细胞外基质蛋白1在多房棘球蚴感染小鼠肝纤维化过程中的作用

目的探讨多房棘球蚴不同感染时间小鼠肝组织细胞外基质蛋白1 (extracellular matrix protein 1,ECM1)的表达水平以及与肝纤维化过程的相关性。方法将40只雌性C57BL/6小鼠随机分为多房棘球蚴感染组和对照组(20只/组),感染组经肝门静脉接种2 000个原头节/鼠,对照组注射等量生理盐水。分别于感染后第1、 6、 12和24周,两组各取5只小鼠采集肝组织,切片后HE染色观察多房棘球蚴感染后小鼠肝组织病理学变化;天狼星红染色以及α-SMA染色检测小鼠肝纤维化程度;免疫组织化学染色检测肝组织中ECM1的表达水平与分布。采用Pearson相关系数法分析ECM1表达水平与肝纤维化水平的相关性。结果 HE染色结果显示,多房棘球蚴感染后6周,小鼠肝组织内可见具有明显生发层结构的病灶形成,周围炎性细胞浸润明显且伴有纤维组织增生。天狼星红染色结果显示,感染组小鼠肝组织内病灶周围胶原沉积面积占比在感染后1、 6、 12和24周分别为(6.97±0.07)%、(10.39±0.02)%、(17.31±1.78)%和(22.24±1.07)%,均高于对照组(P 0.05);α-SMA染色结果显示,感染组肝组织病灶周围α-SMA阳性染色面积占比在感染后1、 6、 12和24周分别为(5.31±0.39)%、(9.97±1.3)%、(16.16±0.17)%和(19.01±0.49)%,均高于对照组(P 0.05);且随着多房棘球蚴感染小鼠时间的延长病灶周围肝纤维化程度不断加重。免疫组化检测结果显示,ECM1主要表达在病灶周围的炎性细胞带中,少量表达在肝窦;感染组ECM1阳性染色面积占比在感染后1、 6、 12和24周分别为(8.60±0.44)%、(13.90±0.57)%、(16.37±0.77)%和(19.50±0.50)%,均高于对照组(P 0.05)。Pearson相关性分析显示,ECM1表达水平与天狼星红阳性染色区域面积(r=0.900, P 0.01)及α-SMA阳性染色区域面积占比(r=0.941, P 0.01)均呈正相关。结论 ECM1在多房棘球蚴不同感染时间小鼠肝组织的表达均较对照组升高,并与肝纤维化程度呈正相关。     

LAG3缺陷对多房棘球蚴感染小鼠自然杀伤细胞功能及肝纤维化的影响

目的 探讨淋巴细胞活化基因3(lymphocyte activation gene 3,LAG3)缺陷(LAG3^-/-)对多房棘球蚴感染小鼠自然杀伤(natural killer,NK)细胞功能及肝纤维化的影响。方法 取体质量为(20±2) g的C57BL/6小鼠,分为LAG3^-/-组和野生型(wild type,WT)组,分别经肝门静脉接种3 000个多房棘球蚴原头节。感染后12周,取小鼠肝脏制备肝脏组织切片,天狼星红染色观察肝脏病灶和纤维化程度。分离小鼠肝脏和脾脏淋巴细胞,流式细胞术检测小鼠肝脏和脾脏NK细胞比例,NK细胞表面CD25、CD44和CD69分子表达水平,以及γ干扰素(interferon γ,IFN-γ)、肿瘤坏死因子-α(tumor necrosis factor α,TNF-α)、白细胞介素(interleukin,IL)-4、IL-10、IL-17A等相关细胞因子分泌水平。结果 天狼星红染色结果显示,与WT组相比,LAG3^-/-组小鼠肝脏病灶周围炎性细胞带增宽、纤维化结缔组织增生,且呈现较多胶原纤...     

