role in growth control, cell differentiation and programmed cell death. These studies focus on the JNK and p38 MAP kinase cascades and their roles in cellular regulation and specific gene induction. 4) The IKK/NF-kB signaling pathway and its physiological and pathophysiological functions. We are most interested in studying IKK and NF-kB as important links between chronic inflammation and cancer. These studies utilize biochemical as well as whole animal approaches. 5) The regulation of mRNA turnover. In addition to gene transcription, an important control point, is mRNA turnover. We are studying both the general mechanisms responsible for rapid mRNA degradation in mammals and the control of protooncogene and cytokine mRNA turnover by extracellular signals.

Dr. Karin made seminal contributions to the discipline of signal transduction describing how extracellular stimuli, including growth factors, cytokines, tumor promoters and UV radiation, regulate gene expression in eukaryotic cells. Starting with cloning of the human metallothionein IIA gene and analysis of its promoter, Karin and coworkers were the first to identify cis elements that mediate induction of cellular genes by stress signals, glucocorticoids and tumor promoters. This resulted in identification of several transcription factors, including AP-1, that recognize these cis elements. AP-1 was subsequently shown by Karin and coworkers to be composed of Jun and Fos proteins. This provided one of the first demonstrations that nuclear protooncoproteins function as transcription factors. Analysis of the mechanisms by which growth factors and UV radiation induce AP-1 activity led to identification of a major signaling pathway (the JNK MAP kinase cascade), elucidation of the mechanisms by which protein phosphorylation controls transcription factor activity and an explanation for the ability of membrane associated oncoproteins, such as Ras, to modulate gene transcription. Karin and coworkers have also described how proinflammatory stimuli regulate the activity of transcription factor NF-kB and identified the IkB kinase (IKK) complex, which they have shown to be a major regulator of innate immunity and inflammation. Genetic analysis of IKK function resulted in identification of a novel signaling pathway that controls development of the mammalian epidermis. Karin and coworkers were also the first to biochemically identify a cell type specific transcription factor (GHF-1/Pit1), demonstrate its kinship to homeodomain proteins and provide important insights to the mechanism of tissue specific gene expression.

References

References From PubMed (NCBI)

 

相关基因：

IL6 白细胞介素6 白介素6

Official Symbol IL6 and Name: interleukin 6 (interferon, beta 2) [Homo sapiens]
Other Aliases: BSF2, HGF, HSF, IFNB2, IL-6
Chromosome: 7; Location: 7p21
Annotation: Chromosome 7, NC_000007.12 (22733344..22738140)
MIM: 147620
GeneID: 3569

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PowderMed’s Therapeutic DNA Vaccine for Chronic Hepatitis B Enters
Phase I Clinical Trials in Patients
Oxford, UK, 29 March 2006 …PowderMed, the immunotherapeutics company focused on the
development and manufacture of DNA-based vaccines for viral diseases and cancer, has
announced that its dual-antigen encoding immunotherapeutic for Hepatitis B (HBV) has received
US IND approval together with approval from the Singapore, Hong Kong and Taiwanese
Regulatory Authorities and has entered Phase I Clinical Trials. The study will primarily evaluate
the safety and tolerability of the HBV immunotherapeutic, pdpSC18, administered by PMED™,
PowderMed’s needle-free delivery technology, in patients with chronic hepatitis B infection, both
in combination with lamivudine and as monotherapy. Additionally, assessments of
immunogenicity and clinical response will be made.
Worldwide HBV affects 350 million people and there are no commercially available therapeutic
vaccines for the treatment of chronic HBV infection. Chronic infection occurs in 98% of newborn
children infected by vertical transmission from the mother (the most common means of
transmission in Asia-Pacific), and in 5% of individuals infected after 2 years of age. About 25% of
these patients will progress to cirrhosis and 20% of this subgroup will develop hepatocellular
carcinoma – one of the most common cancers worldwide.
Welcoming this study and the potential for a novel therapeutic vaccine to HBV, Dr Antonio
Bertoletti, of the Center for Molecular Medicine, Singapore, said:
“Patients with chronic hepatitis B show a state of relative hypo-responsiveness of HBV-specific T
cells compared with that demonstrated in patients who control the virus replication after acute
infection. Therapeutic induction and/or activation of the T-cell response for HBV core and surface
proteins may have the potential to control infection. It has been shown that Hepatitis B surface
(HBsAg) and core antigen (HBcAg) induces envelope-and core specific CD4+ and CD8+ T-cell
responses and that the response against the Hepatitis B core antigen (HBcAg), is often
associated with viral control. The combination of these two genes in PowderMed’s pdpSC18 HBV
therapeutic DNA vaccine, thus provides a potential mechanism to both clear the virus via the
CD8+ response and to overcome unresponsiveness in chronically infected patients via the CD4+
response.”
This Phase I, First Time in Human Study will enrol patients at seven sites in SE Asia (Singapore,
Taiwan, Hong Kong) and the USA. Since the immunological response and hepatic tolerability of
the hepatitis B immunotherapeutic would be expected to differ considerably between non-infected
subjects and subjects with active hepatitis B disease, the Phase I clinical study will enrol subjects
with active hepatitis B disease in order to specifically address both safety and immunogenicity in
the most predictive manner possible. Each subject will participate in the study for a period of up
to 27 weeks, plus a 4-week run-in and screening period. Allowing for the planned safety reviews
between dosing cohorts and a 4-month recruitment window, results can be expected during 2007.
Commenting on the trial, Dr John Beadle, PowderMed’s Chief Medical Officer, said

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慢性乙型肝炎与Toll样受体2的关系 郭云蔚 尉秀清 杨绍基

