TLR4通路诱导上皮间质转化在原发性胆汁性肝硬化中的作用研究
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摘要
原发性胆汁性肝硬化（primary biliary cirrhosis, PBC）是一种以肝内胆管上皮细胞损伤为主要特征的慢性、自身免疫性、胆汁淤积性肝病。该病以中老年女性多发，病因和发病机制尚不完全清楚。大量证据表明，PBC是以遗传为基础，由遗传和环境相互作用而产生的。一些临床和流行病学研究提示，泌尿道感染等因素可能与PBC的发病密切相关，是PBC发病的危险因素，但现有的研究结果并不完全一致，甚至个别研究的结果截然相反。因此，关于泌尿道感染与PBC的关系有待进一步确认。
毫无疑问，大多数PBC最终会发展成为肝纤维化和肝硬化，但能否进展成为肝癌，或者说PBC患者肝癌的患病风险是否较一般人群显著升高尚存在很大争议。其原因主要是个体研究的方法学、样本数量、统计效能等问题。循证医学研究可以对当前的所有相关研究进行系统评价和荟萃分析，研究结果可以为临床提供最高层次和最具可信性的证据。但是，到目前为止，关于PBC与肝癌的相关性尚没有进行循证医学研究。
如果能够证实泌尿道感染是PBC的一个病因，而PBC又是发生肝癌的高危因素，那么感染在PBC向肝硬化、肝癌发展过程中的具体作用机制就是一个急需解决的问题。有研究提示，PBC患者肝内胆管上皮细胞（intrahepatic biliary epithelialcells，IBECs）TLR4表达明显上调，可能参与PBC的发病。泌尿道感染主要是革兰阴性细菌，其主要成分为脂多糖（LPS）。因此，我们有理由推测，LPS-TLR4通路在PBC向肝硬化、肝癌发展过程中具有重要作用。
为进一步证实以上推测，我们进行了下面的研究：一、通过循证医学手段证实泌尿道感染与PBC的关系；二、通过循证医学手段证实PBC是肝癌的一个高危因素；三、从临床水平探讨TLR4与EMT（该过程是PBC向肝纤维化、硬化发展的一个重要步骤）的相关性及在PBC中的意义；四、探讨LPS-TLR4通路对IBEC发生EMT的诱导作用及其具体机制。
第一部分原发性胆汁性肝硬化危险因素的循证医学研究
我们主要通过PUBMED、EMBASE、Cochrane图书馆和中国各种医学搜索数据库进行文献检索，按照以下标准纳入相关文献：（1）报告了相对危险度RR和OR，并且提供了95%可信区间的观察性研究；（2）病例对照研究和队列研究；（3）独立的研究，没有重复报告相同人群。应用随机或固定效应模型进行荟萃分析，探讨各种危险因素与PBC的相关性。
经过筛查鉴定，我们纳入5篇相关的文献进行系统评价和荟萃分析。5篇文献均为英文文献，包含1913例PBC患者和4697例健康对照。5项研究均涉及吸烟与PBC的相关性，4项研究涉及泌尿道感染与PBC的相关性，3项研究涉及家族史和甲状腺疾病与PBC的相关性，2项研究涉及饮酒、染发、扁桃体切除手术、怀孕年龄、激素治疗、银屑病、类风湿性关节炎和湿疹与PBC的相关性。荟萃分析结果显示，泌尿道感染、吸烟和家族史与PBC的发生密切相关，泌尿道感染患者患PBC的危险度OR=2.02（95%CI：1.4-2.65）；吸烟患PBC的危险度OR=1.67（95%CI：1.41-1.92）；具有PBC家族史人群患PBC的危险度OR=7.56（95%CI：1.9-13.22）。而甲状腺疾病与PBC的发病没有显著相关性OR=3.08（95%CI：0.84-5.32）。为进一步确认研究结果的稳定性，我们对纳入的研究进行了出版偏倚评价。Egger检验和Begger s funnel图分析结果均表明，纳入的研究不存在出版偏倚，提示研究结果还是比较稳定的。这部分研究结果提示，泌尿道感染是PBC的一个重要病因。
第二部分原发性胆汁性肝硬化与肝癌相关性的循证医学研究
为探讨PBC与肝癌的关系，证实PBC是肝癌的一个重要危险因素，我们在本部分应用系统评价和荟萃分析的方法探讨了PBC与肝癌的相关性。我们主要通过PUBMED、EMBASE和Cochrane图书馆进行文献检索，按照以下标准纳入相关文献：（1）队列研究或病例对照研究；（2）将PBC作为一个暴露因素的研究；（3）将肿瘤作为一个结果的研究；（4）给出RR值及其95%可信区间或能够计算出二者的相关数据的研究；（5）独立的没有重复的研究。如果多个研究报道相同人群或部分相同人群，我们仅纳入最新或数据最全的研究。
我们从3510篇文献中筛选、纳入16篇文献，包括17个个体研究，其中有一篇文献报告了两组队列研究，一组是西班牙人群，另一组是意大利人群。我们对17个个体研究进行了系统评价和荟萃分析。这17个个体研究共包含16368名PBC患者。9个研究报告了PBC与所有肿瘤的危险关系；12个研究报告了PBC与肝癌危险的关系，9个研究报告了PBC与乳腺癌危险的关系；5个研究报告了与肾癌危险的关系；5个研究报告了与结肠癌危险的关系。结果显示，与一般人群相比，PBC患者患所有肿瘤的相对危险度RR值为1.55（95%CI：1.28-1.83），即PBC患者患所有肿瘤的危险比一般人群要高55%。PBC患者患肝癌的危险度RR值为18.8（95%CI：10.81-26.79），即PBC患者患肝癌的风险比一般健康人群高17.8倍。通过亚组分析，我们发现在各个亚组PBC均可以显著增高肝癌的患病风险。只有两个亚组例外，分别是美国人群和以人群为基础的研究。虽然在这两个亚组中，PBC患者患肝癌的风险仍然是一般人群的8倍以上（美国人群是23.88倍，以人群为基础的研究是8.61倍），但统计学差异并不显著，这种情况的出现可能主要是因为这两个亚组所包含的研究数量太少（均为3篇），而且异质性很高，缺少统计效能。此外，与其他肿瘤发病危险性的荟萃分析结果显示，PBC与乳腺癌、肾癌等肿瘤的发生无明显相关性，对乳腺癌进行亚组分析的结果显示在各亚组中，PBC均与乳腺癌的发生没有密切关系。
这部分研究结果表明，PBC与肝癌的发生密切相关，是肝癌发生的一个高危因素。由于肝纤维化和肝硬化是肝癌的重要原因，而上皮间质转化在纤维化疾病中具有重要作用。结合第一部分的研究，LPS-TLR4通路异常活化是否诱导肝内胆管上皮细胞间质转化？其具体机制如何？我们将在第三、四部分进行探讨。
第三部分TLR4及上皮间质转化相关蛋白在原发性胆汁性肝硬化患者的表达及临床意义
为进一步探讨TLR4和肝内胆管上皮细胞EMT与PBC向纤维化进展的关系以及二者在PBC进展过程中的关联，我们在本部分研究中应用免疫组化的方法从临床水平检测了TLR4及EMT相关蛋白在PBC患者肝组织门管区胆管上皮细胞的表达。我们收集PBC患者肝穿和肝活检标本共11例，其中病理1期2例，2期3例，3期2例，4期4例，同时收集5例供肝者肝组织切片标本作为正常对照。采用免疫组化的方法检测各组细胞角蛋白CK19、TLR4、E-钙粘蛋白及波形蛋白的表达水平，结果显示CK19在正常人及PBC患者胆管上皮细胞中均有表达，且阳性率随着PBC分期增高而明显降低。TLR4和波形蛋白在正常人肝内胆管上皮细胞几乎无表达，1期PBC患者阳性表达率分别为28.6%和30.0%，2期患者平均阳性率为41.6%和40.1%,3期患者平均阳性率为64%和73.7%,4期患者平均阳性率为73.1%和77.0%。两两比较的结果显示，除1,2期及3,4期外，其余各组间阳性细胞数差异均有统计学意义（P=0.001）。E-钙粘蛋白在正常人肝内胆管上皮细胞表达，而在PBC患者表达明显降低，1期PBC患者E-钙粘蛋白平均阳性率为84%，2期患者平均阳性率为48.1%,3期患者平均阳性率为23%,4期患者平均阳性率为19%。两两比较的结果显示，除正常对照与1期以及3,4期外，其余各组间阳性细胞数差异均有统计学意义（P=0.001）。此外，PBC患者TLR4阳性表达率与E-钙粘蛋白阳性表达率呈负相关（相关系数rs=-0.927，P<0.01）,TLR4与波形蛋白阳性表达率呈正相关（相关系数rs=0.918，P<0.01），波形蛋白与E-钙粘蛋白阳性表达率呈负相关（相关系数rs=-0.918，P<0.01）。
因此，PBC患者肝组织门管区肝内胆管上皮细胞TLR4的表达明显增高，而且按照PBC的组织学分期，TLR4随着PBC纤维化严重程度的加深而表达增强。同时，我们发现PBC患者肝组织门管区肝内胆管上皮细胞E-钙粘蛋白表达明显降低、波形蛋白表达明显增高，这种变化也是随着PBC纤维化严重程度的加重而更加明显，提示在PBC向纤维化方向发展的过程中会出现肝内胆管上皮细胞EMT现象，且与TLR4表达密切相关。
那么，在PBC的进展过程中，TLR4对肝内胆管上皮细胞EMT是否具有诱导作用及其具体机制，我们将在第四部分通过体外培养肝内胆管上皮细胞进行深入探讨。
第四部分TLR4通路对肝内胆管上皮细胞EMT的作用机制
为进一步阐明TLR4通路异常活化在PBC进展过程中作用机制，我们拟在本部分研究中体外培养肝内胆管上皮细胞，利用刮痕实验、趋化实验、细胞免疫化学、实时荧光定量PCR、MyD88及Snail小干扰RNA、抑制剂阻断、Western blot、萤光素酶报告等实验方法深入探讨LPS－TLR4信号通路活化对肝内胆管上皮细胞EMT的诱导作用及其具体分子机制。结果显示10ug/ml LPS刺激肝内胆管上皮细胞24小时后，细胞迁移明显高于对照组，经过Transwell小室迁移的细胞数为110.4±9.17，明显高于对照组（75.8±7.78），差异均有统计学意义（P<0.01）。RT-PCR结果显示，10ug/ml,50ug/ml及100ug/ml LPS作用24小时，与对照组比较，E-钙粘蛋白基因表达明显降低，S100A4基因表达明显升高，差异有统计学意义（P<0.01），细胞免疫化学结果显示10ug/ml LPS作用24小时，E－钙粘蛋白表达明显降低，S100A4蛋白表达明显升高。转染MyD88SiRNA后，经10ug/ml LPS刺激24h,刮痕实验显示细胞迁移相对距离明显低于单独LPS刺激组，Transwell小室实验结果显示迁移细胞数为85.6±8.97，明显低于LPS刺激组，且转染MyD88SiRNA组与单独加入LPS组比较，E-钙粘蛋白基因和蛋白表达明显升高，S100A4基因和蛋白表达明显降低，差异有统计学意义（P<0.01）。
应用抑制剂PDTC、SB203580及SP600125预刺激后加入10ug/ml LPS刺激24小时，与单独加入LPS组比较，E-钙粘蛋白基因表达明显增高，差异有统计学意义（P<0.01）；抑制剂SB203580预刺激后，S100A4及Snail基因表达比单独加LPS组明显降低，差异有统计学意义（P<0.01），各组Slug基因表达无明显变化。
经10ug/ml LPS刺激24小时后，转染Snail SiRNA组E-钙粘蛋白基因表达比未转染组明显增高，S100A4基因表达比未转染组明显降低，差异有统计学意义（P<0.01）。萤光素酶报告实验结果显示10ug/ml LPS刺激转染野生型载体的肝内胆管上皮细胞，相对萤光值比对照组显著升高，差异有统计学意义（P<0.01）,加入AP-1抑制剂100uM6-姜酚后，相对萤光值明显降低，而转染突变型载体组差异无明显变化。
通过本部分研究，可以推测LPS通过TLR4-MyD88-P38-AP1-Snail的途径诱导肝内胆管上皮细胞发生EMT，为PBC向纤维化、硬化发展的机理提供了一种可能的解释，为进一步探讨如何逆转PBC向纤维化方向发展提供一种可能的潜在靶点。
综上所述，通过以上四部分的研究，我们初步提出PBC发生发展的一个可能机制，即：泌尿道感染-LPS-TLR4通路异常活化-肝内胆管上皮细胞EMT-肝纤维化、硬化-肝癌。这对于有效预防或避免PBC的危险因素，寻找治疗PBC的潜在靶点，实现对PBC的合理预防、有效治疗或病情逆转具有重要的参考价值和借鉴意义。
Primary biliary cirrhosis (PBC) is a chronic progressive, autoimmune, cholestatic liverdisease of unknown etiology and pathogenesis, characterized by inflammatory destructionof intrahepatic bile ducts, which predominantly affects middle aged women. A large ofevidence indicated that urinary tract infection (UTI), as a risk factor, may be associatedwith PBC development. However, so far, the results have remained inconclusive.Therefore, the association between them needs to be further confirmed.
Without question, the majority of PBC patients may develop to liver fibrosis andcirrhosis in final. However, it remained controversial whether PBC can increase the risk ofliver cancer. The differences noted between the studies may be explained partially by themethodology, sample sizes, statistical power, and so on. Evidence-based study can be usedto assess all the published studies by the systematic review and meta-analysis, which canprovide high-level and believable evidence for the clinics. However, by far, there has beenno evidence-based study on the association between PBC and the risk of liver cancer.
If UTI was proved to be a risk factor of PBC development, which can increase the riskof liver cancer, the detailed mechanism of UTI in progression of PBC to liver cirrhosis orcancer should be explored as soon as possible. It has been revealed that the TLR4expression is significantly upregulated on the intrahepatic biliary epithelial cells (IBECs)from PBC patients, which suggested that TLR4may play a role in the pathogenesis of PBC.UTI was mainly caused by Gram-negative bacteria, the most components of which is LPS.Accordingly, we can propose that LPS-TLR4signaling pathway man play an importantrole in the progression of PBC to liver cirrhosis and cancer.
To confirm the above speculation, we have conducted studies as follows:1) to confirmthe association between UTI and PBC development by evidence-based method;2) toconfirm if PBC is a high risk factor for liver cancer by evidence-based method;3). toexplore the association between the expression of TLR4and EMT and the clinicalsignificance in PBC;4) to explore the detailed mechanism by which LPS-TLR4inducedIBEC EMT, which is a critical step of PBC to liver fibrosis and cirrhosis.