TGF⁃β1 p38MAPK及BMP⁃7蛋白在肝多房棘球蚴病患者肝组织中的表达及意义

目的　检测肝多房棘球蚴病患者肝组织中转化生长因子⁃β1（transforming growth factor⁃β1, TGF⁃β1）、p38MAPK及骨形态发生蛋白⁃7（bone morphogenetic protein⁃7, BMP⁃7）表达水平,探讨其在肝多房棘球蚴病肝纤维化中的潜在作用。方法　以20例肝多房棘球蚴病患者为研究对象,分别采集距肝脏病灶0.5 cm内（A组）、距肝脏病灶0.5～1.5 cm（B组）肝组织及距肝脏病灶2 cm及以上的正常肝组织（C组）。肝组织标本分别行HE和Masson染色观察纤维化病理变化,采用Western blotting检测肝组织中TGF⁃β1、p38MAPK及BMP⁃7蛋白表达水平,分析TGF⁃β1、p38MAPK及BMP⁃7蛋白表达与肝纤维化的相关性。结果　HE染色结果显示,A组和B组肝组织中肝细胞结构排列紊乱、肝小叶结构有不同程度破坏,可见不同程度肝细胞变性、萎缩、坏死及纤维组织增生,并有嗜酸性粒细胞浸润;C组肝组织无异常病理改变,肝细胞结构形态正常、大小均匀,未见明显排列紊乱,肝小叶结构清晰,无或轻度细胞变性、坏死及炎性细胞浸润。Masson染色结果显示,A组和B组肝组织可见汇管区较多纤维结缔组织增生,出现不同程度小叶内纤维化;C组肝组织无明显异常病理改变。A、B、C组肝组织中TGF⁃β1（P < 0.001）、p38MAPK（P < 0.01）及BMP⁃7蛋白（P < 0.05）表达水平差异均有统计学意义,A组和B组TGF⁃β1、p38MAPK及BMP⁃7蛋白表达水平均显著高于C组（P均< 0.05）,B组TGF⁃β1、p38MAPK及BMP⁃7蛋白表达水平亦显著高于C组（P均< 0.05）。TGF⁃β1、p38MAPK及BMP⁃7蛋白表达水平均与肝纤维化程度呈正相关（r = 0.866、0.702、0.801,P均< 0.05）,不同纤维化程度肝组织中TGF⁃β1（F = 72.580,P < 0.01）、p38MAPK（[χ2] = 31.705,P < 0.01）及BMP⁃7蛋白（[χ2] = 48.388,P < 0.01）表达水平差异均有统计学意义。TGF⁃β1蛋白与p38MAPK、BMP⁃7蛋白表达水平均呈显著正相关（r = 0.607、0.702,P均 < 0.001）,BMP⁃7与p38MAPK蛋白表达水平亦呈显著正相关（r = 0.456,P < 0.001）。结论　TGF⁃β1、p38MAPK和BMP⁃7蛋白通过相互作用、共同介导了肝多房棘球蚴病肝纤维化发生。     

快速诊断多房棘球蚴病胶体金免疫层析试条方法的建立与评价

目的建立快速、简便诊断多房棘球蚴病的胶体金免疫层析试条方法。方法提取多房棘球蚴原头节总RNA,通过RT-PCR获得编码Em18基因片段并克隆入pGEX-3X表达载体,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达得到重组蛋白;采用柠檬酸三钠还原法制备胶体金,标记抗人IgG单克隆抗体;将重组Em18抗原包被于硝酸纤维素膜适当位置,制成检测特异抗体的胶体金免疫层析试条。用该试条检测多房棘球蚴病(56份)、细粒棘球蚴病(87份)、囊尾蚴病(30份)、日本血吸虫病(10份)和弓形虫病(10份)患者血清,以及健康人(50份)血清,以评价其检测的敏感性和特异性,同时用ELISA法进行平行检测,以评价该试条的诊断性能。结果以重组蛋白Em18为抗原胶体金免疫层析试条检测多房棘球蚴病患者血清,敏感性为92.9%(52/56)。与细粒棘球蚴病和囊尾蚴病患者血清分别存在9.2%(8/87)和3.3%(1/30)的交叉反应,与健康人血清存在8.0%(4/50)的假阳性率,与日本血吸虫病和弓形虫病患者血清则无交叉反应,特异性为93.0%(174/187)。ELISA法检测的敏感性和特异性分别为94.6%(54/56)和92%(172/187),与试条法比较两者差异均无统计学意义(P>0.05)。kappa分析结果显示,试条法与ELISA法检测多房棘球蚴病患者血清的结果高度一致(κ=0.98)。结论以重组Em18抗原建立的快速诊断胶体金免疫层析试条,检测多房棘球蚴病的敏感性和特异性均较高。     