www.heporg.com/pnews/detail.asp?systemid=684&ord=37
【摘要】  目的  探讨CHB与Toll样受体2（TLR2）的相关性。 方法  采用Elivision二步法检测CHB、慢性重型肝炎患者及健康对照组肝组织TLR2的表达。采用直接免疫荧光流式细胞术检测TLR2在肝癌细胞株HepG2、HepG2-X及HepG2.2.15上表达的平均荧光强度（MFI）及阳性细胞率。 结果  TLR2在CHB及慢性重型肝炎肝组织上的表达明显高于健康对照组，差异有统计学意义，P<0.01，主要表达于肝细胞质及部分胞膜。在CHB中，TLR2的表达强度与肝组织炎症活动度分级（G）呈显著正相关（r＝0.597，P<0.01），各级炎症患者肝组织TLR2阳性表达强度患者的血清TBil值,差异有统计学意义（P<0.05）。在慢性乙型重型肝炎患者中，TLR2主要呈小灶性表达。TLR2在肝癌细胞株HepG2.2.15上表达的平均荧光强度为10.7±2.8，阳性细胞率为16.3%±7.0%；HepG2的平均荧光强度为1.0±0.3，阳性细胞率为0.4%±0.1%，两组比较t值分别为11.92和7.92，P值均<0.01。而HepG2和HepG2-X之间，差异无统计学意义。 结论  CHB患者肝组织中TLR2的表达明显升高，与肝组织的炎症活动密切相关。
【关键词】  肝炎，乙型，慢性； 免疫组织化学； 细胞培养； Toll样受体
A close relationship between viral hepatitis B and Toll-like receptor 2    GUO Yun-wei, WEI Xiu-qing, YANG Shao-ji. Department of Gastroenterology, Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510630, China
Corresponding author: WEI Xiu-qing, Email: weixiuqing1975@ yahoo.com.cn
【Abstract】    Objective    To investigate the relationship of Toll-like receptor 2 (TLR2) and viral hepatitis B through testing the expression of TLR2 in liver tissues of patients infected with HBV and in HepG2 and HepG2.2.15 cell lines. Methods    Expression of TLR2 was determined by Elivision immunohistochemistry in liver tissues from patients with chronic viral hepatitis B (CHB), chronic severe hepatitis (CSH), from healthy controls and from cells of HepG2 and HepG2.2.15 hepatocellular carcinoma cell lines. Direct immunofluorescence flow cytometry was used to detect TLR2+ cell percentage and mean fluorescence intensity (MFI). Results    The intensity of TLR2 expression in liver tissues of CHB and CSH was significantly higher than that of the healthy controls (P < 0.01). The positive staining was mainly located in the cytoplasm and on some cell membranes. In CHB, the intensity of TLR2 expression was positively correlated with the grade of necroinflammatory activity (r = 0.597, P < 0.01). There were significant differences of serum total bilirubin levels with different grades of positive staining of TLR2 (P < 0.05). In liver tissues of the CHB patients, the positive staining of TLR2 was shown in small foci. MFI of TLR2 (10.7±2.8) and TLR2+ cell percentage (16.3%±7.0%) of HepG2.2.15 cells were both significantly higher than those of HepG2 cells (1.0±0.3, 0.4%±0.1%, P < 0.01). Conclusions    Expression of TLR2 is closely correlated with hepatitis B, especially to the grades of its necroinflammatory activity.
【Key words】  Hepatitis B, chronic; Immunohistochemistry; Cell culture; Toll-like receptor
Toll样受体（toll-like receptor，TLR）目前已发现10余个家族成员，它可通过识别病原相关的分子模式（pathogen-associated molecular patterns，PAMPs），引发信号转导导致炎症介质的释放，在天然免疫防御中起重要作用，并最终激活获得性免疫系统，TLR2是其中功能研究得相对清楚的成员之一。HBV侵入人体后，免疫介导的肝损伤是乙型肝炎发病的主要机制，TLR2信号途径是否参与其中，目前尚不清楚。本研究通过检测CHB患者肝组织，肝癌细胞株HepG2和HepG2.2.15细胞TLR2的表达，探讨TLR2与乙型肝炎的相关性。
资料与方法
一、资料
1. 研究对象：2003年6月至2006年5月中山大学附属第三医院住院患者90例，男84例，女6例，中位年龄35岁，其中CHB组75例，为肝穿刺标本，慢性乙型重型肝炎（chronic severe hepatitis, CSH）组15例，为肝移植病肝切除标本。另设健康对照组10例，标本取自肝移植供肝常规留取标本。均行常规石蜡包埋切片。诊断符合2005年《慢性乙型肝炎防治指南》及2000年《病毒性肝炎防治方案》[1，2]。
2. 细胞株：肝癌细胞株HepG2及HepG2.2.15（HepG2转染HBV全基因组形成）为中山大学附属第三医院传染科实验室保存，HepG2-X为转染HBV X基因组的HepG2，已由中山大学附属第三医院传染科杨林教授前期建立并保存。
3. 主要试剂及仪器：鼠源性FITC-anti-human TLR2、FITC-mouse IgG 2a，kappa购自美国Ebioscience公司，anti-TLR2 PcAb（兔源性）购自美国Santa Cruz公司，Elivision 二步法试剂盒购自福州迈新公司，Trizol试剂购自美国Invitrogen公司、实时荧光定量逆转录-PCR试剂盒SuperScriptTM Ⅲ Platinum  SYBR  Green One-Step qRT-PCR Kit购自日本TaKaRa公司，ABI 7000 real Time PCR扩增仪购自美国ABI公司。DMEM、新生牛血清购自美国Gibco公司，BD FACS Calibur流式细胞仪购自美国BD公司。
二、方法
1. 细胞培养及细胞总RNA提取：细胞培养液为含10%灭活新生牛血清的DMEM培养基，HepG2.2.15细胞培养液另加G418，常规培养肝癌细胞株HepG2、HepG2-X及HepG2.2.15并传代，然后取对数生长细胞以104/ml接种于6孔板中，至细胞贴壁融合90%以上时每株各取4孔消化收集细胞，检测TLR2的表达，余2孔用Trizol法进行总RNA提取，核酸蛋白紫外分析仪检测RNA质量和浓度，要求A260/A280在1.8～2.0，并调整RNA浓度至1ng/μl。以上实验重复2次。
2. 细胞TLR2蛋白水平表达的检测：采用直接免疫荧光流式细胞术。将待测细胞浓度调为（1～5）×106/ml，PBS洗涤细胞，分别加入FITC-anti-human TLR2 10μl和细胞100μl，每管设同型对照，以FITC-mouse IgG 2a代替FITC-anti-human TLR2室温、避光孵育20min。PBS洗涤后，调整细胞浓度为1×106/ml，用FCM及Cellquest软件分析TLR2的平均荧光强度（MFI）及阳性细胞率，以上实验重复2次。
3. 引物设计合成：TLR2：5′-GAAAGCTCC CAGCAGGAACATC-3′，5′-GAATGAAGTCCC GCTTATGAAGACA-3′（146bp）；内参照β-actin：5′-TGGCACCCAGCACAATGAA-3′，5′-CTAA GTCATAGTCCGCCTAGAAGCA-3′（186bp），由TaKaRa生物技术有限公司设计并合成。
4. 标准曲线的构建：取中山大学附属第三医院传染科实验室建立并保存的pBluescript-HBV质粒，核酸蛋白紫外分析仪定量核酸，1∶10倍比稀释作为标准品，与待测样本同时进行荧光定量PCR反应，以其Ct（threshold cycle）值与不同浓度标准品的对数值拟合作图，建立标准曲线。