Part1Evidence-based study on the risk factors for primary biliarycirrhosis
We performed the published work search using PubMed, EMBASE, the CochraneLibrary and China National Knowledge Infrastructure. Reports fulfilling the followinginclusion criteria were included in the meta-analysis:(1) Observational studies that reported the relative risk (RR) or odds ratio (OR) with95%confidence interval;(2)case-control and cohort studies published as original articles;(3) independent studieswithout repeat reports on the same population or subpopulation. Based on fixed-orrandom-effects model, the meta-analysis was carried out to explore the associationbetween various risk factors and PBC development.
After screening and identification, a total of5studies were included in the finalsystematic review and meta-analysis. All of the included studies were in English, involving1913PBC cases and4697controls. The association of PBC with smoking was reported inall of these five publications, with UTI in four, with family history of PBC and thyroiddisease in three, and with alcohol intake, hair dye, tonsillectomy, age of pregnancy,hormone therapy, psoriasis, eczema and rheumatoid arthritis in only two. The results ofmeta-analysis showed UTI, smoking, and family history of PBC may be risk factors for thedevelopment of PBC, with a pooled OR of2.02(95%CI=1.40–2.65),1.67(95%CI=1.41–1.92) and7.56(95%CI=1.90–13.22) respectively. The pooled OR for thyroiddisease was3.08(95%CI=0.84–5.32). To further confirm the stability of the results, weperformed the evaluation on publication bias of included studies. No evidence forpublication bias was found for any of these four factors by means of Begger and Eggertests, which indicated that the results were stable.
Part2Evidence-based study on the association between PBC and livercancer
To confirm whether PBC is a vital risk factor for the development of liver cancer, wecarried out a systematic review and meta-analysis to explore the association between PBCand the development of liver cancer. A literature search of the Pubmed, EMBASE and theCochrane Library was conducted and studies fulfilling the following inclusion criteria wereincluded in the meta-analysis:1) a cohort or case-control design;2) PBC as one of theexposure interests;3) cancer as one of the outcome of interests;4) rate ratio, hazard ratioor standardized incidence rate (SIR) with their95%CIs (or data to calculate them)available;5) independent studies. In case of multiple reports on the same population orsubpopulation, we included only data from the recent or the most complete studies with thelargest numbers of cases and controls.
Of3510publications identified,16publications involving17studies which met theinclusion criteria were included in the systematic review and meta-analysis. Notably, therewas one publication which involved two cohort studies, one for Spanish patients and the other for Italian. The17studies involved a total of16368PBC patients. Nine studies wereconducted for relative risk of overall malignancy,12for hepatocellular carcinoma,9forbreast cancer,5for kidney cancer, and5for colon cancer. The results of overallmalignancy showed that the pooled RR with95%CI was1.55(95%CI,1.28-1.83) in arandom-effect model for PBC patients compared with general population, indicating thatPBC patients have a55%increased risk for overall cancer. The pooled RR of liver cancerwith95%CI was18.80(95%CI,10.81-26.79) for PBC patients compared with generalpopulation. When conducting subgroup meta-analyses by these factors, PBC still remainedsignificantly associated with an increased risk of HCC in various subgroup-analyses withthe exception of two, one for the studies in the USA population (pooled RR23.88,95%CI,-9.14-56.89), and the other for the population-based studies (pooled RR8.61,95%CI,-4.18-21.40), which might result from too small number of studies (only three studies foreach subgroup) with significant heterogeneity. In addition, the results of meta-analysisabout the association of PBC with risks of other cancers indicated that PBC was not withincreased risk of breast cancer, kidney cancer, etc. For breast cancer, we also conductedsubgroup-analyses by region, case ascertainment, type of effect size and age. The resultsshowing no significant association with increased risk of breast cancer did not change invarious subgroups.
The results in this part indicated that PBC, as a high risk factor, is significantlyassociated with increased risk of liver cancer. Since EMT plays a key role in liver fibrosiswhich is an important cause for liver cancer, we will explore, in Part3and4, the detailedmechanisms by which EMT of IBEC is induced by LPS-TLR4signaling pathway.Part3Expression of TLR4and EMT-related proteins and their clinicalsignificance in PBC patients
To further explore the role of TLR4and IBEC EMT with the progression of PBC toliver fibrosis, as well as correlation between TLR4and EMT in PBC progression,immunohistochemical method was used to detect the expression of TLR4and EMT-relatedproteins in portal IBECs from PBC liver specimens in this part. A total of11samples fromliver biopsy of PBC patients were collected, of whom2patients were in histological stage1,3in stage2,2in stage3and4in stage4. Additionally,5specimens, as normal controls,were collected from liver biopsy of donors.
Immunohistochemistry was used to detect the expression of CK19, TLR4, E-cadherinand vimentin in IBECs. The results showed that CK19was detected in IBECs of both PBC patients and normal controls. The level of CK19in IBECs was significantly decreased withthe progression of PBC histological stages. TLR4and vimentin were hardly detected inIBECs of normal controls. In contrast, TLR4and vimentin were detected respectively in28.6%and30.0%of IBECs of PBC patients with histological stage1, in41.6%and40.1%with stage2,64.0%and73.7%with stage3and73.1%and77.0%with stage4.Furthermore, the results showed significant differences of TLR4and vimentin expressionin IBECs between any two groups of PBC patients with different stages (P=0.001), exceptbetween PBC patients with stage1and stage2as well as stage3and stage4. E-cadherinwas detected in almost all of IBECs of normal individuals. However, its expression wassignificantly decreased in PBC patients. It was detected in84%of IBECs of PBC patientswith histological stage1, in48.1%with stage2, in23%with stage3and19%with stage4.The results showed significant differences of E-cadherin expression in IBECs between anytwo groups of PBC patients with different stages(P=0.001), except between PBC patientswith stage1and normal controls as well as stage3and stage4. Furthermore, PBC patientsshowed significantly inverse correlation between TLR4and E-cadherin(rs=-0.927，P<0.01）, positive correlation between TLR4and vimentin (rs=0.918，P<0.01) and inversecorrelation between E-cadherin and vimentin (rs=-0.918，P<0.01).
Collectively, the expression of IBEC TLR4is significantly upregulated in PBCpatients. Furthermore, the expression is increased significantly with advanced fibrosis ofPBC. Also, the downregulation of E-cadherin and upregulation of vimentin in PBCpatients is significantly associated with fibrosis severity of PBC. These results suggestedthat EMT of IBEC may occur in the progression of PBC to liver fibrosis and may haveclose association with TLR4.
Then, can TLR4signaling pathway induce EMT of IBEC in the progression of PBC?What are detailed mechanisms? These questions will be explored in vitro in Part4.
Part4Mechanisms of the role of TLR4signaling pathway in EMT ofIBEC
To further clarify the detailed mechanisms of abnormal activation of TLR4signalingpathway in PBC progression, IBEC culture, scratching assay, motility assay, cellularimmunochemistry, real-time RT-PCR, siRNA, inhibitors, western blot, luciferase reportassay, etc. were used to explore the role of TLR4signaling pathway in IBEC EMT as wellas its detailed mechanisms. The results indicated that cell motility was significantly fasterand the numbers of migrating cells through Transwell chamber were significantly more (110.4±9.17)24hours after stimulation by LPS, compared with controls (P<0.01). TheRT-PCR results showed that the expression of E-cadherin gene was significantly decreased24hours after stimulation respectively by10ug/ml,50ug/ml and100ug/ml of LPS, butS100A4increased (P<0.01).Cellular immunochemistry showed the same results.
The motility of IBECs with MyD88SiRNA silence was significantly slower than thatwithout MyD88SiRNA silence24hours after stimulation by10ug/ml of LPS. The numberof migrating cells through Transwell chamber was significantly smaller in IBECs withMyD88SiRNA silence than those without MyD88SiRNA silence24hours afterstimulation by10ug/ml of LPS. Also, E-cadherin level was significantly increased, butS100A4decreased, in IBECs with MyD88SiRNA silence than those without MyD88SiRNA silence24hours after stimulation by10ug/ml of LPS.
The expression of E-cadherin gene was significantly increased in IBECs withpretreatment by PDTC, SB203580and SP600125compared with those withoutpretreatment24hours after stimulation by10ug/ml of LPS (P<0.01). In contrast, theexpression levels of S100A4gene and Snail gene were significantly decreased in IBECspretreated by SB203580than not24hours after stimulation by10ug/ml of LPS (P<0.01).
The expression of E-cadherin gene was significantly increased in IBECs with SnailSiRNA silence than in those without, but S100A4decreased,24hours after stimulation by10ug/ml of LPS (P<0.01).The luciferase report assay showed that RLU value wassignificantly increased in IBECs transfected by wild-type vector after stimulation by10ug/ml of LPS (P<0.01). However, the RLU value became decreased after addition of100uM6-Gingerol which is the inhibitor of AP-1. In contrast, the change of RLU valuewas not significant when IBECs were transfected by mutant vector.
This part demonstrated that LPS can induce IBEC EMT byTLR4-MyD88-P38-AP1-Snail pathway. The present study provided a possible explanationfor the mechanisms of the progression of PBC to liver fibrosis and cirrhosis and a potentialtarget for further exploring how the process of PBC to liver fibrosis can be inversed.
In summary, the above four parts of study demonstrated a possible mechanism of PBCdevelopment and progression as follows: urinary tract infection-LPS-abnormal activationof TLR4signaling pathway-IBEC EMT-liver fibrosis and cirrhosis-liver cancer.
毫无疑问，大多数PBC最终会发展成为肝纤维化和肝硬化，但能否进展成为肝癌，或者说PBC患者肝癌的患病风险是否较一般人群显著升高尚存在很大争议。其原因主要是个体研究的方法学、样本数量、统计效能等问题。循证医学研究可以对当前的所有相关研究进行系统评价和荟萃分析，研究结果可以为临床提供最高层次和最具可信性的证据。但是，到目前为止，关于PBC与肝癌的相关性尚没有进行循证医学研究。
如果能够证实泌尿道感染是PBC的一个病因，而PBC又是发生肝癌的高危因素，那么感染在PBC向肝硬化、肝癌发展过程中的具体作用机制就是一个急需解决的问题。有研究提示，PBC患者肝内胆管上皮细胞（intrahepatic biliary epithelialcells，IBECs）TLR4表达明显上调，可能参与PBC的发病。泌尿道感染主要是革兰阴性细菌，其主要成分为脂多糖（LPS）。因此，我们有理由推测，LPS-TLR4通路在PBC向肝硬化、肝癌发展过程中具有重要作用。
为进一步证实以上推测，我们进行了下面的研究：一、通过循证医学手段证实泌尿道感染与PBC的关系；二、通过循证医学手段证实PBC是肝癌的一个高危因素；三、从临床水平探讨TLR4与EMT（该过程是PBC向肝纤维化、硬化发展的一个重要步骤）的相关性及在PBC中的意义；四、探讨LPS-TLR4通路对IBEC发生EMT的诱导作用及其具体机制。
第一部分原发性胆汁性肝硬化危险因素的循证医学研究
我们主要通过PUBMED、EMBASE、Cochrane图书馆和中国各种医学搜索数据库进行文献检索，按照以下标准纳入相关文献：（1）报告了相对危险度RR和OR，并且提供了95%可信区间的观察性研究；（2）病例对照研究和队列研究；（3）独立的研究，没有重复报告相同人群。应用随机或固定效应模型进行荟萃分析，探讨各种危险因素与PBC的相关性。
经过筛查鉴定，我们纳入5篇相关的文献进行系统评价和荟萃分析。5篇文献均为英文文献，包含1913例PBC患者和4697例健康对照。5项研究均涉及吸烟与PBC的相关性，4项研究涉及泌尿道感染与PBC的相关性，3项研究涉及家族史和甲状腺疾病与PBC的相关性，2项研究涉及饮酒、染发、扁桃体切除手术、怀孕年龄、激素治疗、银屑病、类风湿性关节炎和湿疹与PBC的相关性。荟萃分析结果显示，泌尿道感染、吸烟和家族史与PBC的发生密切相关，泌尿道感染患者患PBC的危险度OR=2.02（95%CI：1.4-2.65）；吸烟患PBC的危险度OR=1.67（95%CI：1.41-1.92）；具有PBC家族史人群患PBC的危险度OR=7.56（95%CI：1.9-13.22）。而甲状腺疾病与PBC的发病没有显著相关性OR=3.08（95%CI：0.84-5.32）。为进一步确认研究结果的稳定性，我们对纳入的研究进行了出版偏倚评价。Egger检验和Begger s funnel图分析结果均表明，纳入的研究不存在出版偏倚，提示研究结果还是比较稳定的。这部分研究结果提示，泌尿道感染是PBC的一个重要病因。
第二部分原发性胆汁性肝硬化与肝癌相关性的循证医学研究
为探讨PBC与肝癌的关系，证实PBC是肝癌的一个重要危险因素，我们在本部分应用系统评价和荟萃分析的方法探讨了PBC与肝癌的相关性。我们主要通过PUBMED、EMBASE和Cochrane图书馆进行文献检索，按照以下标准纳入相关文献：（1）队列研究或病例对照研究；（2）将PBC作为一个暴露因素的研究；（3）将肿瘤作为一个结果的研究；（4）给出RR值及其95%可信区间或能够计算出二者的相关数据的研究；（5）独立的没有重复的研究。如果多个研究报道相同人群或部分相同人群，我们仅纳入最新或数据最全的研究。