小鼠肝细粒棘球蚴和多房棘球蚴病不同时期病灶周围组织纤维化对比

目的 本实验通过研究对比不同时间两种肝棘球蚴病灶周围组织纤维化情况,进一步了解肝棘球蚴病的病理生理发展过程,为肝棘球蚴病的诊治提供参考。方法 建立动物模型,使用HE,Masson染色以及COL1,COL3、α-SMA、TGF-β1免疫组化染色对比观察两种肝棘球蚴病在不同时间纤维化情况的不同。结果 随着时间的变化肝细粒棘球蚴病灶周围纤维化由弥漫到聚集,可形成连续致密的纤维外膜;肝多房棘球蚴病灶周围组织纤维化始终为弥漫性,无法形成连续质密的纤维外膜。细粒棘球蚴组病灶周围COL1(r=-0.768,P<0.05)、COL3(r=-0.781,P<0.05)、α-SMA(r=-0.867,P<0.05)、TGF-β1(r=-0.854,P<0.05)的表达强度与时间呈负相关,多房棘球蚴组病灶周围COL1(r=-0.349,P>0.05)、COL3(r=-0.037,P>0.05)、α-SMA(r=-0.107,P>0.05)、TGF-β1(r=-0.148,P>0.05)的表达强度与时间无相关性。 无相关性同时观察到两种包虫周围细胞外基质胶原含量不同,细粒棘球蚴组I、III型胶原比高于多房棘球蚴组(Z=-3.23,P<0.05)。结论 相较于多房棘球蚴,细粒棘球蚴病灶周围可产生连续致密的纤维外囊。细粒棘球蚴在外囊形成后纤维化进程减弱或停止,多房棘球蚴在整个病程中均有活跃的纤维化反应。细粒棘球蚴相较于多房棘球蚴外囊的I/III型胶原比值较高。     

基于非依赖性全扫描采集蛋白组学技术初步研究继发性多房棘球蚴病小鼠差异表达蛋白

目的　通过高分辨质谱数据非依赖性全扫描采集（data independent acquisition,DIA）蛋白组学技术鉴定多房棘球蚴病小鼠不同肝脏组织的差异表达蛋白,寻找其中可能与多房棘球蚴病致病相关的关键蛋白。方法　分离多房棘球蚴病青海田鼠体内原头节,用原头节感染雌性昆明小鼠（6～8周龄）进行造模。小鼠分为实验组与对照组,实验组注射约3 000个原头节,对照组注射等量生理盐水。两组小鼠培养1年后取实验组和对照组小鼠肝脏组织进行病理学检查,另取实验组小鼠肝脏病灶组织（病灶组）、病旁组织（病旁组）及对照组小鼠正常肝脏组织（正常组）进行蛋白DIA定量分析并筛选差异表达蛋白,对差异蛋白进行生物信息学分析。结果　在病灶组与正常组间检出1 020种差异表达蛋白,其中671种上调蛋白、349种下调蛋白;在病旁组与正常组间检出495种差异表达蛋白,其中327种上调蛋白、168种下调蛋白。京都基因和基因组百科全书（KEGG通路）富集分析显示,这些差异表达蛋白参与过氧化物酶体、过氧化物酶体增殖激活受体（PPAR）信号通路、脂肪酸降解等重要通路。通过文献检索,发现过氧化物酶体及PPAR信号通路与肝损伤相关,在这两条通路中共筛选出脂肪酸转运蛋白1（Fabp1）、肝衰竭与长链脂酰CoA合成酶（Acsl1）、酰基辅酶A氧化酶1（Acox1）、三羟酰辅酶A脱氢酶（Ehhadh）、乙酰辅酶A酰基转移酶1b（Acaa1b）等几种可能与多房棘球蚴病致病相关的差异表达蛋白,这些差异蛋白在实验组小鼠体内表达量均显著下调。结论　多房棘球蚴病小鼠肝脏内有大量蛋白质差异表达,其中Fabp1、Acsl1、Acox1、Ehhadh及Acaa1b等蛋白可能与多房棘球蚴病发病相关。     