5. 细胞TLR2 mRNA的定量检测：采用SYBR  GreenⅠ嵌合荧光法进行实时一步逆转录-PCR反应。反应体系50μl，包括上、下游引物各4μl（浓度0.2μmol/L），待测样本总RNA或标准品4μl，加入96孔板，每个待测样本设3个复孔，于ABI 7000实时定量PCR扩增仪进行逆转录及PCR反应。逆转录反应：42℃ 5min，95℃ 2min；PCR反应：95℃ 5s，60℃ 31s，40个循环。根据标准曲线，软件自动计算出待测样本中TLR2和β-actin的mRNA含量，以TLR2和内参照β-actin mRNA含量的比值作为评价TLR2表达水平的指标。
6. 肝组织病理切片的TLR2表达检测：采用Elivision二步法进行免疫组织化学染色，具体步骤参照试剂盒推荐步骤进行，抗体稀释度为1∶100，DAB显色，用柠檬酸高温高压法进行抗原修复，设立阴性对照，以PBS代替第一抗体。
7. 病理诊断：病理切片均行HE、网状纤维染色及HBsAg、HBeAg免疫组织化学染色（SABC法）以协助诊断。CHB肝组织炎症活动度的分级(G)、纤维化程度的分期(S)参照2000年《病毒性肝炎防治方案》[2]。
8. 免疫组织化学结果判断：采用综合评分法，由研究者及1名病理医师分别阅读病理片。在高倍镜下随机计数5个视野，计算阳性细胞所占百分比，根据阳性细胞占所有细胞的比例进行评分：≤10%为0分，11%～25%为1分，26%～50%为2分，51%～75%为3分，≥76%为4分；凡细胞质或细胞核浅棕色为1分，棕色为2分，深棕色为3分，不着色为0分。根据上述2项指标的积分相乘分为4级：0分为阴性(-)、1～4分为弱阳性(+)、5～8分为中等阳性(++)、9～12分为强阳性(+++)。
9. 收集乙型肝炎患者以下临床资料：血清HBV标志物、HBV DNA定量、肝功能、凝血酶原时间及凝血酶原时间活动度、肝纤维化标志物。
10. 统计学分析：采用SPSS12.0统计软件对数据进行统计，各组间的多重比较采用列联表分析，相关分析采用Spearman等级相关分析，两组间计量资料采用t检验和卡方检验。
结    果
1. TLR2在HepG2及HepG2.2.15上的表达：TLR2在HepG2.2.15（n＝12）、HepG2（n＝12）和HepG2-X（n＝12）上表达的MFI分别为10.7±2.8、1.0±0.3和1.2±0.5，HepG2.2.15与HepG2和HepG2-X比较，t值分别为11.94和11.52，P值均<0.01；TLR2阳性率：HepG2.2.15、HepG2和HepG2-X分别为16.3%±7.0%、0.4%±0.1%和1.7%±1.2%，t值分别为7.92和10.24，P值均<0.01。HepG2.2.15细胞上TLR2表达的平均荧光强度及细胞阳性率均明显高于HepG2和HepG2-X，而HepG2和HepG2-X两者之间的表达，差异无统计学意义。实时荧光定量PCR扩增曲线分析显示样本扩增效率良好，溶解曲线分析曲线呈单峰状并基本重叠，说明扩增出单一目的物质。根据标准曲线和内参照β-actin，得出各细胞株TLR2 mRNA的表达值，结果显示，HepG2.2.15细胞上TLR2 mRNA含量明显高于HepG2和HepG2-X（P<0.01），而HepG2和HepG2-X两者的含量之间，差异无统计学意义，见图1（略）。
2. TLR2在乙型肝炎患者肝组织上的表达：TLR2在CHB肝组织上的表达与健康对照组相比明显增强（图2A，B略），免疫组织化学染色强度与肝组织炎症活动度的分级（G）呈显著正相关，r＝0.597，P<0.01。在CSH肝组织上，TLR2主要呈小灶性表达，大片坏死区基本无表达，与健康对照组相比，TLR2的表达亦明显增强（图2C略），CSH组免疫组织化学染色强度评分低于CHB组的G3、G4级，与CHB组的G1、G2级比较，差异无统计学意义，见表1（略）。在细胞水平上，TLR2表达主要定位于肝细胞质及部分胞膜上（图2D略），细胞核无表达，淋巴细胞、汇管区细胞均未见表达。
表1 (略)
 讨    论
TLR2属于TLRs家族，为Ⅰ型跨膜蛋白，在单核细胞、中性粒细胞以及DC大量表达，它通过识别PAMPs引发信号转导而参与机体免疫反应，是天然免疫及获得性免疫系统之间的重要桥梁。除了已知的促进细胞因子的合成与释放、促使免疫细胞成熟、分化和功能化等作用外，TLR2及其他TLRs家族成员在调节机体对入侵病原体的免疫应答于适度水平及抗病毒方面，近期逐渐受到重视[3]。
免疫介导的肝损伤是多种慢性肝病发病的主要机制，近来的研究显示，TLRs信号系统可能在肝脏疾病包括丙型肝炎的发生及免疫介导的肝细胞损伤中起重要作用，至于TLR与乙型肝炎的关系如何，目前尚不清楚。TLR2在人体正常肝组织表达较低或无表达，但某些炎症或前炎性因子可上调TLR2的表达，Matsumura等[4]报道IL-lα，TGFβ可以上调鼠原代培养肝细胞TLR2的表达；而在正常肝组织中，随着脂多糖刺激浓度的升高，TLR2的表达增加[5]。Machida等[6]发现，丙型肝炎患者的外周血单个核细胞中，TLR2及TLR4均有明显的升高，并认为HCV感染后B细胞分泌IL-6及TNFβ的能力明显加强，而这一功能是由TLRs介导的。至于丙型肝炎患者肝组织TLR2的表达情况，Mozer-Lisewska等[7]在对感染HCV的儿童患者的研究中发现，病肝组织上有TLR2及TLR4的表达，而正常肝组织则无，认为TLR在儿童丙型肝炎免疫清除及损伤中起重要作用。
在我们的研究中，CHB及CSH肝组织上TLR2的表达均明显高于健康对照组，并且在CHB组中，TLR2的表达强度，有随肝组织炎症活动度的分级的升高而增强的趋势，这至少说明TLR2参与了CHB和CSH的炎症反应。而HBV本身是否与TLR2的变化有关，从细胞实验结果可以看出。HepG2.2.15细胞株转染了乙型肝炎全基因组，TLR2在HepG2.2.15细胞上表达的平均荧光强度和阳性细胞率均明显高于HepG2，故可推测乙型肝炎病毒直接参与了TLR2的上调。
在细胞水平上，TLR2的表达主要定位于肝细胞质及部分胞膜上，这与Mozer-Lisewska等[7]的研究一致，未见淋巴细胞、汇管区细胞的表达。过强的免疫反应及炎症反应是CSH的重要发病机制，而在我们的研究病例中，TLR2在CSH肝组织上的表达并不非常的强，免疫组织化学染色强度评分低于CHB组的G3、G4级，与G1、G2级差异无统计学意义。其原因可能是因为我们行肝移植的病例基本是末期CSH，肝组织存在大量的坏死区，残存的肝细胞功能明显降低，进而合成TLR2分子的能力下降。
TBil是反映CHB肝损害严重程度最重要的临床指标之一，我们的研究显示，在CHB组，随着肝组织TLR2染色强度的增加，血清TBil随之升高，提示在CHB中，肝损害越严重，肝组织TLR2的表达越强。TLR2与病毒性肝炎关系密切并且可能在病毒性肝炎免疫清除及损伤中起重要作用。至于HBV具体是哪一结构参与细胞TLR2的上调，TLR2是如何参与乙型肝炎的免疫反应，干扰TLR2信号途径，对病毒性肝炎免疫清除及损伤过程有何影响，需要进一步研究。
参  考  文  献
[1]Chinese Society of Hepatology and Chinese Society of Infectious Diseases, Chinese Medical Association. The guideline of prevention and treatment for chronic hepatitis B. Zhonghua Ganzangbing Zazhi, 2005, 13: 881-891.
中华医学会肝病学分会、感染病学分会.慢性乙型肝炎防治指南.中华肝脏病杂志,2005,13:881-891.
[2]Chinese Society of Infectious Diseases and Parasitology and Chinese Society of Hepatology of Chinese Medical Association. The progamme of prevention and cure for viral hepatitis. Zhonghua Ganzangbing Zazhi, 2000, 8: 324-329.
中华医学会传染病与寄生虫病学分会、肝病学分会.病毒性肝炎防治方案.中华肝脏病杂志,2000,8:324-329.
[3]Bieback K, Lien E, Klagge IM, et al. Hemagglutinin protein of wild-type measles virus activates toll-like receptor 2 signaling. J Virol, 2002, 76: 8729-8736.
[4]Matsumura T, Hayashi H, Takii T. TGF-beta down-regnlates lL-l alpha-induced TLR2 expression in murine hepatocytes. J Leukoc Biol, 2004, 75: 1056-l061.
[5]Wan X, Wang PX, ZHOU L, et al. Gene expression of Toll-like receptors in the liver, lungs and spleen in mice after endotoxin challenge. Zhongguo Weizhongbing Jijiu Yixue, 2004, 16: 73-76.
万幸，王培训，周联，等．脂多糖刺激前后小鼠肺肝脾组织中Toll样等受体基因表达情况.中国危重病急救医学,2004,16:73-76．
[6]Machida K, Cheng KT, Sung VM, et al. Hepatitis C virus induces toll-like receptor 4 expression, leading to enhanced production of beta interferon and interleukin-6. J Virol, 2006, 80: 866-874.
[7]Mozer-Lisewska I, Sluzewski W, Kackzmarek M, et al. Tissue localization of Toll-like receptors in biopsy specimens of liver from children infected with hepatitis C virus. Scand J Immunol, 2005, 62: 407-412.
 《中华肝脏病杂志》版权