我们从3510篇文献中筛选、纳入16篇文献，包括17个个体研究，其中有一篇文献报告了两组队列研究，一组是西班牙人群，另一组是意大利人群。我们对17个个体研究进行了系统评价和荟萃分析。这17个个体研究共包含16368名PBC患者。9个研究报告了PBC与所有肿瘤的危险关系；12个研究报告了PBC与肝癌危险的关系，9个研究报告了PBC与乳腺癌危险的关系；5个研究报告了与肾癌危险的关系；5个研究报告了与结肠癌危险的关系。结果显示，与一般人群相比，PBC患者患所有肿瘤的相对危险度RR值为1.55（95%CI：1.28-1.83），即PBC患者患所有肿瘤的危险比一般人群要高55%。PBC患者患肝癌的危险度RR值为18.8（95%CI：10.81-26.79），即PBC患者患肝癌的风险比一般健康人群高17.8倍。通过亚组分析，我们发现在各个亚组PBC均可以显著增高肝癌的患病风险。只有两个亚组例外，分别是美国人群和以人群为基础的研究。虽然在这两个亚组中，PBC患者患肝癌的风险仍然是一般人群的8倍以上（美国人群是23.88倍，以人群为基础的研究是8.61倍），但统计学差异并不显著，这种情况的出现可能主要是因为这两个亚组所包含的研究数量太少（均为3篇），而且异质性很高，缺少统计效能。此外，与其他肿瘤发病危险性的荟萃分析结果显示，PBC与乳腺癌、肾癌等肿瘤的发生无明显相关性，对乳腺癌进行亚组分析的结果显示在各亚组中，PBC均与乳腺癌的发生没有密切关系。
这部分研究结果表明，PBC与肝癌的发生密切相关，是肝癌发生的一个高危因素。由于肝纤维化和肝硬化是肝癌的重要原因，而上皮间质转化在纤维化疾病中具有重要作用。结合第一部分的研究，LPS-TLR4通路异常活化是否诱导肝内胆管上皮细胞间质转化？其具体机制如何？我们将在第三、四部分进行探讨。
第三部分TLR4及上皮间质转化相关蛋白在原发性胆汁性肝硬化患者的表达及临床意义
为进一步探讨TLR4和肝内胆管上皮细胞EMT与PBC向纤维化进展的关系以及二者在PBC进展过程中的关联，我们在本部分研究中应用免疫组化的方法从临床水平检测了TLR4及EMT相关蛋白在PBC患者肝组织门管区胆管上皮细胞的表达。我们收集PBC患者肝穿和肝活检标本共11例，其中病理1期2例，2期3例，3期2例，4期4例，同时收集5例供肝者肝组织切片标本作为正常对照。采用免疫组化的方法检测各组细胞角蛋白CK19、TLR4、E-钙粘蛋白及波形蛋白的表达水平，结果显示CK19在正常人及PBC患者胆管上皮细胞中均有表达，且阳性率随着PBC分期增高而明显降低。TLR4和波形蛋白在正常人肝内胆管上皮细胞几乎无表达，1期PBC患者阳性表达率分别为28.6%和30.0%，2期患者平均阳性率为41.6%和40.1%,3期患者平均阳性率为64%和73.7%,4期患者平均阳性率为73.1%和77.0%。两两比较的结果显示，除1,2期及3,4期外，其余各组间阳性细胞数差异均有统计学意义（P=0.001）。E-钙粘蛋白在正常人肝内胆管上皮细胞表达，而在PBC患者表达明显降低，1期PBC患者E-钙粘蛋白平均阳性率为84%，2期患者平均阳性率为48.1%,3期患者平均阳性率为23%,4期患者平均阳性率为19%。两两比较的结果显示，除正常对照与1期以及3,4期外，其余各组间阳性细胞数差异均有统计学意义（P=0.001）。此外，PBC患者TLR4阳性表达率与E-钙粘蛋白阳性表达率呈负相关（相关系数rs=-0.927，P<0.01）,TLR4与波形蛋白阳性表达率呈正相关（相关系数rs=0.918，P<0.01），波形蛋白与E-钙粘蛋白阳性表达率呈负相关（相关系数rs=-0.918，P<0.01）。
因此，PBC患者肝组织门管区肝内胆管上皮细胞TLR4的表达明显增高，而且按照PBC的组织学分期，TLR4随着PBC纤维化严重程度的加深而表达增强。同时，我们发现PBC患者肝组织门管区肝内胆管上皮细胞E-钙粘蛋白表达明显降低、波形蛋白表达明显增高，这种变化也是随着PBC纤维化严重程度的加重而更加明显，提示在PBC向纤维化方向发展的过程中会出现肝内胆管上皮细胞EMT现象，且与TLR4表达密切相关。
那么，在PBC的进展过程中，TLR4对肝内胆管上皮细胞EMT是否具有诱导作用及其具体机制，我们将在第四部分通过体外培养肝内胆管上皮细胞进行深入探讨。
第四部分TLR4通路对肝内胆管上皮细胞EMT的作用机制
为进一步阐明TLR4通路异常活化在PBC进展过程中作用机制，我们拟在本部分研究中体外培养肝内胆管上皮细胞，利用刮痕实验、趋化实验、细胞免疫化学、实时荧光定量PCR、MyD88及Snail小干扰RNA、抑制剂阻断、Western blot、萤光素酶报告等实验方法深入探讨LPS－TLR4信号通路活化对肝内胆管上皮细胞EMT的诱导作用及其具体分子机制。结果显示10ug/ml LPS刺激肝内胆管上皮细胞24小时后，细胞迁移明显高于对照组，经过Transwell小室迁移的细胞数为110.4±9.17，明显高于对照组（75.8±7.78），差异均有统计学意义（P<0.01）。RT-PCR结果显示，10ug/ml,50ug/ml及100ug/ml LPS作用24小时，与对照组比较，E-钙粘蛋白基因表达明显降低，S100A4基因表达明显升高，差异有统计学意义（P<0.01），细胞免疫化学结果显示10ug/ml LPS作用24小时，E－钙粘蛋白表达明显降低，S100A4蛋白表达明显升高。转染MyD88SiRNA后，经10ug/ml LPS刺激24h,刮痕实验显示细胞迁移相对距离明显低于单独LPS刺激组，Transwell小室实验结果显示迁移细胞数为85.6±8.97，明显低于LPS刺激组，且转染MyD88SiRNA组与单独加入LPS组比较，E-钙粘蛋白基因和蛋白表达明显升高，S100A4基因和蛋白表达明显降低，差异有统计学意义（P<0.01）。
应用抑制剂PDTC、SB203580及SP600125预刺激后加入10ug/ml LPS刺激24小时，与单独加入LPS组比较，E-钙粘蛋白基因表达明显增高，差异有统计学意义（P<0.01）；抑制剂SB203580预刺激后，S100A4及Snail基因表达比单独加LPS组明显降低，差异有统计学意义（P<0.01），各组Slug基因表达无明显变化。
经10ug/ml LPS刺激24小时后，转染Snail SiRNA组E-钙粘蛋白基因表达比未转染组明显增高，S100A4基因表达比未转染组明显降低，差异有统计学意义（P<0.01）。萤光素酶报告实验结果显示10ug/ml LPS刺激转染野生型载体的肝内胆管上皮细胞，相对萤光值比对照组显著升高，差异有统计学意义（P<0.01）,加入AP-1抑制剂100uM6-姜酚后，相对萤光值明显降低，而转染突变型载体组差异无明显变化。
通过本部分研究，可以推测LPS通过TLR4-MyD88-P38-AP1-Snail的途径诱导肝内胆管上皮细胞发生EMT，为PBC向纤维化、硬化发展的机理提供了一种可能的解释，为进一步探讨如何逆转PBC向纤维化方向发展提供一种可能的潜在靶点。
综上所述，通过以上四部分的研究，我们初步提出PBC发生发展的一个可能机制，即：泌尿道感染-LPS-TLR4通路异常活化-肝内胆管上皮细胞EMT-肝纤维化、硬化-肝癌。这对于有效预防或避免PBC的危险因素，寻找治疗PBC的潜在靶点，实现对PBC的合理预防、有效治疗或病情逆转具有重要的参考价值和借鉴意义。
Primary biliary cirrhosis (PBC) is a chronic progressive, autoimmune, cholestatic liverdisease of unknown etiology and pathogenesis, characterized by inflammatory destructionof intrahepatic bile ducts, which predominantly affects middle aged women. A large ofevidence indicated that urinary tract infection (UTI), as a risk factor, may be associatedwith PBC development. However, so far, the results have remained inconclusive.Therefore, the association between them needs to be further confirmed.
Without question, the majority of PBC patients may develop to liver fibrosis andcirrhosis in final. However, it remained controversial whether PBC can increase the risk ofliver cancer. The differences noted between the studies may be explained partially by themethodology, sample sizes, statistical power, and so on. Evidence-based study can be usedto assess all the published studies by the systematic review and meta-analysis, which canprovide high-level and believable evidence for the clinics. However, by far, there has beenno evidence-based study on the association between PBC and the risk of liver cancer.
If UTI was proved to be a risk factor of PBC development, which can increase the riskof liver cancer, the detailed mechanism of UTI in progression of PBC to liver cirrhosis orcancer should be explored as soon as possible. It has been revealed that the TLR4expression is significantly upregulated on the intrahepatic biliary epithelial cells (IBECs)from PBC patients, which suggested that TLR4may play a role in the pathogenesis of PBC.UTI was mainly caused by Gram-negative bacteria, the most components of which is LPS.Accordingly, we can propose that LPS-TLR4signaling pathway man play an importantrole in the progression of PBC to liver cirrhosis and cancer.
To confirm the above speculation, we have conducted studies as follows:1) to confirmthe association between UTI and PBC development by evidence-based method;2) toconfirm if PBC is a high risk factor for liver cancer by evidence-based method;3). toexplore the association between the expression of TLR4and EMT and the clinicalsignificance in PBC;4) to explore the detailed mechanism by which LPS-TLR4inducedIBEC EMT, which is a critical step of PBC to liver fibrosis and cirrhosis.
Part1Evidence-based study on the risk factors for primary biliarycirrhosis
We performed the published work search using PubMed, EMBASE, the CochraneLibrary and China National Knowledge Infrastructure. Reports fulfilling the followinginclusion criteria were included in the meta-analysis:(1) Observational studies that reported the relative risk (RR) or odds ratio (OR) with95%confidence interval;(2)case-control and cohort studies published as original articles;(3) independent studieswithout repeat reports on the same population or subpopulation. Based on fixed-orrandom-effects model, the meta-analysis was carried out to explore the associationbetween various risk factors and PBC development.
After screening and identification, a total of5studies were included in the finalsystematic review and meta-analysis. All of the included studies were in English, involving1913PBC cases and4697controls. The association of PBC with smoking was reported inall of these five publications, with UTI in four, with family history of PBC and thyroiddisease in three, and with alcohol intake, hair dye, tonsillectomy, age of pregnancy,hormone therapy, psoriasis, eczema and rheumatoid arthritis in only two. The results ofmeta-analysis showed UTI, smoking, and family history of PBC may be risk factors for thedevelopment of PBC, with a pooled OR of2.02(95%CI=1.40–2.65),1.67(95%CI=1.41–1.92) and7.56(95%CI=1.90–13.22) respectively. The pooled OR for thyroiddisease was3.08(95%CI=0.84–5.32). To further confirm the stability of the results, weperformed the evaluation on publication bias of included studies. No evidence forpublication bias was found for any of these four factors by means of Begger and Eggertests, which indicated that the results were stable.
Part2Evidence-based study on the association between PBC and livercancer
To confirm whether PBC is a vital risk factor for the development of liver cancer, wecarried out a systematic review and meta-analysis to explore the association between PBCand the development of liver cancer. A literature search of the Pubmed, EMBASE and theCochrane Library was conducted and studies fulfilling the following inclusion criteria wereincluded in the meta-analysis:1) a cohort or case-control design;2) PBC as one of theexposure interests;3) cancer as one of the outcome of interests;4) rate ratio, hazard ratioor standardized incidence rate (SIR) with their95%CIs (or data to calculate them)available;5) independent studies. In case of multiple reports on the same population orsubpopulation, we included only data from the recent or the most complete studies with thelargest numbers of cases and controls.
Of3510publications identified,16publications involving17studies which met theinclusion criteria were included in the systematic review and meta-analysis. Notably, therewas one publication which involved two cohort studies, one for Spanish patients and the other for Italian. The17studies involved a total of16368PBC patients. Nine studies wereconducted for relative risk of overall malignancy,12for hepatocellular carcinoma,9forbreast cancer,5for kidney cancer, and5for colon cancer. The results of overallmalignancy showed that the pooled RR with95%CI was1.55(95%CI,1.28-1.83) in arandom-effect model for PBC patients compared with general population, indicating thatPBC patients have a55%increased risk for overall cancer. The pooled RR of liver cancerwith95%CI was18.80(95%CI,10.81-26.79) for PBC patients compared with generalpopulation. When conducting subgroup meta-analyses by these factors, PBC still remainedsignificantly associated with an increased risk of HCC in various subgroup-analyses withthe exception of two, one for the studies in the USA population (pooled RR23.88,95%CI,-9.14-56.89), and the other for the population-based studies (pooled RR8.61,95%CI,-4.18-21.40), which might result from too small number of studies (only three studies foreach subgroup) with significant heterogeneity. In addition, the results of meta-analysisabout the association of PBC with risks of other cancers indicated that PBC was not withincreased risk of breast cancer, kidney cancer, etc. For breast cancer, we also conductedsubgroup-analyses by region, case ascertainment, type of effect size and age. The resultsshowing no significant association with increased risk of breast cancer did not change invarious subgroups.
The results in this part indicated that PBC, as a high risk factor, is significantlyassociated with increased risk of liver cancer. Since EMT plays a key role in liver fibrosiswhich is an important cause for liver cancer, we will explore, in Part3and4, the detailedmechanisms by which EMT of IBEC is induced by LPS-TLR4signaling pathway.Part3Expression of TLR4and EMT-related proteins and their clinicalsignificance in PBC patients
To further explore the role of TLR4and IBEC EMT with the progression of PBC toliver fibrosis, as well as correlation between TLR4and EMT in PBC progression,immunohistochemical method was used to detect the expression of TLR4and EMT-relatedproteins in portal IBECs from PBC liver specimens in this part. A total of11samples fromliver biopsy of PBC patients were collected, of whom2patients were in histological stage1,3in stage2,2in stage3and4in stage4. Additionally,5specimens, as normal controls,were collected from liver biopsy of donors.
Immunohistochemistry was used to detect the expression of CK19, TLR4, E-cadherinand vimentin in IBECs. The results showed that CK19was detected in IBECs of both PBC patients and normal controls. The level of CK19in IBECs was significantly decreased withthe progression of PBC histological stages. TLR4and vimentin were hardly detected inIBECs of normal controls. In contrast, TLR4and vimentin were detected respectively in28.6%and30.0%of IBECs of PBC patients with histological stage1, in41.6%and40.1%with stage2,64.0%and73.7%with stage3and73.1%and77.0%with stage4.Furthermore, the results showed significant differences of TLR4and vimentin expressionin IBECs between any two groups of PBC patients with different stages (P=0.001), exceptbetween PBC patients with stage1and stage2as well as stage3and stage4. E-cadherinwas detected in almost all of IBECs of normal individuals. However, its expression wassignificantly decreased in PBC patients. It was detected in84%of IBECs of PBC patientswith histological stage1, in48.1%with stage2, in23%with stage3and19%with stage4.The results showed significant differences of E-cadherin expression in IBECs between anytwo groups of PBC patients with different stages(P=0.001), except between PBC patientswith stage1and normal controls as well as stage3and stage4. Furthermore, PBC patientsshowed significantly inverse correlation between TLR4and E-cadherin(rs=-0.927，P<0.01）, positive correlation between TLR4and vimentin (rs=0.918，P<0.01) and inversecorrelation between E-cadherin and vimentin (rs=-0.918，P<0.01).