三维可视化技术在终末期肝多房棘球蚴病自体肝移植术前评估中的应用价值

目的 探讨三维可视化技术在终末期肝多房棘球蚴病患者行自体肝移植术前评估中的应用价值。方法 收集2013年5月至2017年7月在青海省人民医院接受自体肝移植术的8例终末期肝多房棘球蚴病患者。对8例患者术前行腹部CT平扫及三期动态增强扫描,将患者CT原始数据传输至人体三维可视化虚拟手术系统,通过三维可视化重建测定多房棘球绦虫体积、预切肝体积,并观察病灶与周围组织的关系。对比术中实际所见,评估三维可视化技术对终末期肝多房棘球蚴病行自体肝移植术的应用价值。结果 三维可视化重建模型能清晰显示终末期肝多房棘球蚴病病灶与周围组织毗邻关系,三维可视化重建模型术前测得预切肝体积与术中实际切除的肝体积差异无统计学意义（t = 1.083,[P>0.05]）。结论 三维可视化技术可用于终末期肝多房棘球蚴病行自体肝移植术前制定合理的肝脏切除及血管吻合方案,提高手术成功率、改善患者预后。     

自分泌骨桥蛋白通过EGFR信号通路促进多房棘球蚴生长和转移的研究

目的研究骨桥蛋白(OPN)通过表皮生长因子受体(EGFR)通路在多房棘球蚴生长、侵袭和肝外转移中的作用机制。方法构建多房棘球蚴感染C57小鼠模型。感染后1个月,取100只模型小鼠随机均分为4组,Lv-Em OPN-734组和Lv3-NC组分别注射慢病毒Lv-Em OPN-734和Lv3-NC (空质粒),PBS组注射等量PBS,空白对照组不做处理。取Lv-Em OPN-734组2只慢病毒注射后5 d的小鼠肝组织,制备冰冻切片,于激光共聚焦显微镜下观察慢病毒进入肝组织情况。取各组感染后1、 2、 3、 4个月的小鼠,称体质量、肝质量,计算肝体积,并观察感染多房棘球蚴肝脏组织形态,HE染色观察转移灶情况。qPCR检测OPN mRNA的转录水平,Western blotting分析OPN和EGFR通路相关分子EGFR、蛋白激酶B (AKT)、细胞外蛋白调节激酶(ERK)及其磷酸化分子P-EGFR、 P-AKT、 P-ERK的表达情况。两组间差异的比较采用t检验,多组间差异的比较采用方差分析。结果冰冻切片镜下可见,慢病毒主要分布于肝病灶组织炎性带、外囊与生发层细胞。感染后4个月(病毒注射后3个月)的小鼠肝脏中,Lv-Em OPN-734组无肝外转移且肝内受多房棘球蚴侵蚀较轻,其余3组肝脏均被多房棘球蚴组织包裹表面出现不同程度的血管新生。感染后2～4个月(注射病毒后1～3个月),Lv3-NC组、PBS组和空白对照组的肝质量及体积均高于Lv-Em OPN-734组,差异均有统计意义(P 0.01)。4组小鼠体质量在感染后1～3个月均持续增加,感染后4个月,仅Lv-Em OPN-734组继续增加至(41.3000±1.252) g,其余3组小鼠体质量均下降(P 0.01)。HE染色结果提示,在感染后4个月的Lv3-NC组出现了多房棘球蚴的肺转移。qPCR检测结果显示,Lv-Em OPN-734组的OPN mRNA相对转录水平为1.44±0.54,低于Lv3-NC组(16.62±0.61)、 PBS组(15.45±1.11)和空白对照组(16.36±0.79)(P 0.01)。Western blotting分析结果显示,LvEm OPN-734组多房棘球蚴病灶组织OPN、 P-EGFR、 P-ERK和P-AKT的相对表达量分别为0.18±0.01、 0.30±0.04、 0.33±0.04、 0.25±0.02,均较其余3组低(P 0.01); EGFR、 ERK和AKT的相对表达量与其余3组差异无统计学意义(P 0.05)。结论OPN可能通过EGFR通路促进多房棘球蚴的生长发育,当抑制Em OPN的表达时,多房棘球蚴生长减缓,且不发生远端转移,提示OPN可能是多房棘球蚴病治疗的潜在靶点。     