IC

www.sciencenet.cn/htmlnews/2009/12/226455.shtm
乙肝病毒天然免疫研究获新进展
干扰素β或发挥重要作用
作者：鲁伟 来源：科学时报 发布时间：2009-12-24 9:22:47
人体感染乙肝病毒后，天然免疫系统会第一时间做出应答，发挥抑制和清除病毒的强大作用。因此，有些人感染乙肝病毒后能不治而愈，即形成所谓自限性感染。近日，华中科技大学同济医学院附属同济医院教授杨东亮，与德国同行在乙肝病毒天然免疫方面的合作研究取得新进展，研究发现人体肝脏内Toll样受体3和4所介导的天然免疫应答可以抑制乙肝病毒的复制。这一发现为深入理解早期肝内针对乙肝病毒的免疫应答提供了新线索。

过去的研究表明，由于世界各地乙肝病毒本身的生物学差异和感染者个体免疫遗传学的差异，并不是所有感染乙肝病毒的人都会变成慢性肝炎患者，他们中间有一部分人不需要治疗就可以自愈。

研究人员在急性感染乙肝病毒后自愈的人群中发现，他们身体内针对乙肝病毒的适应性T细胞免疫应答特别强，而慢性感染者体内针对乙肝病毒的适应性T细胞免疫应答很弱。这就说明，适应性T细胞免疫应答是控制乙肝病毒感染的一个重要因素。

“这是大家过去的共识。”杨东亮介绍说，随着人们对免疫学认识的深入，发现相对于天然免疫应答，适应性T细胞免疫应答的发生时间比较晚。那么，更为重要的是，当病毒刚刚进入体内时，早期的免疫应答是如何启动的？作为感染病毒后的最早反应，天然免疫应答在急性和慢性感染者之间是否有区别？

过去几年内，杨东亮带领的研究团队与德国埃森大学的同行合作，针对乙肝病毒感染者肝脏内天然免疫应答展开研究，希望能发现肝脏内天然免疫应答对乙肝病毒复制的影响。经过不懈努力，他们发现，由肝内非实质性肝细胞表面Toll样受体3和4所介导的天然免疫应答可以抑制乙肝病毒的复制，干扰素β在这一应答中发挥重要作用。

杨东亮表示，下一阶段，研究人员可以考虑利用对Toll样受体信号通路的调控，有目的地调节乙肝病毒感染者肝内的免疫应答，从而达到抑制和清除乙肝病毒的目的，进而开发一些新的治疗乙肝病毒感染的药物。

《科学时报》 (2009-12-24 A1 要闻)

Toll样受体3在肝脏疾病中作用的研究进展
www.heporg.com/Pnews/Detail.asp?SystemID=752

[ 本帖最后由 IC 于 2009-12-28 22:59 编辑 ]

IC

www.iderapharma.com/pipeline/imo-2125.php
IMO-2125, a TLR9 agonist, is in clinical development for the treatment of chronic hepatitis C virus (HCV) infection.

IMO-2125 was created by Idera through its chemistry-based approach to making TLR-targeted compounds. IMO-2125 has been shown to induce production of high levels of interferon-alpha and other immune system proteins in preclinical models, both in vitro and in vivo. IMO-2125 induces interferon-alpha and immune system proteins that show activity in HCV replicon cell-based assays. [Press Release]
Ongoing Clinical Trials
At present, Idera is evaluating IMO-2125 in two ongoing clinical trials in two different HCV patient populations:

IMO-2125 Phase 1 Clinical Trial in Non-responder Patients with HCV
Idera is conducting a Phase 1 clinical trial of IMO-2125 in patients with chronic HCV infection who are null responders to the current standard of care treatment. Null responders are defined as patients who have failed to achieve a 2 log10 reduction in viral load during previous 12 to 24 weeks of treatment with pegylated recombinant interferon-alpha plus ribavirin. The primary objective of the trial is to assess the safety of IMO-2125 over an escalating range of dose levels and to determine the effect of IMO-2125 on HCV RNA levels. The target enrollment is ten patients per cohort, with eight randomized to receive IMO-2125 treatment and two randomized to receive placebo treatment. Four dose levels of IMO-2125 have been investigated, with subcutaneous administration once per week for four weeks. Interim results showed that all four dose levels of IMO-2125 were well tolerated for the four weeks of treatment. Additionally, IMO-2125-treated patients showed dose-dependent increases in endogenous interferon-alpha, interferon-inducible protein 10 (IP-10), and 2’,5’-oligoadenylate synthetase (2’,5’-OAS) concentrations. Forty to 75% of patients receiving 0.08 to 0.32 mg/kg IMO-2125 achieved reduction in viral load of > 1 log10 at least once during the treatment period. None of the patients who received placebo or 0.04 mg/kg IMO-2125 showed reduction in viral load of > 1 log10 at any time during the treatment period. Based on these data, the trial has been extended to a fifth dose level and patients are being recruited. The trial is being conducted at six U.S. sites. [Press Release].

IMO-2125 Phase 1 Clinical Trial with Ribavirin in Treatment-naïve Patients with HCV
Idera is also conducting a Phase 1 clinical trial of IMO-2125 in combination with ribavirin in patients with chronic HCV infection who are naïve to treatment with standard of care. The primary objective of the trial is to assess the safety of IMO-2125 over an escalating range of dosages in combination with standard doses of ribavirin and to determine the effect of treatment on HCV RNA levels. The target enrollment is 15 patients per cohort, with 12 randomized to receive IMO-2125 plus ribavirin treatment and three randomized to receive placebo plus ribavirin treatment. IMO-2125 will be administered subcutaneously once per week and ribavirin will be administered orally every day for four weeks. The trial is being conducted at five or more sites in France and Russia. [Press Release]

Idera has formed a Hepatitis C Clinical Advisory Board composed of distinguished members of the global infectious disease community that advises Idera on the development of IMO-2125 for HCV.