Collectively, the expression of IBEC TLR4is significantly upregulated in PBCpatients. Furthermore, the expression is increased significantly with advanced fibrosis ofPBC. Also, the downregulation of E-cadherin and upregulation of vimentin in PBCpatients is significantly associated with fibrosis severity of PBC. These results suggestedthat EMT of IBEC may occur in the progression of PBC to liver fibrosis and may haveclose association with TLR4.
Then, can TLR4signaling pathway induce EMT of IBEC in the progression of PBC?What are detailed mechanisms? These questions will be explored in vitro in Part4.
Part4Mechanisms of the role of TLR4signaling pathway in EMT ofIBEC
To further clarify the detailed mechanisms of abnormal activation of TLR4signalingpathway in PBC progression, IBEC culture, scratching assay, motility assay, cellularimmunochemistry, real-time RT-PCR, siRNA, inhibitors, western blot, luciferase reportassay, etc. were used to explore the role of TLR4signaling pathway in IBEC EMT as wellas its detailed mechanisms. The results indicated that cell motility was significantly fasterand the numbers of migrating cells through Transwell chamber were significantly more (110.4±9.17)24hours after stimulation by LPS, compared with controls (P<0.01). TheRT-PCR results showed that the expression of E-cadherin gene was significantly decreased24hours after stimulation respectively by10ug/ml,50ug/ml and100ug/ml of LPS, butS100A4increased (P<0.01).Cellular immunochemistry showed the same results.
The motility of IBECs with MyD88SiRNA silence was significantly slower than thatwithout MyD88SiRNA silence24hours after stimulation by10ug/ml of LPS. The numberof migrating cells through Transwell chamber was significantly smaller in IBECs withMyD88SiRNA silence than those without MyD88SiRNA silence24hours afterstimulation by10ug/ml of LPS. Also, E-cadherin level was significantly increased, butS100A4decreased, in IBECs with MyD88SiRNA silence than those without MyD88SiRNA silence24hours after stimulation by10ug/ml of LPS.
The expression of E-cadherin gene was significantly increased in IBECs withpretreatment by PDTC, SB203580and SP600125compared with those withoutpretreatment24hours after stimulation by10ug/ml of LPS (P<0.01). In contrast, theexpression levels of S100A4gene and Snail gene were significantly decreased in IBECspretreated by SB203580than not24hours after stimulation by10ug/ml of LPS (P<0.01).
The expression of E-cadherin gene was significantly increased in IBECs with SnailSiRNA silence than in those without, but S100A4decreased,24hours after stimulation by10ug/ml of LPS (P<0.01).The luciferase report assay showed that RLU value wassignificantly increased in IBECs transfected by wild-type vector after stimulation by10ug/ml of LPS (P<0.01). However, the RLU value became decreased after addition of100uM6-Gingerol which is the inhibitor of AP-1. In contrast, the change of RLU valuewas not significant when IBECs were transfected by mutant vector.
This part demonstrated that LPS can induce IBEC EMT byTLR4-MyD88-P38-AP1-Snail pathway. The present study provided a possible explanationfor the mechanisms of the progression of PBC to liver fibrosis and cirrhosis and a potentialtarget for further exploring how the process of PBC to liver fibrosis can be inversed.
In summary, the above four parts of study demonstrated a possible mechanism of PBCdevelopment and progression as follows: urinary tract infection-LPS-abnormal activationof TLR4signaling pathway-IBEC EMT-liver fibrosis and cirrhosis-liver cancer.
引文
[1] Kawai T, Akira S. TLR signaling. Seminars in Immunology.2007:19:24-32.
[2] Wang AP, Migita K, Ito M, et al. Hepatic expression of toll-like receptor4in primarybiliary cirrhosis. J Autoimmun.2005;25:85-91.
[3]Honda Y, Yamagiwa S, Matsuda Y, et al. Altered expression of TLR homolog RP105on monocytes hepersensitive to LPS in patients with primary biliary cirrhosis. J Hepatol.2007;47:404-411.
[4]Yokoyama T, Komori A, Nakamura, et al. Human intrahepatic biliary epithelial cellsfunction in innate immunity by producing IL-6and IL-8via the TLR4-NF-kappaB and-MAPK signaling pathways. Liver Int.2006;26:467-476.
[5] Kalluri R, Neilson EG: Epithelial mesenchymal transformation and its implication forfibrosis. J Clin Invest,2003,112:1776-1784.
[6] Acloque H, Thiery JP, Nieto MA. The physiology and pathology of the EMT. EMBOreports.2008;9:322-326.
[7] Teng YQ, Zeisberg M, Kalluri R. Transcriptional regulation of epithelial-mesenchymaltransition. J Clin Invest.2007;117:304-306.
[8] Gershwin ME, Mackay IR. The causes of primary biliary cirrhosis: convenient andinconvenient truths. Hepatology.2008;47:737–745.
[9] Howel D, Fischbacher CM, Bhopal RS, Gray J, Metcalf JV, James OFW. Anexploratory population-based case-control study of primary biliary cirrhosis.Hepatology2000;31:1055–1060.
[10]Parikh-Patel A, Gold EB, Worman H, et al. Risk factors for primary biliary cirrhosis ina cohort of patients from the United States. Hepatology2001;33:16–21.
[11]Gershwin ME, Slemi C, Worman HJ et al. Risk factors and comorbidities in primarybiliary cirrhosis: a controlled interviewbased study of1032patients. Hepatology2005;42:1194–1202.
[12]Prince MI, Ducker SJ, James OFW. Case-control studies of risk factors for primarybiliary cirrhosis in two United Kingdom populations. Gut2010;59:508–512.
[13]Corpechot C, Chrétien Y, Chazouillères O, Poupon R. Demographic, lifestyle, medicaland familial factors associated with primary biliary cirrhosis. J Hepatol2010;53:162–169.
[14]Zein CO, Beatty K, Post AB, Logan L, Debanne S, McCullough AJ. Smoking andincreased severity of hepatic fibrosis in primary biliary cirrhosis: a cross validatedretrospective assessment. Hepatology2006;44:1564–71.
[15]Selmi C, Bowlus CL, Gershwin ME, et al. Primary biliary cirrhosis. Lancet.2011;377:1600–1609.
[16]Floreani A, Baragiotta A, Baldo V, Menegon T, Farinati F, Naccarato R. Hepatic andextrahepatic malignancies in primary biliary cirrhosis. Hepatology.1999;29:1425-1428.
[17]Krasner P, Johnson PJ, Portmann B, Watkinson G, Macsween RNM, Williams R.Hepatocellular carcinoma in primary biliary cirrhosis: report of four cases. Gut,1979;20:255-258.
[18]Nakanuma Y, Terada T, Doishita K, Miwa A. Hepatocellular carcinoma in primarybiliary cirrhosis: an autopsy study. Hepatology1990;11:1010-1016.
[19]Shimizu A, Koyama M, Okuda Y, Takase K, Nakano T, Tameda Y. Hepatocellularcarcinoma in primary biliary cirrhosis: a case report and review of the Japaneseliterature. Hepatogastroenterology1998;45:2352-2355.
[20]Farinati F, Floreani A, De Maria N, Fagiuoli S, Naccarato R, Chiaramonte M.Hepatocellular carcinoma in primary biliary cirrhosis. J Hepatol1994;21:315-316.
[21]Caballeria L, Pare′s A, Castells A, Gine′s A, Bru C, Rode′s J. Hepatocellularcarcinoma in primary biliary cirrhosis: similar incidence to that in hepatitis Cvirus-related cirrhosis. Am J Gastroenterol.2001;96:1160-1163.
[22]Deutsch M, Papatheodoridis GV, Tzakou A, Hadziyannis SJ. Risk of hepatocellularcarcinoma and extrahepatic malignancies in primary biliary cirrhosis. Eur JGastroenterol Hepatol.2008;20:5-9.
[23]Piscaglia F, Sagrini E. Malignancies in primary biliary cirrhosis. Eur J GastroenterolHepatol.2008;20:1-4.
[24]Silveira MG, Suzuki A, Lindor KD. Surveillance for hepatocellular carcinoma inpatients with primary biliary cirrhosis. Hepatology.2008;48:1149-1156.
[25]Mills PR, Boyle P, Quigley EMM, et al. Primary biliary cirrhosis: an increasedincidence of extrahepatic malignancies? J Clin Pathol.1982;35:541–3.
[26]Goudie BM, Burt AD, Boyle P, Macfarlane G, Birnie GG, Gillis PCR, MacsweenRNM, Watkinson G. Breast cancer in women with primary biliary cirrhosis. BMJ.1985;291:1597–1598
[27]Wolke AM, Schaffner F, Kapelman B, et al. Malignancy in primary biliary cirrhosis:high incidence of breast cancer in affected women. Am J Med.1984;76:1075–1078.
[28]Landgren AM, Landgren O, Gridley G, Dores GM, Linet MS, Morton LM.Autoimmune disease and subsequent risk of developing alimentary tract cancersamong4.5million US male veterans. Cancer.2011;117(6):1163-71.
[29]Wells GA, Shea B, O’Connell D, Peterson J, Welch V, Losos M, Tugwell P. TheNewcastle-Ottawa scale (NOS) for assessing the quality of nonrandomized studies inmeta-analysis. http://www.ohri.ca/programs/clinical_epidemiology/oxford.htm.
[30]Cavazza A, Caballería L, Floreani A, Farinati F, Bruguera M, Caroli D, Parés A.Incidence, risk factors, and survival of hepatocellular carcinoma in primary biliarycirrhosis: comparative analysis from two centers. Hepatology.2009;50(4):1162-1168.
[31]Goldacre MJ, Wotton CJ, Yeates D, Seagroatt V, Collier J. Liver cirrhosis, other liverdiseases, pancreatitis and subsequent cancer: record linkage study. Eur J GastroenterolHepatol.2008,20:384–392.
[32]Jackson H, Solaymani-Dodaran M, Card TR, Aithal GP, Logan R, West J. Influence ofUrsodeoxycholic Acid on the Mortality and Malignancy Associated With PrimaryBiliary Cirrhosis: A Population-Based Cohort Study. Hepatology.2007;46:1131-1137.
[33]Howel D, Metcalf JV, Gray J, Newman WL, Jones DEJ, James OFW. Cancer risk inprimary biliary cirrhosis: a study in northern England. Gut.1999;45:756–760.
[34]Nijhawan PK, Therneau TM, Dickson ER, Boynton J, Lindor KD. Incidence of Cancerin Primary Biliary Cirrhosis: The Mayo Experience. Hepatology.1999;29:1396-1398.
[35]L f L, Adami HO, Sparén P, Danielsson A, Eriksson LS, Hultcrantz R, Lindgren S,Olsson R, Prytz H, Ryden BO, et al. Cancer risk in primary biliary cirrhosis: apopulation-based study from Sweden. Hepatology.1994;20(1Pt1):101-104.
[36]Witt-Sullivan H, Heathcote J, Cauch K, Blendis L, Ghent C, Katz A, Milner R, PappasSC, Rankin J, Wanless IR.The demography of primary biliary cirrhosis in Ontario,Canada. Hepatology.1990;12(1):98-105.
[37]Ngu JH, Gearry RB, Frampton CM, Stedman CA. Mortality and the risk ofmalignancy in autoimmune liver diseases: a population based study in Canterbury,New Zealand. Hepatology.2012;55(2):522-529.
[38]Shibuya A, Tanaka K, Miyakawa H, Shibata M, Takatori M, Sekiyama K, HashimotoN, Amaki S, Komatsu T, Morizane T. Hepatocellular carcinoma and survival inpatients with primary biliary cirrhosis. Hepatology.2002;35(5):1172-1178.
[39]Kuiper EM, Hansen BE, Adang RP, van Nieuwkerk CM, Timmer R, Drenth JP,Spoelstra P, Brouwer HT, Kuyvenhoven JP, van Buuren HR; Dutch PBC Study Group.Relatively high risk for hepatocellular carcinoma in patients with primary biliarycirrhosis not responding to ursodeoxycholic acid. Eur J Gastroenterol Hepatol.2010;22(12):1495-502.
[40]Su CW, Hung HH, Huo TI, Huang YH, Li CP, Lin HC, Lee PC, Lee SD, Wu JC.Natural history and prognostic factors of primary biliary cirrhosis in Taiwan: afollow-up study up to18years. Liver Int.2008;28(9):1305-1313.
[41]Panjala C, Talwalkar JA, Lindor KD. Risk of lymphoma in primary biliary cirrhosis.Clin Gastroenterol Hepatol.2007;5(6):761-764.
[42]Lindor KD, Gershwin ME, Poupon R, et al. Primary biliary cirrhosis. Hepatology.2009;50:291-308.
[43]杨再兴,梁艳,王皓等.原发性胆汁性肝硬化患者外周血单个核细胞Toll样受体4基因的表达及临床意义研究.中国实验诊断学.2008;12:75-77.
[44]Mao TK, Lian ZX, Selmi C, et al. Altered monocyte responses to defined TLR ligandsin patients with primary bilairy cirrhosis. Hepatology.2005;42:802-808.
[45]Honda Y, Yamagiwa S, Matsuda Y, et al. Altered expression of TLR homolog RP105on monocytes hepersensitive to LPS in patients with primary biliary cirrhosis. JHepatol.2007;47:404-411.
[46]Yokoyama T, Komori A, Nakamura, et al. Human intrahepatic biliary epithelial cellsfunction in innate immunity by producing IL-6and IL-8via the TLR4-NF-kappaB and-MAPK signaling pathways. Liver Int.2006;26:467-476.
[47]Mayo MJ, Parkes J, Adams-Huet B, et al. Prediction of clinical outcomes inprimary biliary cirrhosis by serum enhanced liver fibrosis assay. Hepatology.2008;48:1549-1557.
[48]Robertson H, Kirby JA, Yip WW, et al. Biliary epithelial-mesenchymal transition inposttransplantation recurrence of Primary biliary cirrhosis. Hepatology.2007;45:977-981.
[49]Rygiel KA, et al. Epithelial-mesenchymal transition contributes to portal tractfibrogenesis during human chronic liver disease. Lab Invest.2008;88:112-123.
[50]Kaplan MM, Gershwin ME. Primary biliary cirrhosis. N Engl J Med,2005,353:1261-73.
[51]Thiery JP.Epithelial mesenchymal transition in tumor progression. Nat Rev Cancer,2002,2:442-454.