青海省少年儿童肝多房棘球蚴病与胆囊并发症发生的相关因素分析

目的 分析青海省少年儿童肝多房棘球蚴病患者中胆囊相关疾病的分布特征及相关因素。方法 收集2012年1月-2017年12月青海省人民医院住院的未满18周岁的肝多房棘球蚴病患者临床资料,分析经手术治疗的患者中胆囊相关并发症发生的影响因素。结果 共收集51例少年儿童肝多房棘球蚴病患者。按照WHO棘球蚴病PNM影像学分型标准,P1、P2、P3型分别占37.25%（19/51）、41.18%（21/51）和19.60%（10/51）。按照《包虫病诊断标准》（WS 257-2006）诊断标准,51例患者中浸润型、钙化型、液化空洞型分别占66.67%（34/51）、21.57%（11/51）和11.76%（6/51）。患者中术前有胆道系统临床症状者占78.43%（40/51）,有胆囊相关并发症者占58.82% （30/51）。对40例多房棘球蚴病患者实施手术,术后有并发症者占77.50%（31/40）。Logistic回归分析结果表明,WHO影像学分型、《包虫病诊断标准》（WS 257-2006）分型、病灶位置、病灶肝段分布、病灶大小、病灶数量等是少年儿童肝多房棘球蚴病胆囊相关并发症发生的危险因素。结论 青海省少年儿童肝多房棘球蚴病患者中胆囊相关并发症的发生率较高,对患者手术方式、术后并发症的发生及预后影响较大,早期诊断和治疗尤为重要。     

Revolution CT全肝灌注对肝多房棘球蚴病病灶边缘浸润带的影像学评估

目的 通过Revolution CT全肝灌注分析肝多房棘球蚴病病灶边缘区域血供及代谢情况。方法 利用Revolution CT对青海省人民医院就诊的30例肝多房棘球蚴病患者行全肝灌注CT扫描并分析图像,比较病灶内、病灶边缘浸润带及周围正常肝组织灌注血流量、达峰时间、血容量、平均通过时间、肝动脉分数值的差异。结果 肝多房棘球蚴病病灶内、病灶边缘浸润带及周围正常肝组织灌注血流量、达峰时间、血容量和平均通过时间差异有统计学意义（F = 24.579、8.343、20.535、21.843,P均 < 0.05）,但病灶内、病灶周围浸润带及周围正常肝组织肝动脉分数值差异无统计学意义（F = 2.621,P > 0.05）。结论 Revolution CT全肝灌注能够对肝多房棘球绦虫,尤其是其浸润带进行更精确的定量分析,对肝多房棘球蚴病诊断及手术方案的制定具有重要指导意义。     

Wnt信号通路相关分子在肝纤维化中的表达水平变化及其意义

目的 观察Wnt信号通路相关分子在实验性肝纤维化组织中表达水平的变化.探讨其在肝纤维化发生中的作用.方法 建立大鼠肝纤维化动物模型.用Wnt信号通路PCR array功能基因芯片观察Wnt信号通路相关分子在肝纤维化模型中表达水平的变化.以模型组较正常组表达上调或下调2倍为差异表达基因;利用免疫组化及Western印迹观察平滑肌肌动蛋白、Wnt4、Frizzled2、β-钙粘蛋白在肝纤维化组织表达水平的变化.结果 芯片检测发现共有36条基因发生了显著改变,模型组较正常组上调2倍的基因有25个,上调基因主要包括Wnt5a类基因,如Wnt4、Wnt5a、Wnt11等,分别上调了13.9、16.5和2.17倍;较正常组下调2倍的基因有11个,主要为Wnt1、Wnt3等.分别下调了2.32、2.15倍.免疫组化及Western印迹检测发现模型组肝组织Wnt4、Frizzled2的表达水平较正常组显著上升,而磷酸化的β-钙粘蛋白的表达水平下降.结论 经典及非经典Wnt信号通路均可能参与了实验性肝纤维化的发生机制.     

Chitinase 3-like 1 is a profibrogenic factor overexpressed in the aging liver and in patients with liver cirrhosis

Older age at the time of infection with hepatitis viruses is associated with an increased risk of liver fibrosis progression. We hypothesized that the pace of fibrosis progression may reflect changes in gene expression within the aging liver. We compared gene expression in liver specimens from 54 adult donors without evidence of fibrosis, including 36 over 40 y old and 18 between 18 and 40 y old. Chitinase 3-like 1 (CHI3L1), which encodes chitinase-like protein YKL-40/CHI3L1, was identified as the gene with the greatest age-dependent increase in expression in liver tissue. We investigated the cellular source of CHI3L1 in the liver and its function using liver tissue specimens and in vitro models. CHI3L1 expression was significantly higher in livers of patients with cirrhosis of diverse etiologies compared with controls represented by patients who underwent liver resection for hemangioma. The highest intrahepatic CHI3L1 expression was observed in cirrhosis due to hepatitis D virus, followed by hepatitis C virus, hepatitis B virus, and alcohol-induced cirrhosis. In situ hybridization of CHI3L1 messenger RNA (mRNA) identified hepatocytes as the major producers of CHI3L1 in normal liver and in cirrhotic tissue, wherein hepatocytes adjacent to fibrous septa showed higher CHI3L1 expression than did those in more distal areas. In vitro studies showed that recombinant CHI3L1 promotes proliferation and activation of primary human hepatic stellate cells (HSCs), the major drivers of liver fibrosis. These findings collectively demonstrate that CHI3L1 promotes liver fibrogenesis through a direct effect on HSCs and support a role for CHI3L1 in the increased susceptibility of aging livers to fibrosis progression.