IC

http://fedetd.mis.nsysu.edu.tw/F ... amp;index_word=mice
Toll-like receptor 4及一氧化氮在小鼠急性B型肝炎病毒感染模式中所扮演的角色

The role of Toll-like receptor 4 and nitric oxide in the murine model of acute hepatitis B virus infection


作者：張文瑋
畢業學校：國立成功大學
出版單位：國立成功大學
核准日期：2006-06-13
類型：Electronic Thesis or Dissertation
權限：Copyright information available at source archive--National Cheng Kung University....


中文摘要

利用水流動式活體轉染(hydrodynamics-based in vivo transfection)方式將Hepatitis B virus (HBV)質體，pHBV3.6，以靜脈方式注入BALB/c小鼠，發現HBV在小鼠肝臟內能複製，血清中也能夠偵測到HBV抗原及HBV-DNA的存在，小鼠會產生特異性抗體反應，這樣的過程與人類急性HBV感染類似。透過應用此種急性HBV感染小鼠模式我們探討先天性免疫反應在急性HBV感染扮演重要角色。首先我們發現在TLR4正常C3H/HeN小鼠，HBV感染肝細胞周圍的肝臟浸潤白血球表現TLR4。在TLR4突變C3H/HeJ小鼠體內，HBV的複製高於TLR4正常C3H/HeN小鼠。C3H/HeJ小鼠肝臟內HBV特異性T反應、脾臟內Th1反應無法有效被誘發，將來自於TLR4正常C3H/HeN小鼠之脾臟細胞給予TLR4突變的C3H/HeJ小鼠，能夠使得C3H/HeJ小鼠體內HBV的複製被控制至與C3H/HeN小鼠相當的程度。另外，C3H/HeJ小鼠肝臟浸潤白血球無法有效表現iNOS，顯示iNOS可能參與在急性HBV感染的免疫反應。因此我們再比較iNOS缺失小鼠與正常小鼠HBV的感染情形，發現iNOS缺失小鼠肝臟內HBV的複製量、血清中HBV抗原及HBV-DNA的表現都要較正常小鼠高。HBV複製程度的增加可能是由於iNOS缺失小鼠無法有效誘使某些免疫細胞浸潤至肝臟有關，例如CD3+ T細胞、NKT細胞、B細胞及CD4+ T細胞。巨噬細胞及樹突細胞無法正常活化也可能是原因之一。此外，NO釋放化合物能夠透過抑制p38 MAPK磷酸化而抑制肝癌細胞株Huh7 HBV蛋白的分泌，顯示免疫細胞產生的NO能夠直接影響肝細胞內HBV的複製以及p38 MAPK可能參與HBV複製過程中。最後我們利用體外轉染pHBV3.6至Huh7細胞，發現細胞內p38 MAPK及其下游分子ATF2有磷酸化的現象。p38 MAPK特異性抑制劑SB203580可以有效抑制HBV的複製，包括HBV的RNA、細胞內核心蛋白相關DNA (intracellular core-assocaied DNA)及細胞外病毒顆粒相關DNA (extracellualr virion-associated DNA)。目前認為HBV複製重要指標的covantly closed circular DNA，也同樣能夠有效的被SB203580抑制，這顯示HBV感染肝細胞過程造成p38 MAPK的活化，並且p38 MAPK的活化可能是HBV複製所必需。

英文摘要

When pHBV3.6 plasmid containing whole HBV genome was intravenously injected into BALB/c mice by hydrodynamics-based in vivo transfection, HBV replication was observed in the liver, HBV antigens were detected in the circulation, and specific HBV antibody responses were induced post hydrodynamic injection of pHBV3.6. A murine model was established to mimic the human acute HBV infection. Using this murine model, we investigated the involvement of innate immunity during acute HBV infection. First, TLR4-expressing liver infiltrating leukocytes were observed nearby HBsAg-expressing hepatocytes. HBV replication in liver and HBV antigenemia were higher in TLR4 mutation C3H/HeJ than in TLR4 wild type C3H/HeN mice. Impaired HBV-induced IFN-g, TNF-a or IL-12 responses in liver or spleen were observed in C3H/HeJ mice. After adoptive transfer of splenocytes from C3H/HeN mice, the HBV replication in TLR4 mutant C3H/HeJ mice was reduced to the similar level of the C3H/HeN mice. Futhermore, iNOS expression on liver infiltrating leukocytes was impaired in C3H/HeJ mice. This suggested that iNOS might also involve in the immune responses during acute HBV infection. Therefore, the role of iNOS in acute HBV infection was further investigated using iNOS knockout mice. HBV replication in liver, as well as HBV antigenemia and serum HBV-DNA, was higher in iNOS knockout than in wild type mice. Increasing HBV replication might be due to the impaired infiltration of leukocytes, such as CD3+ T cells, CD3+NK1.1+ T cells, CD19+ B cells and CD4+ T cells. Impaired activation of macrophages and dendritic cells might also account for the increasing HBV replication in iNOS knockout mice. Furthermore, NO donor sodium nitroprusside could inhibit HBV secretion in the pHBV3.6-transfeted human hepatoma Huh7 cells through inhibition of the p38 MAPK phosphorylation. These results indicated that NO produced from immune cells could directly influence HBV replication in hepatocytes and p38 MAPK might involve in HBV replication. Finally, using a transient transfection of pHBV3.6 to Huh7 cells, p38 MAPK and its downstream molecule, ATF2, were found to be phosphorylated. SB203580, a selective p38 MAPK inhibitor, could inhibit HBV replication including its RNA, intracellular core-associated DNA and extracellular virion-associated DNA. The colvantly closed circular DNA, a most important index of HBV replication, was also inhibited by SB203580. This suggests that p38 MAPK was activated and might be essential for HBV replication in human hepatocytes.

齐欢畅2

富贵数字

不错啊，就是看不懂

IC

釐清靈芝多醣 活化樹突細胞的作用機制
免疫調節／台灣
www.newmyhealth.url.tw/health4/f3/z1/18.htm
2005 May 13
台灣大學醫學院免疫學研究所教授江伯倫等最近發表在美國《Journal of Leukocyte Biology》
（白血球生物學期刊）的報告指出，樹突細胞能觸動樹突細胞上的Toll-like receptor (TLR)-4
受體，進而啟動樹突細胞的免疫反應。

關於樹突細胞

樹突細胞（dendritic cells）和巨噬細胞一樣，在人類免疫系統裡都屬於第一線的防禦部隊，
當任何病原體進入體內時，樹突細胞將以不亞於巨噬細胞的速度趕抵事故現場，把病原吞入並切
成碎片，再將這些碎片掛在細胞表面呈現給Ｔ細胞看，讓這位免疫兵團的總司確切知道要對付的
是什麼樣的敵人，並擬定作戰計劃，派出適合的兵力迎戰。

於此同時，樹突細胞還會視捕捉到的病原體釋放不同的細胞激素，進而左右Ｔ細胞的作戰計劃。
所以，樹突細胞可說是啟動後續免疫反應的關鍵。


靈芝多醣能活化樹突細胞

原本會讓樹突細胞「採取行動」的是那些會讓我們生病的病原體，不過江伯倫教授等的研究則發
現，靈芝多醣也能啟動樹突細胞，使免疫兵團警戒升級。

江教授使用的靈芝多醣（PS-G）具有β（1→6）側鏈結構，而先前已證實其具有抗腫瘤和活化自
然殺手細胞的特性。

實驗發現，由人類單核細胞分化而來的樹突細胞在加入靈芝多醣之後，其表面的CD80、CD86、CD83
、CD40、CD54等分子標誌和人類白血球抗原DR（HLA-DR）的表達增加，此變化正是樹突細胞活化
的表現，猶如車子從靜止狀態被啟動引擎，蓄勢待發。