[1] Pascual V, Chaussabel D, Banchereau J. A genomic approach to human autoimmunediseases. Annu Rev Immunol.2010;28:535-571.
[2] Planas R, Pujol-Borrell R, Vives-Pi M. Global gene expression changes in type1diabetes: Insights into autoimmuneresponse in the target organ and in the periphery.Immunol Letters.2010;133:55-61.
[3] Han JW, Zheng HF, Cui Y, et al. Genome-wide association study in a Chinese Hanpopulation identifies nine new susceptibility loci for systemic lupus erythematosus. NatGenet,2009,41:1234-1237.
[4] Lessard CJ, Adrianto I, Kelly JA, et al. Identification of a systemic lupus erythematosussusceptibility locus at11p13between PDHX and CD44in a multiethnic study. Am J HumGenet.2011;88(1):83-91
[5] Sandling JK, Garnier S, Siqurdsson S, et al. A candidate gene study of the type Iinterferon pathway implicates IKBKE and IL8as risk loci for SLE. Eur J Hum Genet.2011;19(4):479-484
[6] Ramos PS, Criswell LA, Moser KL, et al. A Comprehensive Analysis of Shared Locibetween Systemic Lupus Erythematosus (SLE) and Sixteen Autoimmune Diseases RevealsLimited Genetic Overlap. PLoS Genet,2011,7:1002-1406.
[7] Freudenberg J, Lee HS, Han BG, et al. Genome-wide association study of rheumatoidarthritis in Koreans: population-specific loci as well as overlap with Europeansusceptibility loci.Arthritis Rheum.2011;63(4):884-93.
[8] Stahl EA, Raychaudhuri S, Remmers EF, et al. Genome-wide association studymeta-analysis identifies seven new rheumatoid arthritis risk loci. Nat Genet,2010,42:508-514.
[9] Lin Z, Bei JX, Shen M, et al. A genome-wide association study in Han Chineseidentifies new susceptibility loci for ankylosing spondylitis. Nat Genet，2011,44:73-77.
[10] Evans DM, Spencer CC, Pointon JJ, et al. Interaction between ERAP1and HLA-B27in ankylosing spondylitis implicates peptide handling in the mechanism for HLA-B27indisease susceptibility. Nat Genet,2011,43:761-767.
[11] Rivas MA, Beaudoin M, Gardet A, et al. Deep resequencing of GWAS loci identifiesindependent rare variants associated with inflammatory bowel disease. Nat Genet,2011,43:1066-1073.
[12] Stuart PE, Nair RP, Ellinghaus E, et al. Genome-wide association analysis identifiesthree psoriasis susceptibility loci. Nat Genet,2010,42:1000-1004.
[13] Australia and New Zealand Multiple Sclerosis Genetics Consortium (ANZgene).Genome-wide association study identifies new multiple sclerosis susceptibility loci onchromosomes12and20. Nat Genet.2009;41(7):824-828.
[14] De Jager PL, Jia X, Wang J, et al. Meta-analysis of genome scans and replicationidentify CD6, IRF8and TNFRSF1A as new multiple sclerosis susceptibility loci. NatGenet.2009;41(7):776-782.
[15] Mells GF, Floyd JA, Morley KI, et al. Genome-wide association study identifies12new susceptibility loci for primary biliary cirrhosis.Nat Genet,2011,43:329-332.
[16] Chu X, Pan CM, Zhao SX,et al. A genome-wide association study identifies two newrisk loci for Graves' disease. Nat Genet,2011,43:897-901.
[17] Costenbader KH, Gay S, Riquelme ME, et al. Genes, epigenetic regulation andenvironmental factors: Which is the most relevant n developing autoimmune diseases?Autoimmun Rev (2011),doi:10.1016/j.autrev.2011.10.022.
[18] Ospelt C, Reedquist K, Gay S, et al. Inflammatory memories: Is epigenetics themissing link to persistent stromal cell activation in rheumatoid arthritis? Autoimmun Rev，2011,10:519-524.
[19] Tomankova T, Petrek M, Gallo J, et al. MicroRNAs: emerging regulators ofimmune-mediated diseases. Scand J Immunol,2012,75(2):129-141.
[20] Dai R, Ahmed SA. MicroRNA, a new paradigm for understanding immunoregulation,inflammation, and autoimmune diseases. Transl Res,2011,157:163-179.
[21] Liang Y, Yang Z, Zhong R. Smoking, family history and urinary tract infection areassociated with primary biliary cirrhosis: A meta-analysis. Hepatol Res,2011,41:572-578.
[22] Gualtierotti R, Biggioggero M, Penatti A.E, et al. Updating on the pathogenesis ofsystemic lupus erythematosus. Autoimmun Rev,2010,10:3-7.
[23] Harel-Meir M, Sherer Y, Shoenfeld Y. Tobacco smoking and autoimmune rheumaticdiseases. Nat Clin Pract Rheumatol,2007,3:707-715.
[24] Brent GA. Environmental exposures and autoimmune thyroid disease. Thyroid,2010,20(7):755-761.
[25]Selmi C, Bowlus CL, Gershwin ME, et al. Primary biliary cirrhosis.Lancet,2011;377(9777):1600-1609.
[26] Smyk DS, Rigopoulou EI, Lleo A, et al. Immunopathogenesis of primary biliarycirrhosis: an old wives' tale. Immun Ageing，2011,8(1):12.
[27] Su KY, Pisetsky DS. The Role of Extracellular DNA in Autoimmunity in SLE. ScandJ Immunol,2009,70:175-183.
[28] J ger A, Kuchroo VK. Effector and regulatory T-cell subsets in autoimmunity andtissue inflammation. Scand J Immunol,2010,72:173-184.
[29] Korn T, Bettelli E, Oukka M, et al. IL-17and Th17Cells. Annu Rev Immunol,2009,27:485-517.
[30] Doreau A, Belot A, Bastid J, et al. Interleukin17acts in synergy with B cell activatingfactor to influence B cell biology and the pathophysiology of systemic lupus erythematosus.Nat Immunol,2009,10:778-785.
[31] Rong G, Zhou Y, Xiong Y, et al. Imbalance between T helper type17and T regulatorycells in patients with primary biliary cirrhosis: the serum cytokine profile and peripheralcell population. Clin Exp Immunol,2009,156:217-225.
[32] Kallenberg CG, Kimura A, Kishimoto T. IL-6: regulator of Treg/Th17balance,2010,40:1830-1835.
[33] Kallenberg CG. Pathogenesis of ANCA-associated vasculitides. Ann Rheum Dis,2011,70Suppl1:59-63.
[34] Annunziato F, Romagnani S. Heterogeneity of human effector CD4+T cells. ArthritisRes Ther,2009,11:257.
[35] King C, Tangye SG, Mackay CR. T follicular helper (TFH) cells in normal anddysregulated immune responses. Annu Rev Immunol,2008,26:741-766.
[36] Nurieva RI, Chung Y, Martinez GJ, et al. Bcl6mediates the development of Tfollicular helper cells. Science,2009,325:1001-1005.
[37] Gómez-Martín D, Díaz-Zamudio M, Romo-Tena J, et al. Follicular helper T cellspoise immune responses to the development of autoimmune pathology. Autoimmun Rev,2011,10:325-330.
[38] Dong W, Zhu P, Wang Y, et al. Follicular helper T cells in systemic lupuserythematosus: a potential therapeutic target. Autoimmu Rev,2011,10:299-304.
[39] Hu N, Westra J, Kallenberg CG. Dysregulated neutrophil--endothelial interaction inantineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides: implications forpathogenesis and disease intervention. Autoimmun Rev,2011,10:536-543.
[40] Blair PA, Norena LY, Flores-Borja F, et al. CD19(+)CD24(hi)CD38(hi) B cells exhibitregulatory capacity in healthy individuals but are functionally impaired in systemic lupuserythematosus patients. Immunity,2010,32:129-140.
[41] Dhirapong A, Lleo A, Yang GX, et al. B cell depletion therapy exacerbates murineprimary biliary cirrhosis. Hepatology,2011,53:527-535.
[42] Mu oz LE, Janko C, Schulze C, et al. Autoimmunity and chronic inflammation-twoclearance-related steps in the etiopathogenesis of SLE. Autoimmun Rev,2010,10:38-42.
[43] Mu oz LE, Lauber K, Schiller M, et al. The role of defective clearance of apoptoticcells in systemic autoimmunity. Nat Rev Rheumatol,2010,6:280-289.
[44] Lande R, Ganguly D, Facchinetti V, et al. Neutrophils activate plasmacytoid dendriticcells by releasing. self-DNA-peptide complexes in systemic lupus erythematosus. SciTransl Med,2011,3(73):73ra19.
[45] Seitz HM, Matsushima GK. Dendritic cells in systemic lupus erythematosus. Int RevImmunol,2010,29:184-209.
[46] Zozulya AL, Clarkson BD, Ortler S, et al. The role of dendritic cells in CNSautoimmunity. J Mol Med (Berl),2010,88:535-544.
[47] Diana J, Gahzarian L, Simoni Y, et al. Innate immunity in type1diabetes. DiscovMed,2011,11:513-520.
[48] Silver KL, Crockford TL, Bouriez-Jones T, et al. MyD88-dependent autoimmunedisease in Lyn-deficient mice. Eur J Immunol,2007,37:2734-2743.
[49] Lau CM, Broughton C, Tabor AS, et al. RNA-associated autoantigens activate B cellsby combined B cell antigen receptor/Toll-like receptor7engagement. J Exp Med,2005,202:1171-1177.
[50] Ehlers M, Fukuyama H, McGaha TL, et al. TLR9/MyD88signaling is required forclass switching to pathogenic IgG2a and2b autoantibodies in SLE. J Exp Med,2006,203:553-561.
[51] Ulloa L, Messmer D. High-mobility group box1(HMGB1) protein: friend and foe.Cytokine Growth Factor Rev,2006,17:189-201.
[52] Chen M, Daha MR, Kallenberg CGM. The complement system in systemicautoimmune disease. J Autoimmun,2010,34:276-286.
[53] Diana J, Gahzarian L, Simoni Y, et al. Innate immunity in type1diabetes. DiscovMed,2011,11:513-520.
[54] Harada K, Nakanuma Y. Biliary innate immunity in the pathogenesis of biliarydiseases. Inflamm Allergy Drug Targets,2010,9:83-90.
[55] Marques R, Boneca IG. Expression and functional importance of innate immunereceptors by intestinal epithelial cells. Cell Mol Life Sci,2011,68:3661-3673.
[56] Kruse K, Janko C, Urbonaviciute V, et al. Inefficient clearance of dying cells inpatients with SLE:anti-dsDNA autoantibodies, MFG-E8, HMGB-1and other players.Apoptosis,2010,15:1098-1113.
[1] Kalluri R, Neilson EG: Epithelial mesenchymal transformation and its implication forfibrosis. J Clin Invest,2003,112:1776-1784.
[2] Yang J, Liu Y. Dissection of key events in tubular epithelial to myofibroblast transitionand its implications in renal interstitial fibrosis. Am J Pathol,2001,159:1465-1475.
[3] Zeisberg M, Neilson EG.Biomarkers for epithelial-mesenchymal transitions. J ClinInvest,2009,119:1429-1437.
[4] Zeisberg M, Maeshima Y, Mosterman B, et al. Renal fibrosis. Extracellular matrixmicroenvironment regulates migratory behavior of activated tubular epithelial cells. Am JPathol,2002,160:2001-2008.
[5] Wang B, Koh P, Winbanks C, et al. miR-200a prevents renal fibrogenesis throughrepression of TGF-beta2expression. Diabetes,2011,60:280-287.
[6] Wang Q, Wang Y, Minto AW, et al. MicroRNA-377is up-regulated and can lead toincreased fibronectin production in diabetic nephropathy. FASEB J2008,22:4126-4135.
[7] Simonson MS: Phenotypic transitions and fibrosis in diabetic nephropathy. KidneyInt,2007,71:846-854.
[8] Rossini M, Cheunsuchon B, Donnert E,et al. Immunolocalization of fibroblast growthfactor-1(FGF-1), its receptor (FGFR-1), and fibroblast-specific protein-1(FSP-1) ininflammatory renal disease. Kidney Int,2005,68:2621-2628.
[9] Bariety J, Hill GS, Mandet C,et al. Glomerular epithelial-mesenchymaltransdifferentiation in pauci-immune crescentic glomerulonephritis. Nephrol DialTransplant,2003,18:1777-1784.
[10]Nishitani Y, Iwano M, Yamaguchi Y, et al. Fibroblast-specific protein1is a specificprognostic marker for renal survival in patients with IgAN. Kidney Int,2005,68:1078-1085.
[11]Hertig A, Anglicheau D, Verine J, et al. Early epithelial phenotypic changes predictgraft fibrosis. J Am Soc Nephrol,2008,19:1584-1591.
[12]Strutz F, et al. Identification and characterization of a fibroblast marker: FSP1. J CellBiol,1995,130(2):393-405.
[13]Kalluri R, Neilson EG. Epithelial-mesenchymal transition and its implications forfibrosis. J Clin Invest,2003,112:1776-1784.
[14]Iwano M, Plieth D, Danoff TM, et al. Evidence that fibroblasts derive from epitheliumduring tissue fibrosis. J Clin Invest,2002,110:341-350.
[15]Li L, Zepeda-Orozco D, Black R, et al. Autophagy is a component of epithelial cellfate in obstructive uropathy. Am J Pathol,2010,176(4):1767-1778.
[16]Humphreys BD, Lin SL, Kobayashi A et al. Fate tracing reveals the pericyte and notepithelial origin of myofibroblasts in kidney fibrosis. Am J Pathol,2010,176(1):85-97.
[17]Liu YH.New insights into epithelial-mesenchymal transition in kidney fibrosis. J AmSoc Nephrol,2010,21:212-222.
[18]Strutz F, et al. Role of basic fibroblast growth factor-2in epithelial-mesenchumaltransformation. Kidney Int,2002,61:1714-1728.
[19]Yang J, et al. Disruption of tissue-type plaminogen activator gene in mice reducesinterstitial fibrosis in obstructive nephropathy. J Clin Invest,2002,110:1525-1538.
[20]Yu L, Border WA, Huang Y, et al. TGF-beta isoforms in renal fibrogenesis. Kidney Int,2003,64:844-856.
[21]Valcourt U, Kowanetz M, Niimi H, et al. TGF-beta and the Smad signaling pathwaysupport transcriptomic reprogramming during epithelial-mesenchymal cell transition. MolBiol Cell,2005,16:1987-2002.
[22]Li J, Qu X, Yao J, et al. Blockade of endothelial-mesenchymal transition by a Smad3inhibitor delays the early development of streptozotocin-indeuced diabetic nephropathy.Diabetes,2010,59:2612-2624.