It is well-established that the incidence of severe liver disease with rapid liver fibrosis progression in humans is increased in the elderly, although the underlying mechanisms remain to be elucidated (1). The role of age has been particularly well documented in patients with hepatitis C virus (HCV) infection, where age at the onset of infection was found to be a major determinant for fibrosis progression and disease severity in immunocompetent subjects (2–6). Likewise, donor age was shown to have a major impact on graft outcome after liver transplantation for end-stage HCV disease: When the donors were younger than 40, the interval to cirrhosis was 10 y, whereas when the donors were 41 to 50 or older, the intervals were 6.7 and 2.7 y, respectively (7). Collectively, these data suggest that age-related changes in liver response to injury play a key role in determining the increased susceptibility of the aging liver to fibrosis (2, 4, 7).We hypothesized that the different rate of liver fibrosis progression in patients over 40 y of age could reflect changes in gene expression in aging livers. To test this hypothesis, we studied a large series of liver specimens from 54 well-characterized liver transplant donors by comparing gene expression between liver donors less than and over 40 y of age. We identified chitinase 3-like 1 (CHI3L1) as the gene with the greatest age-dependent increase in expression. CHI3L1, also known as YKL-40 in humans, is a secreted glycoprotein of ∼40 kDa (8), which has been shown to play a critical role in a variety of human diseases associated with inflammation, tissue remodeling, and injury (9–12). A correlation between serum levels of CHI3L1 with aging was previously documented in a large cohort of healthy individuals in Denmark (13). Elevated levels of CHI3L1 in serum have also been reported as a biomarker of liver fibrosis in patients with chronic liver disease of any etiology (9, 12, 14–18). However, the mechanisms underlying the correlation between increased circulating CHI3L1 levels and liver fibrosis have not yet been determined. There is very limited information on the expression of CHI3L1 in primary liver tissue, since in most previous studies serum was the sole clinical material analyzed. Thus, in this study, we investigated the source of CHI3L1 and the mechanisms linking CHI3L1 with liver fibrosis by using primary liver tissue and in vitro models.     

DNA微阵矩技术研究肝纤维化差异基因表达谱变化

应用DNA微阵矩技术研究乙型肝炎肝硬化组织差异基因表达谱改变,以寻找肝纤维化相关基因,探讨肝纤维化的发病机理。抽提正常肝组织和肝硬化组织中的mRNA来制备探针,经杂交、洗涤后,通过计算机扫描分析正常肝组织和肝硬化组织基因表达谱的差异情况。在10000个候选基因中,筛选出99条差异表达基因,表达上调的有45条,表达下调的有54条。其中未知基因9条。因此基于DNA微阵矩技术的肝纤维化基因表达谱分析能够高通量筛选与肝纤维化发生发展相关的基因。     

基因芯片技术研究肝纤维化相关基因

目的通过对大鼠肝纤维化发生过程中基因表达差异性的分析，探讨肝纤维化的发生机制。方法采用四氯化碳((CC14)诱导大鼠肝纤维化，于用药2周、4周、6周、8周分别取肝组织提取总RNA，分离纯化poly(A)^ RNA，逆转录并用[α-^33P]dATP制备探针，与含1176个基因的大鼠cDNA微阵列杂交及洗涤，信号检测，微阵列图像分析和计算机软件处理数据。结果用药4周时，筛选出的166条差异表达基因中，上调基因156条，主要为细胞骨架和动力蛋白基因、激素受体基因、细胞内蛋白磷酸化酶基因和ras相关蛋白基因等；下调基因主要为P450相关基因、核糖体蛋白基因、脂肪酸结合蛋白基因和生长因子受体基因等。用药8周时，筛选出差异表达基因中上调基因246条，主要为细胞外信息传导基因、生长因子相关基因、细胞周期相关基因、凋亡相关蛋白基因和激素受体基因等；下调基因主要为脂质代谢基因、激素受体基因和氨基酸受体基因等。其中部分差异表达基因用RT—PCR和Northern印迹法加以证实。结论肝纤维化形成过程中存在多个表达水平不同的基因，涉及丝裂原活化蛋白激酶(MAPK)信号通路，同一基因在不同时期表达水平也不一样，值得进一步研究。     