活化的樹突細胞能促進Ｔ細胞的免疫反應

實驗也發現，靈芝多醣能促使樹突細胞分泌IL-12p70、IL-12p40和IL-10，並提高IL-12p35、IL-12p40
和IL-10 的mRNA等基因遺傳訊息的表達，並降低樹突細胞的吞噬作用（此為樹突細胞成熟的表現）。

此外，經靈芝多醣處理的樹突細胞在與Ｔ細胞作用之後，能促使Ｔ細胞增殖，並誘導Ｔ細胞分泌
干擾素γ（INF-γ）與IL-10。


TLR4受體是靈芝多醣啟動樹突細胞的關鍵

上述結果似乎暗示著，靈芝多醣之所以能活化樹突細胞，是它觸動了樹突細胞表面的Toll-like
receptor (TLR)-4受體，因為過去已有研究證實，當突出在樹突細胞上的TLR4受體被觸動時，樹突
細胞會出現類似上述的反應。

為了證實這點，研究人員先以抗體蓋住樹突細胞表面的TLR4受體，然後再加入靈芝多醣，果然不見
樹突細胞分泌IL-12 p40和IL-10。顯然TLR4受體是靈芝多醣啟動樹突細胞的關鍵。  


NF-kappaB和p38 MAPK　是主要的訊息傳遞路徑

研究進一步證實，當靈芝多醣與TLR4受體結合後，會引起樹突細胞內部的蛋白分子產生一連串的化
學反應，包括增加kappaB激抑制物（ IKappaB）、活化NF-kappaB激，以及IKappaB-α和p38 MAPK
等蛋白分子的磷酸化。這些反應最後帶出來的結果，就是促使樹突細胞合成並釋出細胞激素。

如果先在樹突細胞裡加入了NF-kappaB和p38 MAPK抑制劑（helenalin和SB98059），然後再與靈芝
多醣作用，所得到的結果則是樹突細胞表面的CD80、 CD86、CD83、CD40、CD54的分子標記不表達
或低表達（表示無法活化樹突細胞），而 IL-12p70、IL-12p40和IL-10的分泌也明顯受到抑制。

顯然NF-kappaB和p38 MAPK是靈芝多醣調控樹突細胞內部訊息傳遞的主要路徑。
　
〔資料來源〕
Lin YL, Liang YC, Lee SS, Chiang BL.: Polysaccharide purified from Ganoderma lucidum
induced activation and maturation of human monocyte-derived dendritic cells by the
NF-{kappa}B and p38 mitogen-activated protein kinase pathways.
J Leukoc Biol. 2005 May 13。

資料來源：媒體報導
http://www.doublecrane.com.tw/dcweb/mediareport/9502.htm

IC

髓样分化蛋白88在炎症感染相关原发性肝癌发生中的作用
李雅娟 鲁凤民 庄辉
www.heporg.com/pnews/detail.asp?systemid=1668&ord=15

【关键词】  癌，肝细胞； 前C区； 性别因素

The role of MyD88 in inflammatory/infection-related hepatocellular carcinoma    LI Ya-juan, LU Feng-min, ZHUANG Hui.

【Key words】 Carcinoma, hepatocellular;   Precore;   Sex factors

【First author’s address】 Department of Microbiology, Peking University Health Science Center, Beijing 100191, China

Corresponding author: LU Feng-min, Email: lufengmin@ gmail.com

Co-corresponding author: ZHUANG Hui, Email: zhuangbmu@  126.com



原发性肝癌是常见的恶性肿瘤之一，其中90%为肝细胞癌 (HCC)。近年来，人们对HBV、HCV慢性感染等所致肝脏持续炎症反应中Toll样受体 (TLR) 髓样分化蛋白88 (MyD88) 在肝癌发生中作用机制的研究取得了较大进展，增加了对肝癌发生的性别差异分子基础的认识。现就HCC这一炎症相关肿瘤的发病机制及与之相关的宿主遗传因素在HCC发生中的作用的最新研究进展作一综述。

一、与慢性感染及炎症反应相关的病毒因素

HCC是一种典型的炎症相关性肿瘤。HBV和HCV慢性感染导致的HCC均与炎性反应以及炎性信号通路有关。HBV、HCV感染诱发宿主免疫系统产生针对病毒抗原的免疫炎性反应，导致肝细胞损伤，随后出现肝细胞的代偿性再生，可进一步发展为肝纤维化和肝硬化，其中部分肝硬化患者最终将发展为HCC。有研究结果表明，HBV病毒载量、基本核心区启动子、前C区突变与患者肝组织局部炎性反应及肝纤维化等相关[1]。对肝癌组织HBV突变的分析结果表明，肿瘤组织存在前S2/S启动子缺失突变体，导致病毒包膜蛋白S的表达减少，并造成HBV包膜蛋白L折叠异常，此种异常折叠的L蛋白质在细胞内质网膜积聚，可引起内质网应急，对细胞产生严重的长期伤害，引发、加重炎性反应，并通过DNA氧化损伤引起DNA突变[2]。最近HBV拉米夫定耐药突变rtA181T与肝癌的相关性已引起人们的关注[3-4]，该突变可造成S基因所编码的包膜蛋白L、M、S的羧基端截断突变，造成包膜蛋白分泌异常[5]。应开展进一步的临床流行病学及实验室研究，以明确HBV耐药突变所致病毒表面抗原羧基端截断突变体的细胞内异常堆积是否增加患者HCC的发病风险及可能的致癌机制。

二、与慢性感染及炎症反应相关的宿主因素

1. 性别因素：多数HCC是由慢性肝病发展而来的，以男性居多，男性发病率是女性的2～5倍[6]。与女性原发性肝癌患者比较，男性患者预后较差，存活时间较短。动物实验研究结果也表明，在自发或化学物质的诱导下，与雌性啮齿动物相比，雄性啮齿动物更易形成肝癌，且更易发生病毒的慢性感染。在HCC患者中造成这种性别差异的具体机制仍不明确，男性HCC发病率高除与男性肝硬化比例较高及吸烟和饮酒等协同因素有关外，性激素的作用可能是引起性别差异的原因。肝脏是一个激素敏感器官，肝细胞有雄激素受体和雌激素受体。性激素可影响到哺乳动物肝脏的多种功能。雌激素对肝正常生理作用的重要性尚不明确，但雌激素在控制肝细胞的增殖中可能起到重要作用。Lam等[7]研究                结果表明，女性肝癌患者的存活率更高，可能与口服避孕药相关，其中性激素可能发挥作用。动物实验结果表明雌性啮齿动物接受睾丸酮后诱发肿瘤，其发生肿瘤的易感性与雄性动物相似。Tanaka等[8]研究结果表明，增加血清睾丸酮和降低雌激素可促使肝硬化向肝癌发展。Wang等[9]报道，Ras相关雌激素调节生长抑制基因 (Ras-related and estrogen-regulated growth-inhibitory gene，RERG) 可能与HCC发生中性别比例差异有关。87.5%女性患者的肝癌组织中RERG基因表达低于癌旁组织，而57.9%男性患者的肝癌组织中RERG表达高于癌旁组织。RERG表达水平在男性和女性患者间差异有统计学意义。提示RERG在肝肿瘤形成中可能是造成HCC患者性别差异的原因之一。此外，肝癌患者的肿瘤组织和周围肝组织中雄激素的表达和活性增强。雄激素受体的存在与肝癌切除术后增加肝癌复发危险性和降低存活率有关。睾丸酮能增加转化生长因子α水平，促进肝细胞的增殖和肝癌的发生[10]。Rogers等[11]利用小鼠动物模型研究发现，慢性肝炎发展为肝癌可能与性别特异性细胞代谢紊乱有关，而性别特异性细胞代谢的紊乱是由细胞因子介导的。最近一项研究结果表明，微小RNA-18a可抑制雌激素受体 (ER)α的翻译表达，从而抑制雌激素的保护作用，与女性HCC的发生有关[12]。Iavarone等[13]报道，雌激素受体与男性HCC发生率较高有明显的相关性。此外，HBV和HCV基因编码的蛋白可与雌激素受体发生作用。Han等[14]报道HBx蛋白可与ERα作用，通过组蛋白去乙酰化酶抑制ERα转录活性。ERα与HCV NS5B相互作用也可促进HCV的复制[15]，通过病毒蛋白与性激素受体的相互作用影响HCC的发生。