[23]Gagliardini E, Benigni A. Role of anti-TGF-beta antibodies in the treatment of renalinjury. Cytokine Growth Factor Rev,2006,17:89-96.
[24]Yokoi H, Mukoyama M, Nagae T, et al. Reduction in connective tissue growth factorby antisense treatment ameliorates renal tubulointerstitial fibrosis. J Am SocNephrol,2004,15:1430-1440.
[25]Ziyadeh FN, Hoffman BB, Han DC, et al. Long-term prevention of renal insufficiency,excess matrix gene expression, and glomerular mesangial matrix expansion by treatmentwith monoclonal antitransforming growth factor-beta antibody in db/db diabetic mice. ProcNatl Acad Sci USA,2000,97:8015-8020.
[26]Wang W, Wang X, Chun J, et al. Inflammasome-independent NLRP3augmentsTGF-βsignaling in kidney epithelium. J Immunol,2013,190(3):1239-49.
[27]Morishita Y, Watanabe M, Nakazawa E,et al. The interaction of LFA-1onmononuclear cells and ICAM-1on tubular epithelial cells accelerates TGF-β1-inducedrenal epithelial-mesenchymal transition. Plos one,2011,6(8):e23267.
[28]Schlessinger K, Hall A, Tolwinski N.Wnt signaling pathways meet Rho GTPases.Genes Dev,2009,23:265-277.
[29]Zeisberg M, Hanai J, Sugimoto H, et al. BMP-7counteracts TGF-β1inducedepithelial-to-mesenchymal transition and reverse renal injury. Nat Med,2003,9:964-968.
[30]Veerasamy M, Phanish M, Dockrell ME. Smad mediated regulation of inhibitor ofDNA binding2and its role in phenotypic maintenance of human renal proximal tubuleepithelial cells. Plos one,2013,8(1):e51842.
[31]Yang YL, Ju HZ, Liu SF, et al. BMP-2suppresses renal interstitial fibrosis byregulating epithelial-mesenchymal transition. J Cell Biochem,2011,112(9):2558-65.
[32]Sun S, Ning X, Zhang Y, et al. Hypoxia-inducible factor-1alpha induces Twistexpression in tubular epithelial cells subjected to hypoxia,leading toepithelial-to-mesenchymal transition. Kidney Int,2009;75,1278-1287.
[33]Ibrini J, Fadel S, Chana RS, et al. Albumin-induced epithelial mesenchymaltransformation. Nephron Exp Nephrol,2012,120(3): e91-102.
[34]Sun CY, Chang SC, Wu MS. Uremic toxins induce kidney fibrosis by activatingintrarenal renin-angiotension-aldosterone system associated epithelial-to-mesenchymaltransition. Plos one,2012,7(3):e34026.
[35]Lynch J, Nolan S, Slattery C, et al. High-mobility group box protein1: a novelmediator of inflammatory-induced renal epithelial-mesenchymal transition. Am J Nephrol,2010,32(6):590-602.
[36]Boutet A, De Frutos CA, Maxwell PH, et al. Snail activation disrupts tissuehomeostasis and induces fibrosis in the adult kidney. EMBO J,2006,25:5603-5613.
[37]Schramek H, Feifel E, Marschitz I,et al. Loss of active MEK1-ERK1/2restoresepithelial phenotype and morphogenesis in transdifferentiated MDCK cells. Am J PhysiolCell Physiol,2003,285:C652-661.
[38]Leask A. Targeting the jagged/notch pathway: a new treatment for fibrosis? J CellCommun Signal,2010,4:197-198.
[39]Kaimori A, Potter j, Kaimori JY,et al. Transfoming growth fator-beta1induces anepithelial-to-mesenchymal transition state in mouse hepatocytes in vitro. J Biol Chem,2007,282:22089-22101.
[40]Nitta T, Kim JS, Mohuczy D, et al. Murine cirrhosis induces hepatocyte epithelialmesenchymal transition and alterations in survival signaling pathways. Hepatology,2008,48:909-919.
[41]Wang S, Hyun J, Youn B, et al. Hedgehog signaling regulates the repair response inmouse liver damaged by irradiation. Radiat Res,2013,179(1):69-75.
[42]Chen YL, Lv J, Ye XL et al.Sorafenib inhibits transforming growth factorβ1-mediatedepithelial-mesenchymal transition and apoptosis in mouse hepatocytes. Hepatology,2011,53(5):1708-18.
[43]Taura K, Miura K, Iwaisako K, et al. Hepatocytes do not undergoepithelial-mesenchymal transition in liver fibrosis in mice. Hepatology,2010,51(3):1027-36.
[44]Diaz R, Kim JW, Hui JJ, et al. Evidence for the epithelial to mesenchymal transition inbiliary atresia fibrosis. Hum Pathol,2008,39:102-115.
[45]Xia JL, Dai C,Michalopoulos GK, et al. Hepatocyte growth factor attenuates liverfibrosis induced by bile duct ligation. Am J Pathol,2006,168:1500-1512.
[46]Omenetti A, Porrello A, Jung Y, et al. Hedgehog signaling regulatesepithelial-mesenchumal transition during biliary fibrosis in rodents and humans. J ClinInvest,2008,118:3331-3342.
[47]Rygiel KA, Robertson H, Marshall HL, et al. Epithelial-mesenchymal transitioncontributes to portal tract fibrogenesis during human chronic liver disease. LabInvest,2008,88:112-123.
[48]Scholten D, Scholten A, Brenner DA, et al. Epithelial-to-mesenchymal transition(EMT)in cholangiocytes does not contribute to liver fibrosis. Hepatology,2009,50:818A.
[49]Chu AS, Diaz R, Hui JJ,et al. Lineage tracing demonstrates no evidence ofcholangiocyte epithelial-to-mesenchymal transition in murine models of hepatic fibrosis.Hepatology,2011,53(5):1685-95.
[50]Hashimoto N, Phan SH, Imaizumi K, et al. Endothelial-mesenchymal transition inbleomycin-induced pulmonary fibrosis.Am J Respir Cell Mol Biol,2010,43:161-172.
[51]Zhao J, Shi W, Wang YL,et al. Smad3deficiency attenuates bleomycin-inducedpulmonary fibrosis in mice. Am J Physiol Lung Cell Mol Physiol,2002,282:L585-L593.
[52]Willis BC, Liebler JM, Luby-Phelps K, et al. Induction of epithelial-mesenchymaltransition in alveolar epithelial cells by transforming growth factor-beta1: potential role inidiopathic pulmonary fibrosis. Am J Pathol,2005,166:1321-1332.
[53]Mikami Y, Yamauchi Y, Horie M, et al. Tumor necrosis factor superfamily memberLIGHT induces epithelial-mesenchymal transition in A549human alveolar epithelial cells.Biochem Biophys Res Commun,2012,428(4):451-457.
[54]Chen HH, Zhou XL, Shi YL, et al. Roles of p38MAPK and JNK in TGF-β1-inducedhuman alveolar epithelial to mesenchymal transition. Arch Med Res,2013,[Epub ahead ofprint].
[55]Kondo Y, Higa-Nakamine S, Noguchi N, et al. Induction of epithelial-mesenchymaltransition by flagellin in cultured lung epithelial cells. Am J Physiol Lung Cell Mol Physiol,2012,303(12):L1057-69.
[56]Schneider DJ, Wu M, Le TT, et al. Cadherin-11contributes to pulmonary fibrosis:potential role in TGF-β production and epithelial to mesenchymal transition. FASEB J,2012,26(2):503-12.
[57]Wettstein G, Bellaye PS, Kolb M, et al. Inhibition of HSP27blocks fibrosisdevelopment and EMT features by promoting Snail degradation. FASEB J,2013,[Epubahead of print]
[58]Balli D, Ustiyan V, Zhang Y, et al. Foxm1transcription factor is required for lungfibrosis and epithelial-to-mesenchymal transition. EMBO J,2013,32(2):231-44.
[59]Zeisberg EM, Tarnavski O, Zeisberg M,et al. Endothelial-to-mesenchymal transitioncontributes to cardiac fibrosis.Nat Med,2007,13:952-961.
[60]Goumans M, van Zonneveld AJ, ten Dijke P: Transforming growth factor β-inducedendothelial-to-mesenchymal transition: a switch to cardiac fibrosis? Trends CardiovascMed,2008,18:293-298.
[61]Widyantoro B, Emoto N, Nakayama K, et al. Endothelial cell-derived endothelin-1promotes cardiac fibrosis in diabetic hearts through stuimulation ofendothelial-to-mesenchymal transition.Circulation2010,121:2407-2418.
[62]Bronnum H, Andersen DC, Schneider M, et al. Islet-1is a dual regulator of fibrogenicepithelial-to-mesenchynal transition in epicardial mesothelial cells. Exp Cell Res,2013,319(4):424-435.
[63]Yanjia H, Xinchun J. The role of epithelial-mesenchymal transition in oral squamouscell carcinoma and oral submucous fibrosis. Clin Chim Acta,2007,383:51-56.
[64]Scharl M, Frei S, Pesch T, et al. Interleukin-13and transforming growthfactorβsynergise in the pathogenesis of human intestinal fistulae. Gut,2013,62(1):63-72.
[2] Wang AP, Migita K, Ito M, et al. Hepatic expression of toll-like receptor4in primarybiliary cirrhosis. J Autoimmun.2005;25:85-91.
[3]Honda Y, Yamagiwa S, Matsuda Y, et al. Altered expression of TLR homolog RP105on monocytes hepersensitive to LPS in patients with primary biliary cirrhosis. J Hepatol.2007;47:404-411.
[4]Yokoyama T, Komori A, Nakamura, et al. Human intrahepatic biliary epithelial cellsfunction in innate immunity by producing IL-6and IL-8via the TLR4-NF-kappaB and-MAPK signaling pathways. Liver Int.2006;26:467-476.
[5] Kalluri R, Neilson EG: Epithelial mesenchymal transformation and its implication forfibrosis. J Clin Invest,2003,112:1776-1784.
[6] Acloque H, Thiery JP, Nieto MA. The physiology and pathology of the EMT. EMBOreports.2008;9:322-326.
[7] Teng YQ, Zeisberg M, Kalluri R. Transcriptional regulation of epithelial-mesenchymaltransition. J Clin Invest.2007;117:304-306.
[8] Gershwin ME, Mackay IR. The causes of primary biliary cirrhosis: convenient andinconvenient truths. Hepatology.2008;47:737–745.
[9] Howel D, Fischbacher CM, Bhopal RS, Gray J, Metcalf JV, James OFW. Anexploratory population-based case-control study of primary biliary cirrhosis.Hepatology2000;31:1055–1060.
[10]Parikh-Patel A, Gold EB, Worman H, et al. Risk factors for primary biliary cirrhosis ina cohort of patients from the United States. Hepatology2001;33:16–21.
[11]Gershwin ME, Slemi C, Worman HJ et al. Risk factors and comorbidities in primarybiliary cirrhosis: a controlled interviewbased study of1032patients. Hepatology2005;42:1194–1202.
[12]Prince MI, Ducker SJ, James OFW. Case-control studies of risk factors for primarybiliary cirrhosis in two United Kingdom populations. Gut2010;59:508–512.
[13]Corpechot C, Chrétien Y, Chazouillères O, Poupon R. Demographic, lifestyle, medicaland familial factors associated with primary biliary cirrhosis. J Hepatol2010;53:162–169.
[14]Zein CO, Beatty K, Post AB, Logan L, Debanne S, McCullough AJ. Smoking andincreased severity of hepatic fibrosis in primary biliary cirrhosis: a cross validatedretrospective assessment. Hepatology2006;44:1564–71.
[15]Selmi C, Bowlus CL, Gershwin ME, et al. Primary biliary cirrhosis. Lancet.2011;377:1600–1609.
[16]Floreani A, Baragiotta A, Baldo V, Menegon T, Farinati F, Naccarato R. Hepatic andextrahepatic malignancies in primary biliary cirrhosis. Hepatology.1999;29:1425-1428.
[17]Krasner P, Johnson PJ, Portmann B, Watkinson G, Macsween RNM, Williams R.Hepatocellular carcinoma in primary biliary cirrhosis: report of four cases. Gut,1979;20:255-258.
[18]Nakanuma Y, Terada T, Doishita K, Miwa A. Hepatocellular carcinoma in primarybiliary cirrhosis: an autopsy study. Hepatology1990;11:1010-1016.
[19]Shimizu A, Koyama M, Okuda Y, Takase K, Nakano T, Tameda Y. Hepatocellularcarcinoma in primary biliary cirrhosis: a case report and review of the Japaneseliterature. Hepatogastroenterology1998;45:2352-2355.
[20]Farinati F, Floreani A, De Maria N, Fagiuoli S, Naccarato R, Chiaramonte M.Hepatocellular carcinoma in primary biliary cirrhosis. J Hepatol1994;21:315-316.
[21]Caballeria L, Pare′s A, Castells A, Gine′s A, Bru C, Rode′s J. Hepatocellularcarcinoma in primary biliary cirrhosis: similar incidence to that in hepatitis Cvirus-related cirrhosis. Am J Gastroenterol.2001;96:1160-1163.
[22]Deutsch M, Papatheodoridis GV, Tzakou A, Hadziyannis SJ. Risk of hepatocellularcarcinoma and extrahepatic malignancies in primary biliary cirrhosis. Eur JGastroenterol Hepatol.2008;20:5-9.
[23]Piscaglia F, Sagrini E. Malignancies in primary biliary cirrhosis. Eur J GastroenterolHepatol.2008;20:1-4.
[24]Silveira MG, Suzuki A, Lindor KD. Surveillance for hepatocellular carcinoma inpatients with primary biliary cirrhosis. Hepatology.2008;48:1149-1156.
[25]Mills PR, Boyle P, Quigley EMM, et al. Primary biliary cirrhosis: an increasedincidence of extrahepatic malignancies? J Clin Pathol.1982;35:541–3.
[26]Goudie BM, Burt AD, Boyle P, Macfarlane G, Birnie GG, Gillis PCR, MacsweenRNM, Watkinson G. Breast cancer in women with primary biliary cirrhosis. BMJ.1985;291:1597–1598
[27]Wolke AM, Schaffner F, Kapelman B, et al. Malignancy in primary biliary cirrhosis:high incidence of breast cancer in affected women. Am J Med.1984;76:1075–1078.
[28]Landgren AM, Landgren O, Gridley G, Dores GM, Linet MS, Morton LM.Autoimmune disease and subsequent risk of developing alimentary tract cancersamong4.5million US male veterans. Cancer.2011;117(6):1163-71.