自身免疫性肝炎小鼠模型差异表达基因的筛查及柴胡皂苷D对部分差异表达基因表达的影响

目的对自身免疫性肝炎(AIH)小鼠模型的差异表达基因进行基因芯片筛查,并观察柴胡皂苷D(SS-d)对部分差异表达基因表达的影响,探讨AIH的发病机制及SS-d对该病的治疗作用机制。方法健康、雌性SPF级C57BL/6小鼠40只[体质量(20±2)g],分为基因芯片组(n=8)和SS-d治疗组(n=32)。基因芯片组小鼠又分为正常对照组(n=4)和模型组(n=4),正常对照组常规饲养,模型组小鼠按15 mg/kg剂量给予尾静脉注射刀豆蛋白A(Con A),12 h后处死并提取肝组织,按Agilent芯片说明书进行差异表达基因的筛选,采用荧光定量PCR技术对部分差异表达基因进行验证。SS-d治疗组小鼠随机分为正常组(常规饲养)、模型组(按15 mg/kg剂量给予尾静脉注射Con A)、SS-d低剂量组和SS-d高剂量组(分别按2.5 mg/kg和5.0 mg/kg剂量给予腹腔注射SS-d预处理,8 h后按15 mg/kg剂量给予尾静脉注射Con A)(每组8只)。12 h后收集各组小鼠静脉血,ELISA法检测血清ALT、AST。无菌条件下提取小鼠肝组织,部分多聚甲醛固定后切片,并进行HE染色;部分肝组织用于提取RNA,采用荧光定量PCR技术检测部分差异表达基因(IFNγ、IL-4、IL-5、IL-13和IL-17)的mRNA水平变化。多组间比较采用单因素方差分析,进一步两两比较采用LSD-t法检验;两组间比较采用t检验。结果基因芯片组小鼠共筛查差异表达基因11512个,其中上调5189个,下调6323个,显著富集于138条信号通路;荧光定量PCR验证结果显示,与正常对照组比较,模型组小鼠IFNγmRNA和IL-17 mRNA的表达升高(P值均<0.01),而IL-4 mRNA、IL-5 mRNA和IL-13 mRNA的表达下调(P值均<0.01),与芯片筛查结果一致。在SS-d治疗组中,与正常对照组比较,模型组小鼠血清ALT、AST水平均升高(P值均<0.01);肝组织内可见大量淋巴细胞浸润;IFNγmRNA和IL-17 mRNA的表达水平明显升高(P值均<0.05),而IL-4 mRNA、IL-5 mRNA和IL-13 mRNA的表达水平明显降低(P值均<0.05)。与模型组比较,SS-d低剂量组和SS-d高剂量组小鼠血清ALT、AST水平均降低(P值均<0.05),肝组织内淋巴细胞浸润程度减轻;IFNγmRNA和IL-17 mRNA的表达水平均降低(P值均<0.05),IL-4 mRNA、IL-5 mRNA和IL-13 mRNA的表达水平均升高(P值均<0.05),上述基因的含量变化差异在SS-d高剂量组与SS-d低剂量组之间具有统计学意义(P<0.05)。结论AIH的发生发展系大量基因异常表达的共同作用结果。其中IFNγ、IL-4、IL-5、IL-13、IL-17与AIH的发病密切相关,同时也是SS-d发挥免疫调节及肝损伤保护作用的生物学靶点。     

Molecular features of non-B, non-C hepatocellular carcinoma: a PCR-array gene expression profiling study

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) usually develops following chronic liver inflammation caused by hepatitis C or B virus. Through expression profiling in a rare type of HCC, for which the causes are unknown, we sought to find key genes responsible for each step of hepatocarcinogenesis in the absence of viral influence. METHODS: We used 68 non-B, non-C liver tissues (20 HCC, 17 non-tumor, 31 normal liver) for expression profiling with PCR-array carrying 3072 genes known to be expressed in liver tissues. To select the differentially expressed genes, we performed random permutation testing. A weighted voting classification algorithm was used to confirm the reliability of gene selection. We then compared these genes with the results of previous expression profiling studies. RESULTS: A total of 220 differentially expressed genes were selected by random permutation tests. The classification accuracies using these genes were 91.8, 92.0 and 100.0% by a leave-one-out cross-validation, an additional PCR-array dataset and a Stanford DNA microarray dataset, respectively. By comparing our results with previous reports on virus-infected HCC, four genes (ALB, A2M, ECHS1 and IGFBP3) were commonly selected in some studies. CONCLUSIONS: The 220 differentially expressed genes selected by PCR-array are potentially responsible for hepatocarcinogenesis in the absence of viral influence.     