2. TLR-MyD88的作用：Toll样受体类家族是在免疫系统特异性识别微生物病原体抗原中发挥重要调控作用的受体家族。在病毒感染后，机体通过TLR-MyD88途径进行免疫应答发挥抗病毒效应。MyD88是TLR介导的先天免疫反应以及白细胞介素 (IL) 1受体和IL-8受体介导的信号通路中一个重要的蛋白分子，存在于细胞质中，具有Toll结构域，可与Toll结合，在启动核因子 (NF) -κB依赖的信号级联反应中发挥重要的作用[16]，NF-κB在天然免疫应答和获得性免疫应答中均起关键作用，它能调节多种炎症和免疫反应相关基因的表达，如细胞炎症因子、趋化因子和黏附分子等。TLR识别病原相关分子模式，TLR与配体和IL-1 (Toll样受体与IL-1受体的胞内部分有高度同源性) 作用后，使受体二聚化，可与MyD88蛋白结合，激活下游丝/苏氨酸蛋白激酶，使蛋白激酶自身磷酸化，活化两条信号转导途径，c-Jun N末端激酶和p38丝裂原蛋白激酶家族，进一步激活NF-κB，后者活化后转位进核，引起特定基因表达，从而产生原发性致炎因子，如肿瘤坏死因子（TNF）α、IL-1和IL-6等，完成炎症的信号传导过程[17]。

Naugler等[18]和Greten等[19]的动物实验研究结果证明，MyD88涉及到天然免疫应答，在炎症相关肿瘤的发生中通过激活NF-κB，促进炎性细胞因子IL-6的产生和分泌而发挥作用。IL-6是一种炎症因子，当存在肝损伤或感染时，IL-6能通过激活信号转导与转录激活因子3和NF-κB信号通路减少肝细胞凋亡、促进周围细胞的增殖[20]。肝细胞的炎性坏死和再生将加速细胞遗传变异的积累，最终导致肿瘤的形成。Naugler等[18]在动物实验中发现，雄性小鼠发生肝癌率明显高于雌性小鼠，雄性小鼠产生IL-6的量远多于雌性小鼠，雄性小鼠IL-6水平增高与血清转氨酶升高，以及肝细胞凋亡和增殖有关。给予雄性小鼠雌性激素，可抑制IL-6的产生，能有效抑制二乙基亚硝胺 (DEN) 对小鼠的致癌作用。IL-6敲除的雄性小鼠，经DEN处理后，血清转氨酶降低，肝细胞凋亡和增殖也减弱，不易发生DEN诱导的肝癌。性别因素可能影响到小鼠对DEN刺激发生的炎症反应，雄性小鼠肝损伤和增殖性修复更明显，可见性别因素和IL-6对肝损伤和修复有着重要的影响。MyD88作为必须的衔接蛋白，在炎症反应导致肝癌的过程中，MyD88缺陷的雄性小鼠，不但阻断了DEN对雄性小鼠的致炎作用，而且也抑制了雄性小鼠肝癌的形成。在雌性小鼠中，雌激素可以减弱NF-κB和CCAAT增强子结合蛋白β的活性，进而抑制肝脏枯否细胞IL-6启动子活性，减弱IL-6下游信号，从而阻断肝癌的形成。以上结果提示，肝癌易感性存在性别差异与MyD88依赖IL-6的产生有关。

除了动物实验以外，在对人HBV或HCV慢性感染引发肝癌方面也有研究。Dolganiuc等[21]研究发现，HCV慢性感染者的单核细胞中MyD88表达水平高于非感染者，且在单核细胞和T淋巴细胞中Toll样受体 2、6、7、8、9和10的 mRNA表达水平均上调。可见HBV或HCV感染后，机体的天然免疫系统 (单核细胞) 和获得性免疫 (T淋巴细胞) 发挥抗病毒的免疫作用引发炎性反应，依赖于TLR和MyD88途径，使免疫细胞产生TNFα可有效抵抗HCV。由于HCV慢性感染的存在，在HCV核心蛋白的长期刺激作用下，导致单核细胞产生过量的TNFα，并增加MyD88/IL-1受体相关激酶复合物的产生，增强NF-κB的活性，进而增加炎性细胞因子的产生，导致免疫炎性损伤。在细胞生长因子作用下，肝细胞的修复增殖异常，可能导致肝癌。可见慢性感染引起严重炎症反应是细胞转化、肿瘤发生和肿瘤进展的重要影响因素。然而，天然免疫和获得性免疫的整体效果既能消除化学物质诱导的原发性肿瘤或自发性肿瘤，又能通过减弱肿瘤免疫原性或减弱保护性抗肿瘤应答，促进肿瘤的生长，整个过程称为“肿瘤免疫编辑”[22]。Swann等[23]通过动物实验模型证实，经MyD88途径，对感染原产生炎性应答反应，同时MyD88在炎症诱发致癌中也发挥重要的作用,即由MyD88介导的炎症依赖性肿瘤与由MyD88 (TNFα和干扰素α/β) 调控细胞因子引发的肿瘤免疫监视/免疫编辑同时存在。因此，免疫系统和肿瘤发生的复杂作用产生多种效应结果，有些是无法控制的，免疫应答既可促进又可抑制肿瘤的发生，如使肿瘤免疫原性消失，从而使肿瘤逃避免疫控制。