[29]Wells GA, Shea B, O’Connell D, Peterson J, Welch V, Losos M, Tugwell P. TheNewcastle-Ottawa scale (NOS) for assessing the quality of nonrandomized studies inmeta-analysis. http://www.ohri.ca/programs/clinical_epidemiology/oxford.htm.
[30]Cavazza A, Caballería L, Floreani A, Farinati F, Bruguera M, Caroli D, Parés A.Incidence, risk factors, and survival of hepatocellular carcinoma in primary biliarycirrhosis: comparative analysis from two centers. Hepatology.2009;50(4):1162-1168.
[31]Goldacre MJ, Wotton CJ, Yeates D, Seagroatt V, Collier J. Liver cirrhosis, other liverdiseases, pancreatitis and subsequent cancer: record linkage study. Eur J GastroenterolHepatol.2008,20:384–392.
[32]Jackson H, Solaymani-Dodaran M, Card TR, Aithal GP, Logan R, West J. Influence ofUrsodeoxycholic Acid on the Mortality and Malignancy Associated With PrimaryBiliary Cirrhosis: A Population-Based Cohort Study. Hepatology.2007;46:1131-1137.
[33]Howel D, Metcalf JV, Gray J, Newman WL, Jones DEJ, James OFW. Cancer risk inprimary biliary cirrhosis: a study in northern England. Gut.1999;45:756–760.
[34]Nijhawan PK, Therneau TM, Dickson ER, Boynton J, Lindor KD. Incidence of Cancerin Primary Biliary Cirrhosis: The Mayo Experience. Hepatology.1999;29:1396-1398.
[35]L f L, Adami HO, Sparén P, Danielsson A, Eriksson LS, Hultcrantz R, Lindgren S,Olsson R, Prytz H, Ryden BO, et al. Cancer risk in primary biliary cirrhosis: apopulation-based study from Sweden. Hepatology.1994;20(1Pt1):101-104.
[36]Witt-Sullivan H, Heathcote J, Cauch K, Blendis L, Ghent C, Katz A, Milner R, PappasSC, Rankin J, Wanless IR.The demography of primary biliary cirrhosis in Ontario,Canada. Hepatology.1990;12(1):98-105.
[37]Ngu JH, Gearry RB, Frampton CM, Stedman CA. Mortality and the risk ofmalignancy in autoimmune liver diseases: a population based study in Canterbury,New Zealand. Hepatology.2012;55(2):522-529.
[38]Shibuya A, Tanaka K, Miyakawa H, Shibata M, Takatori M, Sekiyama K, HashimotoN, Amaki S, Komatsu T, Morizane T. Hepatocellular carcinoma and survival inpatients with primary biliary cirrhosis. Hepatology.2002;35(5):1172-1178.
[39]Kuiper EM, Hansen BE, Adang RP, van Nieuwkerk CM, Timmer R, Drenth JP,Spoelstra P, Brouwer HT, Kuyvenhoven JP, van Buuren HR; Dutch PBC Study Group.Relatively high risk for hepatocellular carcinoma in patients with primary biliarycirrhosis not responding to ursodeoxycholic acid. Eur J Gastroenterol Hepatol.2010;22(12):1495-502.
[40]Su CW, Hung HH, Huo TI, Huang YH, Li CP, Lin HC, Lee PC, Lee SD, Wu JC.Natural history and prognostic factors of primary biliary cirrhosis in Taiwan: afollow-up study up to18years. Liver Int.2008;28(9):1305-1313.
[41]Panjala C, Talwalkar JA, Lindor KD. Risk of lymphoma in primary biliary cirrhosis.Clin Gastroenterol Hepatol.2007;5(6):761-764.
[42]Lindor KD, Gershwin ME, Poupon R, et al. Primary biliary cirrhosis. Hepatology.2009;50:291-308.
[43]杨再兴,梁艳,王皓等.原发性胆汁性肝硬化患者外周血单个核细胞Toll样受体4基因的表达及临床意义研究.中国实验诊断学.2008;12:75-77.
[44]Mao TK, Lian ZX, Selmi C, et al. Altered monocyte responses to defined TLR ligandsin patients with primary bilairy cirrhosis. Hepatology.2005;42:802-808.
[45]Honda Y, Yamagiwa S, Matsuda Y, et al. Altered expression of TLR homolog RP105on monocytes hepersensitive to LPS in patients with primary biliary cirrhosis. JHepatol.2007;47:404-411.
[46]Yokoyama T, Komori A, Nakamura, et al. Human intrahepatic biliary epithelial cellsfunction in innate immunity by producing IL-6and IL-8via the TLR4-NF-kappaB and-MAPK signaling pathways. Liver Int.2006;26:467-476.
[47]Mayo MJ, Parkes J, Adams-Huet B, et al. Prediction of clinical outcomes inprimary biliary cirrhosis by serum enhanced liver fibrosis assay. Hepatology.2008;48:1549-1557.
[48]Robertson H, Kirby JA, Yip WW, et al. Biliary epithelial-mesenchymal transition inposttransplantation recurrence of Primary biliary cirrhosis. Hepatology.2007;45:977-981.
[49]Rygiel KA, et al. Epithelial-mesenchymal transition contributes to portal tractfibrogenesis during human chronic liver disease. Lab Invest.2008;88:112-123.
[50]Kaplan MM, Gershwin ME. Primary biliary cirrhosis. N Engl J Med,2005,353:1261-73.
[51]Thiery JP.Epithelial mesenchymal transition in tumor progression. Nat Rev Cancer,2002,2:442-454.
[1] Pascual V, Chaussabel D, Banchereau J. A genomic approach to human autoimmunediseases. Annu Rev Immunol.2010;28:535-571.
[2] Planas R, Pujol-Borrell R, Vives-Pi M. Global gene expression changes in type1diabetes: Insights into autoimmuneresponse in the target organ and in the periphery.Immunol Letters.2010;133:55-61.
[3] Han JW, Zheng HF, Cui Y, et al. Genome-wide association study in a Chinese Hanpopulation identifies nine new susceptibility loci for systemic lupus erythematosus. NatGenet,2009,41:1234-1237.
[4] Lessard CJ, Adrianto I, Kelly JA, et al. Identification of a systemic lupus erythematosussusceptibility locus at11p13between PDHX and CD44in a multiethnic study. Am J HumGenet.2011;88(1):83-91
[5] Sandling JK, Garnier S, Siqurdsson S, et al. A candidate gene study of the type Iinterferon pathway implicates IKBKE and IL8as risk loci for SLE. Eur J Hum Genet.2011;19(4):479-484
[6] Ramos PS, Criswell LA, Moser KL, et al. A Comprehensive Analysis of Shared Locibetween Systemic Lupus Erythematosus (SLE) and Sixteen Autoimmune Diseases RevealsLimited Genetic Overlap. PLoS Genet,2011,7:1002-1406.
[7] Freudenberg J, Lee HS, Han BG, et al. Genome-wide association study of rheumatoidarthritis in Koreans: population-specific loci as well as overlap with Europeansusceptibility loci.Arthritis Rheum.2011;63(4):884-93.
[8] Stahl EA, Raychaudhuri S, Remmers EF, et al. Genome-wide association studymeta-analysis identifies seven new rheumatoid arthritis risk loci. Nat Genet,2010,42:508-514.
[9] Lin Z, Bei JX, Shen M, et al. A genome-wide association study in Han Chineseidentifies new susceptibility loci for ankylosing spondylitis. Nat Genet，2011,44:73-77.
[10] Evans DM, Spencer CC, Pointon JJ, et al. Interaction between ERAP1and HLA-B27in ankylosing spondylitis implicates peptide handling in the mechanism for HLA-B27indisease susceptibility. Nat Genet,2011,43:761-767.
[11] Rivas MA, Beaudoin M, Gardet A, et al. Deep resequencing of GWAS loci identifiesindependent rare variants associated with inflammatory bowel disease. Nat Genet,2011,43:1066-1073.
[12] Stuart PE, Nair RP, Ellinghaus E, et al. Genome-wide association analysis identifiesthree psoriasis susceptibility loci. Nat Genet,2010,42:1000-1004.
[13] Australia and New Zealand Multiple Sclerosis Genetics Consortium (ANZgene).Genome-wide association study identifies new multiple sclerosis susceptibility loci onchromosomes12and20. Nat Genet.2009;41(7):824-828.
[14] De Jager PL, Jia X, Wang J, et al. Meta-analysis of genome scans and replicationidentify CD6, IRF8and TNFRSF1A as new multiple sclerosis susceptibility loci. NatGenet.2009;41(7):776-782.
[15] Mells GF, Floyd JA, Morley KI, et al. Genome-wide association study identifies12new susceptibility loci for primary biliary cirrhosis.Nat Genet,2011,43:329-332.
[16] Chu X, Pan CM, Zhao SX,et al. A genome-wide association study identifies two newrisk loci for Graves' disease. Nat Genet,2011,43:897-901.
[17] Costenbader KH, Gay S, Riquelme ME, et al. Genes, epigenetic regulation andenvironmental factors: Which is the most relevant n developing autoimmune diseases?Autoimmun Rev (2011),doi:10.1016/j.autrev.2011.10.022.
[18] Ospelt C, Reedquist K, Gay S, et al. Inflammatory memories: Is epigenetics themissing link to persistent stromal cell activation in rheumatoid arthritis? Autoimmun Rev，2011,10:519-524.
[19] Tomankova T, Petrek M, Gallo J, et al. MicroRNAs: emerging regulators ofimmune-mediated diseases. Scand J Immunol,2012,75(2):129-141.
[20] Dai R, Ahmed SA. MicroRNA, a new paradigm for understanding immunoregulation,inflammation, and autoimmune diseases. Transl Res,2011,157:163-179.
[21] Liang Y, Yang Z, Zhong R. Smoking, family history and urinary tract infection areassociated with primary biliary cirrhosis: A meta-analysis. Hepatol Res,2011,41:572-578.
[22] Gualtierotti R, Biggioggero M, Penatti A.E, et al. Updating on the pathogenesis ofsystemic lupus erythematosus. Autoimmun Rev,2010,10:3-7.
[23] Harel-Meir M, Sherer Y, Shoenfeld Y. Tobacco smoking and autoimmune rheumaticdiseases. Nat Clin Pract Rheumatol,2007,3:707-715.
[24] Brent GA. Environmental exposures and autoimmune thyroid disease. Thyroid,2010,20(7):755-761.
[25]Selmi C, Bowlus CL, Gershwin ME, et al. Primary biliary cirrhosis.Lancet,2011;377(9777):1600-1609.
[26] Smyk DS, Rigopoulou EI, Lleo A, et al. Immunopathogenesis of primary biliarycirrhosis: an old wives' tale. Immun Ageing，2011,8(1):12.
[27] Su KY, Pisetsky DS. The Role of Extracellular DNA in Autoimmunity in SLE. ScandJ Immunol,2009,70:175-183.
[28] J ger A, Kuchroo VK. Effector and regulatory T-cell subsets in autoimmunity andtissue inflammation. Scand J Immunol,2010,72:173-184.
[29] Korn T, Bettelli E, Oukka M, et al. IL-17and Th17Cells. Annu Rev Immunol,2009,27:485-517.
[30] Doreau A, Belot A, Bastid J, et al. Interleukin17acts in synergy with B cell activatingfactor to influence B cell biology and the pathophysiology of systemic lupus erythematosus.Nat Immunol,2009,10:778-785.
[31] Rong G, Zhou Y, Xiong Y, et al. Imbalance between T helper type17and T regulatorycells in patients with primary biliary cirrhosis: the serum cytokine profile and peripheralcell population. Clin Exp Immunol,2009,156:217-225.
[32] Kallenberg CG, Kimura A, Kishimoto T. IL-6: regulator of Treg/Th17balance,2010,40:1830-1835.
[33] Kallenberg CG. Pathogenesis of ANCA-associated vasculitides. Ann Rheum Dis,2011,70Suppl1:59-63.
[34] Annunziato F, Romagnani S. Heterogeneity of human effector CD4+T cells. ArthritisRes Ther,2009,11:257.
[35] King C, Tangye SG, Mackay CR. T follicular helper (TFH) cells in normal anddysregulated immune responses. Annu Rev Immunol,2008,26:741-766.
[36] Nurieva RI, Chung Y, Martinez GJ, et al. Bcl6mediates the development of Tfollicular helper cells. Science,2009,325:1001-1005.
[37] Gómez-Martín D, Díaz-Zamudio M, Romo-Tena J, et al. Follicular helper T cellspoise immune responses to the development of autoimmune pathology. Autoimmun Rev,2011,10:325-330.
[38] Dong W, Zhu P, Wang Y, et al. Follicular helper T cells in systemic lupuserythematosus: a potential therapeutic target. Autoimmu Rev,2011,10:299-304.
[39] Hu N, Westra J, Kallenberg CG. Dysregulated neutrophil--endothelial interaction inantineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides: implications forpathogenesis and disease intervention. Autoimmun Rev,2011,10:536-543.
[40] Blair PA, Norena LY, Flores-Borja F, et al. CD19(+)CD24(hi)CD38(hi) B cells exhibitregulatory capacity in healthy individuals but are functionally impaired in systemic lupuserythematosus patients. Immunity,2010,32:129-140.
[41] Dhirapong A, Lleo A, Yang GX, et al. B cell depletion therapy exacerbates murineprimary biliary cirrhosis. Hepatology,2011,53:527-535.
[42] Mu oz LE, Janko C, Schulze C, et al. Autoimmunity and chronic inflammation-twoclearance-related steps in the etiopathogenesis of SLE. Autoimmun Rev,2010,10:38-42.
[43] Mu oz LE, Lauber K, Schiller M, et al. The role of defective clearance of apoptoticcells in systemic autoimmunity. Nat Rev Rheumatol,2010,6:280-289.
[44] Lande R, Ganguly D, Facchinetti V, et al. Neutrophils activate plasmacytoid dendriticcells by releasing. self-DNA-peptide complexes in systemic lupus erythematosus. SciTransl Med,2011,3(73):73ra19.
[45] Seitz HM, Matsushima GK. Dendritic cells in systemic lupus erythematosus. Int RevImmunol,2010,29:184-209.
[46] Zozulya AL, Clarkson BD, Ortler S, et al. The role of dendritic cells in CNSautoimmunity. J Mol Med (Berl),2010,88:535-544.
[47] Diana J, Gahzarian L, Simoni Y, et al. Innate immunity in type1diabetes. DiscovMed,2011,11:513-520.
[48] Silver KL, Crockford TL, Bouriez-Jones T, et al. MyD88-dependent autoimmunedisease in Lyn-deficient mice. Eur J Immunol,2007,37:2734-2743.