Hepatic gene expression profiles associated with fibrosis progression and hepatocarcinogenesis in hepatitis C patients

AIM: To determine fibrosis progression and hepatocellular carcinoma (HCC), using simultaneous gene expression analysis. METHODS: Total RNA samples were extracted from liver biopsies from 19 patients with hepatitis C virus (HCV) infection and 3 patients without HCV infection. Among the 19 HCV-infected patients, 7 and 12 patients had grade Fl-2 and F3-4 fibrosis, respectively. Of the 12 patients with F3-4 fibrosis, 8 had HCC. Gene expression in the liver samples was determined using an oligonucleotide microarray. The following comparisons were performed: normal livers vs HCV-infected livers; F1-2 vs F3-4; and F3-4 with HCC vs F3-4 without HCC. Genes that were differentially expressed between these groups were identified based on signal-to-noise ratios. RESULTS: In the HCV-infected livers, genes involved in immune responses were highly expressed. Expression levels of genes for plasma proteins and drug-metabolizing enzymes were decreased and those of genes involved in the cell cycle and oncogenesis were increased in the F3-4 cases as compared to the F1-2 cases. Among the F3-4 cases, genes involved in carbohydrate metabolism tended to be more highly expressed in patients with HCC than in patients without HCC. CONCLUSION: We identified genes that are associated with fibrosis progression and hepatocarcinogenesis. This information may be used to detect increased carcinogenic potential in the livers of patients with HCV infection.     

Liver gene expression signature of mild fibrosis in patients with chronic hepatitis C

BACKGROUND & AIMS: The molecular mechanisms of hepatocellular carcinoma have been studied, but little is known of the changes in liver gene expression during the different stages of chronic hepatitis C virus (HCV) infection, in particular the transition from mild to moderate fibrosis. METHODS: We used real-time quantitative RT PCR to study the messenger RNA expression of 240 selected genes in 2 pools of liver specimens according to the stages of fibrosis (Metavir score; mild fibrosis = F1 and septal fibrosis = F2). Genes whose expression differed between pools (F2 vs F1) by at least 2-fold were selected. In addition, the expression level of these selected genes then was assessed in each of the 62 individual samples (F4, n = 6; F3, n = 17; F2, n = 21; vs F1, n = 18). RESULTS: The 22 genes that were up-regulated in the 21 F2 samples relative to the 18 F1 samples mainly encoded genes involved in cytoskeleton (KRT 19 and SCG 10), growth factors/cytokines (CXCL6, interleukin 8 [IL8], IL1A, IL2, and CXCL10), or growth factor receptors (CCR2, CXCR3, and CXCR4), or were involved in extracellular matrix production (COL1A1, CHI3L, and SPP1), in extracellular matrix remodeling (TIMP1, MMP7, and MMP9), and in cell junction (ITGA2 and CLDN 4). When hierarchically clustering the F2 and F1 samples according to the expression of the 11 most discriminatory genes (KRT 19, COL1A1, STMN2, CXCL6, CCR2, TIMP1, IL8, IL1A, ITGA2, CLDN 4, and IL2), the patient population was categorized into 2 subgroups: F1 and F2. Specifically, 15 of 18 F1 (83%) and 19 of 21 F2 (90%) were classified correctly (P < 10(-5)). We also studied the messenger RNA expression of these 240 selected genes in normal liver in comparison with F1. Genes dysregulated in the transition from normal liver to F1 mainly were interferon-inducible genes, and therefore were very different from those dysregulated in the transition from F1 to F2. CONCLUSIONS: Genes involved in extracellular matrix turnover and immune response are implicated in the transition from mild to moderate fibrosis. Eleven of the genes could form the basis for the gene expression signature of mild versus moderate fibrosis in patients with chronic hepatitis C.