三、与慢性炎症反应相关的环境因素  

肝脏是重要的解毒器官，药物、酒精和其他毒素类物质都需经过肝脏的代谢。大量的毒素类物质可能影响到肝脏的正常生理功能。酗酒和吸烟可能是HBV或HCV慢性感染引发肝癌的协同因素。有研究结果显示，长期大量饮酒可使HCC的危险性增加。在HBV或HCV感染的同时，重度吸烟产生的毒素，可引起肝脏坏死性炎症并增加肝损害 (肝纤维化和肝硬化等)的严重程度，增加慢性肝病患者发展为HCC的风险。El-Zayadi等[24]报道，重度吸烟者的肝细胞内可积累过量的铁，这也可能是诱导肝纤维化和HCC发生的原因。在HBV或HCV慢性携带者中，肥胖和糖尿病使HCC发生的风险增加100倍以上，这可能是由于氧化应激和（或）脂质过氧化作用，增加了炎症状态下的肝损伤，导致肝纤维化和肝硬化[25]。此外，与HCC发生有关的其他慢性疾病，还包括自身免疫性肝炎、肝豆状核变性、α1-抗胰蛋白酶缺乏症以及原发性胆汁性肝硬化等。因TLR不仅可识别病原相关分子模式，而且可结合损伤相关分子模式，如肝细胞坏死产物。因此，TLR通过MyD88信号途径，不仅在HBV、HCV等病毒感染情况下，能诱导炎性细胞因子产生和组织损伤应答有关基因的表达，而在酒精、药物等造成肝细胞的坏死等非感染状态下能发挥同样重要的作用，而这些细胞因子和基因表达产物可能在肿瘤形成中起关键作用。

根据上述炎性相关肝癌的发病过程，可使用抑制TLR-MyD88途径的化合物，或保护肝细胞和抗炎特性的细胞因子、生长因子，将有助于降低慢性肝病患者发展为HCC的风险。如果上述动物实验中，DEN诱导肝癌的发病机制与人类肝癌发病过程相似，IL-6水平增高是发生肝癌的危险因素，则使用雌激素或者雌激素类似化合物抑制IL-6的产生，从而抑制慢性肝病的恶性进展，可能具有重要的应用价值。

综上所述，虽已确定HBV和HCV慢性感染是导致HCC发生的主要因素，并且同时存在其他协同致癌危险因素，但仍有某些致癌机制仍不明确。特别是对炎症相关肝癌的发病机制仍需深入研究。目前，加强人群HBV疫苗的免疫接种，以及其他措施减少HBV或HCV引起的慢性感染，对预防肝癌具有更重要的意义。

参  考  文  献

[1]Yang YH, Xie Q, Wang H, et al. Viral factors influencing histological changes of HBeAg-negative chronic hepatitis B patients with persistently normal serum ALT levels. Zhonghua Ganzangbing Zazhi, 2009, 17: 434-439. (in Chinese)

杨艳华,谢青,王晖,等.病毒学因素对ALT持续正常的e抗原阴性慢性乙型肝炎病毒感染者肝脏组织学改变的影响.中华肝脏病杂志,2009,17:434-439.

[2]Hsieh YH, Su IJ, Wang HC, et al. Pre-S mutant surface antigens in chronic hepatitis B virus infection induce oxidative stress and DNA damage. Carcinogenesis, 2004, 25: 2023-2032.

[3]Lai MW, Yeh CT. The oncogenic potential of hepatitis B virus rtA181T/ surface truncation mutant. Antivir Ther, 2008, 13: 875-879.

[4]Lai MW, Huang SF, Hsu CW, et al. Identification of nonsense mutations in hepatitis B virus S gene in patients with hepatocellular carcinoma developed after lamivudine therapy. Antivir Ther, 2009, 14: 249-261.

[5]Warner N, Locarnini S. The antiviral drug selected hepatitis B virus rtA181T/sW172* mutant has a dominant negative secretion defect and alters the typical profile of viral rebound. Hepatology, 2008, 48: 88-98.

[6]El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology, 2007, 132: 2557-2576.

[7]Lam CM, Yong JL, Chan AO, et al. Better survival in female patients with hepatocellular carcinoma: oral contraceptive pills related? J Clin Gastroenterol, 2005, 39:533-539.

[8]Tanaka K, Sakai H, Hashizume M, et al. Serum testosterone: estradiol ratio and the development of hepatocellular carcinoma among male cirrhotic patients. Cancer Res, 2000, 60: 5106-5110.

[9]Wang AG, Fang W, Han YH, et al. Expression of the RERG gene is gender-dependent in hepatocellular carcinoma and regulated by histone deacetyltransferases. J Korean Med Sci, 2006, 21: 891-896.

[10]Matsumoto T, Takagi H, Mori M. Androgen dependency of hepatocarcinogenesis in TGFalpha transgenic mice. Liver, 2000, 20:228-233.

[11]Rogers AB, Theve EJ, Feng Y, et al. Hepatocellular carcinoma associated with liver-gender disruption in male mice. Cancer Res, 2007, 67: 11536-11546.

[12]Liu WH, Yeh SH, Lu CC, et al. MicroRNA-18a prevents estrogen receptor-alpha expression, promoting proliferation of hepatocellular carcinoma cells. Gastroenterology, 2009, 136: 683-693.

[13]Iavarone M, Lampertico P, Seletti C, et al. The clinical and pathogenetic significance of estrogen receptor-beta expression in chronic liver diseases and liver carcinoma. Cancer, 2003, 98: 529-534.

[14]Han J, Ding L, Yuan B, et al. Hepatitis B virus X protein and the estrogen receptor variant lacking exon 5 inhibit estrogen receptor signaling in hepatoma cells. Nucleic Acids Res, 2006, 34: 3095-3106.

[15]Watashi K, Inoue D, Hijikata M, et al. Anti-hepatitis C virus activity of tamoxifen reveals the functional association of estrogen receptor with viral RNA polymerase NS5B. J Biol Chem, 2007, 282: 32765-32772.

[16]Horng T, Barton GM, Flavell RA, et al. The adaptor molecule TIRAP provides signalling specificity for Toll-like receptors. Nature, 2002, 420: 329-333.

[17]Bowie AG, Haga IR. The role of Toll-like receptors in the host response to viruses. Mol Immunol, 2005, 42: 859-867.

[18]Naugler WE, Sakurai T, Kim S, et al. Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production. Science, 2007, 317: 121-124.

[19]Greten FR, Eckmann L, Greten TF, et al. IKKbeta links inflammation and tumorigenesis in a mouse model of colitis-associated cancer. Cell, 2004, 118: 285-296.

[20]Ogata H, Kobayashi T, Chinen T, et al. Deletion of the SOCS3 gene in liver parenchymal cells promotes hepatitis-induced hepatocarcinogenesis. Gastroenterology, 2006, 131: 179-193.

[21]Dolganiuc A, Norkina O, Kodys K, et al. Viral and host factors induce macrophage activation and loss of Toll-like receptor tolerance in chronic HCV infection. Gastroenterology, 2007, 133: 1627-1636.  

[22]Smyth MJ, Dunn GP, Schreiber RD. Cancer immunosurveillance and immunoediting: the roles of immunity in suppressing tumor development and shaping tumor immunogenicity. Adv Immunol, 2006, 90: 1-50.

[23]Swann JB, Vesely MD, Silva A, et al. Demonstration of inflammation-induced cancer and cancer immunoediting during primary tumorigenesis. Proc Natl Acad Sci USA, 2008, 105: 652-656.

[24]El-Zayadi AR, Selim O, Hamdy H, et al. Heavy cigarette smoking induces hypoxic polycythemia (erythrocytosis) and hyperuricemia in chronic hepatitis C patients with reversal of clinical symptoms and laboratory parameters with therapeutic phlebotomy. Am J Gastroenterol, 2002, 97: 1264-1265.

[25]Yu MC, Yuan JM. Environmental factors and risk for hepatocellular carcinoma. Gastroenterology, 2004, 127(5 Suppl 1): S72-78.

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