[49] Lau CM, Broughton C, Tabor AS, et al. RNA-associated autoantigens activate B cellsby combined B cell antigen receptor/Toll-like receptor7engagement. J Exp Med,2005,202:1171-1177.
[50] Ehlers M, Fukuyama H, McGaha TL, et al. TLR9/MyD88signaling is required forclass switching to pathogenic IgG2a and2b autoantibodies in SLE. J Exp Med,2006,203:553-561.
[51] Ulloa L, Messmer D. High-mobility group box1(HMGB1) protein: friend and foe.Cytokine Growth Factor Rev,2006,17:189-201.
[52] Chen M, Daha MR, Kallenberg CGM. The complement system in systemicautoimmune disease. J Autoimmun,2010,34:276-286.
[53] Diana J, Gahzarian L, Simoni Y, et al. Innate immunity in type1diabetes. DiscovMed,2011,11:513-520.
[54] Harada K, Nakanuma Y. Biliary innate immunity in the pathogenesis of biliarydiseases. Inflamm Allergy Drug Targets,2010,9:83-90.
[55] Marques R, Boneca IG. Expression and functional importance of innate immunereceptors by intestinal epithelial cells. Cell Mol Life Sci,2011,68:3661-3673.
[56] Kruse K, Janko C, Urbonaviciute V, et al. Inefficient clearance of dying cells inpatients with SLE:anti-dsDNA autoantibodies, MFG-E8, HMGB-1and other players.Apoptosis,2010,15:1098-1113.
[1] Kalluri R, Neilson EG: Epithelial mesenchymal transformation and its implication forfibrosis. J Clin Invest,2003,112:1776-1784.
[2] Yang J, Liu Y. Dissection of key events in tubular epithelial to myofibroblast transitionand its implications in renal interstitial fibrosis. Am J Pathol,2001,159:1465-1475.
[3] Zeisberg M, Neilson EG.Biomarkers for epithelial-mesenchymal transitions. J ClinInvest,2009,119:1429-1437.
[4] Zeisberg M, Maeshima Y, Mosterman B, et al. Renal fibrosis. Extracellular matrixmicroenvironment regulates migratory behavior of activated tubular epithelial cells. Am JPathol,2002,160:2001-2008.
[5] Wang B, Koh P, Winbanks C, et al. miR-200a prevents renal fibrogenesis throughrepression of TGF-beta2expression. Diabetes,2011,60:280-287.
[6] Wang Q, Wang Y, Minto AW, et al. MicroRNA-377is up-regulated and can lead toincreased fibronectin production in diabetic nephropathy. FASEB J2008,22:4126-4135.
[7] Simonson MS: Phenotypic transitions and fibrosis in diabetic nephropathy. KidneyInt,2007,71:846-854.
[8] Rossini M, Cheunsuchon B, Donnert E,et al. Immunolocalization of fibroblast growthfactor-1(FGF-1), its receptor (FGFR-1), and fibroblast-specific protein-1(FSP-1) ininflammatory renal disease. Kidney Int,2005,68:2621-2628.
[9] Bariety J, Hill GS, Mandet C,et al. Glomerular epithelial-mesenchymaltransdifferentiation in pauci-immune crescentic glomerulonephritis. Nephrol DialTransplant,2003,18:1777-1784.
[10]Nishitani Y, Iwano M, Yamaguchi Y, et al. Fibroblast-specific protein1is a specificprognostic marker for renal survival in patients with IgAN. Kidney Int,2005,68:1078-1085.
[11]Hertig A, Anglicheau D, Verine J, et al. Early epithelial phenotypic changes predictgraft fibrosis. J Am Soc Nephrol,2008,19:1584-1591.
[12]Strutz F, et al. Identification and characterization of a fibroblast marker: FSP1. J CellBiol,1995,130(2):393-405.
[13]Kalluri R, Neilson EG. Epithelial-mesenchymal transition and its implications forfibrosis. J Clin Invest,2003,112:1776-1784.
[14]Iwano M, Plieth D, Danoff TM, et al. Evidence that fibroblasts derive from epitheliumduring tissue fibrosis. J Clin Invest,2002,110:341-350.
[15]Li L, Zepeda-Orozco D, Black R, et al. Autophagy is a component of epithelial cellfate in obstructive uropathy. Am J Pathol,2010,176(4):1767-1778.
[16]Humphreys BD, Lin SL, Kobayashi A et al. Fate tracing reveals the pericyte and notepithelial origin of myofibroblasts in kidney fibrosis. Am J Pathol,2010,176(1):85-97.
[17]Liu YH.New insights into epithelial-mesenchymal transition in kidney fibrosis. J AmSoc Nephrol,2010,21:212-222.
[18]Strutz F, et al. Role of basic fibroblast growth factor-2in epithelial-mesenchumaltransformation. Kidney Int,2002,61:1714-1728.
[19]Yang J, et al. Disruption of tissue-type plaminogen activator gene in mice reducesinterstitial fibrosis in obstructive nephropathy. J Clin Invest,2002,110:1525-1538.
[20]Yu L, Border WA, Huang Y, et al. TGF-beta isoforms in renal fibrogenesis. Kidney Int,2003,64:844-856.
[21]Valcourt U, Kowanetz M, Niimi H, et al. TGF-beta and the Smad signaling pathwaysupport transcriptomic reprogramming during epithelial-mesenchymal cell transition. MolBiol Cell,2005,16:1987-2002.
[22]Li J, Qu X, Yao J, et al. Blockade of endothelial-mesenchymal transition by a Smad3inhibitor delays the early development of streptozotocin-indeuced diabetic nephropathy.Diabetes,2010,59:2612-2624.
[23]Gagliardini E, Benigni A. Role of anti-TGF-beta antibodies in the treatment of renalinjury. Cytokine Growth Factor Rev,2006,17:89-96.
[24]Yokoi H, Mukoyama M, Nagae T, et al. Reduction in connective tissue growth factorby antisense treatment ameliorates renal tubulointerstitial fibrosis. J Am SocNephrol,2004,15:1430-1440.
[25]Ziyadeh FN, Hoffman BB, Han DC, et al. Long-term prevention of renal insufficiency,excess matrix gene expression, and glomerular mesangial matrix expansion by treatmentwith monoclonal antitransforming growth factor-beta antibody in db/db diabetic mice. ProcNatl Acad Sci USA,2000,97:8015-8020.
[26]Wang W, Wang X, Chun J, et al. Inflammasome-independent NLRP3augmentsTGF-βsignaling in kidney epithelium. J Immunol,2013,190(3):1239-49.
[27]Morishita Y, Watanabe M, Nakazawa E,et al. The interaction of LFA-1onmononuclear cells and ICAM-1on tubular epithelial cells accelerates TGF-β1-inducedrenal epithelial-mesenchymal transition. Plos one,2011,6(8):e23267.
[28]Schlessinger K, Hall A, Tolwinski N.Wnt signaling pathways meet Rho GTPases.Genes Dev,2009,23:265-277.
[29]Zeisberg M, Hanai J, Sugimoto H, et al. BMP-7counteracts TGF-β1inducedepithelial-to-mesenchymal transition and reverse renal injury. Nat Med,2003,9:964-968.
[30]Veerasamy M, Phanish M, Dockrell ME. Smad mediated regulation of inhibitor ofDNA binding2and its role in phenotypic maintenance of human renal proximal tubuleepithelial cells. Plos one,2013,8(1):e51842.
[31]Yang YL, Ju HZ, Liu SF, et al. BMP-2suppresses renal interstitial fibrosis byregulating epithelial-mesenchymal transition. J Cell Biochem,2011,112(9):2558-65.
[32]Sun S, Ning X, Zhang Y, et al. Hypoxia-inducible factor-1alpha induces Twistexpression in tubular epithelial cells subjected to hypoxia,leading toepithelial-to-mesenchymal transition. Kidney Int,2009;75,1278-1287.
[33]Ibrini J, Fadel S, Chana RS, et al. Albumin-induced epithelial mesenchymaltransformation. Nephron Exp Nephrol,2012,120(3): e91-102.
[34]Sun CY, Chang SC, Wu MS. Uremic toxins induce kidney fibrosis by activatingintrarenal renin-angiotension-aldosterone system associated epithelial-to-mesenchymaltransition. Plos one,2012,7(3):e34026.
[35]Lynch J, Nolan S, Slattery C, et al. High-mobility group box protein1: a novelmediator of inflammatory-induced renal epithelial-mesenchymal transition. Am J Nephrol,2010,32(6):590-602.
[36]Boutet A, De Frutos CA, Maxwell PH, et al. Snail activation disrupts tissuehomeostasis and induces fibrosis in the adult kidney. EMBO J,2006,25:5603-5613.
[37]Schramek H, Feifel E, Marschitz I,et al. Loss of active MEK1-ERK1/2restoresepithelial phenotype and morphogenesis in transdifferentiated MDCK cells. Am J PhysiolCell Physiol,2003,285:C652-661.
[38]Leask A. Targeting the jagged/notch pathway: a new treatment for fibrosis? J CellCommun Signal,2010,4:197-198.
[39]Kaimori A, Potter j, Kaimori JY,et al. Transfoming growth fator-beta1induces anepithelial-to-mesenchymal transition state in mouse hepatocytes in vitro. J Biol Chem,2007,282:22089-22101.
[40]Nitta T, Kim JS, Mohuczy D, et al. Murine cirrhosis induces hepatocyte epithelialmesenchymal transition and alterations in survival signaling pathways. Hepatology,2008,48:909-919.
[41]Wang S, Hyun J, Youn B, et al. Hedgehog signaling regulates the repair response inmouse liver damaged by irradiation. Radiat Res,2013,179(1):69-75.
[42]Chen YL, Lv J, Ye XL et al.Sorafenib inhibits transforming growth factorβ1-mediatedepithelial-mesenchymal transition and apoptosis in mouse hepatocytes. Hepatology,2011,53(5):1708-18.
[43]Taura K, Miura K, Iwaisako K, et al. Hepatocytes do not undergoepithelial-mesenchymal transition in liver fibrosis in mice. Hepatology,2010,51(3):1027-36.
[44]Diaz R, Kim JW, Hui JJ, et al. Evidence for the epithelial to mesenchymal transition inbiliary atresia fibrosis. Hum Pathol,2008,39:102-115.
[45]Xia JL, Dai C,Michalopoulos GK, et al. Hepatocyte growth factor attenuates liverfibrosis induced by bile duct ligation. Am J Pathol,2006,168:1500-1512.
[46]Omenetti A, Porrello A, Jung Y, et al. Hedgehog signaling regulatesepithelial-mesenchumal transition during biliary fibrosis in rodents and humans. J ClinInvest,2008,118:3331-3342.
[47]Rygiel KA, Robertson H, Marshall HL, et al. Epithelial-mesenchymal transitioncontributes to portal tract fibrogenesis during human chronic liver disease. LabInvest,2008,88:112-123.
[48]Scholten D, Scholten A, Brenner DA, et al. Epithelial-to-mesenchymal transition(EMT)in cholangiocytes does not contribute to liver fibrosis. Hepatology,2009,50:818A.
[49]Chu AS, Diaz R, Hui JJ,et al. Lineage tracing demonstrates no evidence ofcholangiocyte epithelial-to-mesenchymal transition in murine models of hepatic fibrosis.Hepatology,2011,53(5):1685-95.
[50]Hashimoto N, Phan SH, Imaizumi K, et al. Endothelial-mesenchymal transition inbleomycin-induced pulmonary fibrosis.Am J Respir Cell Mol Biol,2010,43:161-172.
[51]Zhao J, Shi W, Wang YL,et al. Smad3deficiency attenuates bleomycin-inducedpulmonary fibrosis in mice. Am J Physiol Lung Cell Mol Physiol,2002,282:L585-L593.
[52]Willis BC, Liebler JM, Luby-Phelps K, et al. Induction of epithelial-mesenchymaltransition in alveolar epithelial cells by transforming growth factor-beta1: potential role inidiopathic pulmonary fibrosis. Am J Pathol,2005,166:1321-1332.
[53]Mikami Y, Yamauchi Y, Horie M, et al. Tumor necrosis factor superfamily memberLIGHT induces epithelial-mesenchymal transition in A549human alveolar epithelial cells.Biochem Biophys Res Commun,2012,428(4):451-457.
[54]Chen HH, Zhou XL, Shi YL, et al. Roles of p38MAPK and JNK in TGF-β1-inducedhuman alveolar epithelial to mesenchymal transition. Arch Med Res,2013,[Epub ahead ofprint].
[55]Kondo Y, Higa-Nakamine S, Noguchi N, et al. Induction of epithelial-mesenchymaltransition by flagellin in cultured lung epithelial cells. Am J Physiol Lung Cell Mol Physiol,2012,303(12):L1057-69.
[56]Schneider DJ, Wu M, Le TT, et al. Cadherin-11contributes to pulmonary fibrosis:potential role in TGF-β production and epithelial to mesenchymal transition. FASEB J,2012,26(2):503-12.
[57]Wettstein G, Bellaye PS, Kolb M, et al. Inhibition of HSP27blocks fibrosisdevelopment and EMT features by promoting Snail degradation. FASEB J,2013,[Epubahead of print]
[58]Balli D, Ustiyan V, Zhang Y, et al. Foxm1transcription factor is required for lungfibrosis and epithelial-to-mesenchymal transition. EMBO J,2013,32(2):231-44.
[59]Zeisberg EM, Tarnavski O, Zeisberg M,et al. Endothelial-to-mesenchymal transitioncontributes to cardiac fibrosis.Nat Med,2007,13:952-961.
[60]Goumans M, van Zonneveld AJ, ten Dijke P: Transforming growth factor β-inducedendothelial-to-mesenchymal transition: a switch to cardiac fibrosis? Trends CardiovascMed,2008,18:293-298.
[61]Widyantoro B, Emoto N, Nakayama K, et al. Endothelial cell-derived endothelin-1promotes cardiac fibrosis in diabetic hearts through stuimulation ofendothelial-to-mesenchymal transition.Circulation2010,121:2407-2418.
[62]Bronnum H, Andersen DC, Schneider M, et al. Islet-1is a dual regulator of fibrogenicepithelial-to-mesenchynal transition in epicardial mesothelial cells. Exp Cell Res,2013,319(4):424-435.
[63]Yanjia H, Xinchun J. The role of epithelial-mesenchymal transition in oral squamouscell carcinoma and oral submucous fibrosis. Clin Chim Acta,2007,383:51-56.
[64]Scharl M, Frei S, Pesch T, et al. Interleukin-13and transforming growthfactorβsynergise in the pathogenesis of human intestinal fistulae. Gut,2013,62(1):63-72.