Thy1.1阳性肝脏卵圆细胞在大鼠肝硬化形成与消减过程中的动态表达

目的 肝脏卵圆细胞（HOC）在二甲基亚硝胺（DMN）致大鼠肝硬化形成与消减过程中表达的动态变化，探讨其病理生理意义。方法 应用DMN腹腔注射（4周12次）制备大鼠肝硬化模型，分别于造模后3d、2、4周及终止造模后2、4周处死大鼠取肝组织，进行常规组织学观察，透射电镜下观察HOC超微结构，免疫组织化学和western blot方法检测Thy1．1的表达变化，应用图像分析法对所标记的HOC进行半定量测定以及光镜下计数细胞数量。结果 DMN造模4周大鼠已形成典型的肝硬化；终止造模后2周时肝组织病变较造模4周时有所减轻，终止造模后4周时炎症明显减轻并以不完全纤维间隔为主。Thy1．1阳性染色细胞正常大鼠肝组织未见到，造模后3d时可见微量表达，2周时呈散在分布，4周时明显增多，见于纤维间隔周围，终止造模后2周、即第6周时阳性染色显著强于造模4周，大量出现于汇管区，8周时较6周有所减少，表达量基本与4周时相等。阳性染色图像分析、光镜下阳染细胞计数及western blot三者的结果呈一致性。Western blot结果与免疫组织化学观察结果也具一致性。电镜下显示HOC具有形态小，核以卯圆型为主，高的核／浆比例等特点。结论 Thy1．1阳性的HOC可能与肝硬化的消减或逆转有关。     

肝脏卵圆细胞在二甲基亚硝胺致大鼠肝硬化形成过程中表达的动态变化及其意义

目的:观察肝脏卵圆细胞(HOC)在二甲基亚硝胺(DMN)致大鼠肝硬化过程中表达的动态变化,探讨其病理生理意义。方法:应用DMN造成大鼠肝硬化动态模型,进行常规组织学观察,透射电镜观察HOC超微结构,免疫组化方法检测Thy1.1在模型不同时间点的表达变化,应用图象分析法对所标记的HOC进行半定量测定以及光镜下计数细胞数量。结果:于造模 4 周肝纤维化最重,形成假小叶,伴大面积出血坏死, 终止造模2周后,即6周时开始缓解,8周炎症明显减轻并以不完全纤维间隔为主。Thy1.1阳性染色细胞2周开始散在分布,4周增多于纤维间隔周围, 6周大量出现于汇管区,8周开始减少。图象分析与光镜下细胞计数结果相一致,均显示Thy1.1染色细胞数量于6周最高,8周开始下降。超微电镜显示HOC具有形态小,核以卵圆型为主,核/浆比例高等特点。结论:在DMN大鼠肝硬化形成与消减过程中,Thy1.1 染色的肝脏卵圆细胞呈现显著的阳性表达并有一定的动态变化特征,在肝硬化消减过程中可能具有重要的作用。     

Dimethylnitrosamine-induced cirrhosis. Evidence for an immunological mechanism

The present study is concerned with the early events associated with the development of cirrhosis induced by dimethylnitrosamine (DMN). The antigenic expression of MHC class II components (Ia) and of some intermediate filament proteins (vimentin and desmin) have been studied by immunohistochemistry and the findings correlated with ultrastructural data. Micronodular cirrhosis developed after 3 weeks of treatment with DMN but enhanced expression of Ia antigen on macrophages and on infiltrating lymphocytes was observed after 1 week, before the formation of septa, suggesting that immune-mediated mechanisms are involved in the response to DMN-induced liver injury. The expression of vimentin and of desmin also increased at an early stage and at 3 weeks the septa were outlined by cellular elements showing positivity for both intermediate filament proteins. In keeping with these observations, ultrastructural data showed active division of macrophages in situ, infiltration of the parenchyma by T and B lymphocytes, activation of lipocytes (Ito cells) showing evidence of mitosis, and the presence of transitional elements between lipocytes, myofibroblasts and fibroblasts. This experimental model may be helpful in understanding the relationship between immune-mediated response to liver injury and development of hepatic fibrosis.     

实验性肝硬化形成过程中层粘连蛋白的动态变化

目的探讨复合致病因子所致大鼠实验性肝硬化形成过程中肝脏层粘连蛋白的动态变化。方法取大鼠８０只，应用复合致病因子复制实验性肝硬化大鼠模型，于实验第二、四、六、八周肝硬化形成不同时期处死动物，测定肝脏胶原含量，应用免疫组化及电镜技术观察肝脏层粘连蛋白分布及结蛋白阳性细胞的变化。结果肝纤维化早期肝细胞坏死区域层粘连蛋白即呈阳性染色，沿坏死区域形成间隔样分布，并随肝硬化的形成其在纤维间隔内的沉积呈进行性增加，且肝窦壁亦出现连续强染。肝纤维化形成时期（实验第四周末）肝内层粘连蛋白的分布面积和结蛋白阳性细胞数呈显著正相关（ｒ＝０．９５５，Ｐ＜０．０１）。结论复合致病因子所致大鼠肝硬化形成过程中肝内层粘连蛋白分布呈进行性增加，与肝硬化的发生发展密切相关。     

Intrahepatic transplantation of hepatic oval cells for fulminant hepatic failure in rats

AIM： To evaluate the effect of intrahepatic transplantation of hepatic oval cells （HOC） on fulminant hepatic failure （FHF） in rats.
METHODS： HOC obtained from rats were labeled with green fluocescent protein （GFP） or 5, 6- carboxyfluorescein diacetate succinmidyl ester （CFDASE）. Cell fluorescence was observed under fluorescent microscope at 6, 24, 48 and 72 h after labeling. CFDA- SE labeled HOC （5 × 10^6 cells each rat） were injected into livers of rats with FHF induced by D-galactosamine. Serum albumin （ALB）, alanine aminotransferase （ALT）, aspartate aminotransferase （AST） and total bilirubin （TBil） levels were measured at different time points. Liver function of rats was examined on days 3, 7, 14 and 21 after HOC transplantation.
RESULTS： The positive rate of GFP and CFDA-SE labeled HOC was 10% and 90%, respectively, with no significant change in cell viabilities. The survival rate was higher in HOC transplantation group than in control group, especially 48 （9/15 vs 6/15） and 72 h （9/15 vs 4/15） after HOC transplantation. The serum ALT, AST and TBil levels were decreased while the serum AIb level was increased after HOC transplantation. Fluorescence became faded and diffused in liver tissues, suggesting that proliferation and differentiation occur in transplanted HOC.
CONCLUSION： CFDA-SE is superior to GFP in labeling HOC, although fluorescence intensity is decreased progressively with cell division. HOC transplantation can improve the liver function and increase the survival rate of recipients.     

肝纤维化过程中胶原、基质金属蛋白酶及其抑制因子表达的动态变化及相互关系

目的研究在整个肝纤维化形成过程中肝组织Ⅰ、Ⅲ型胶原,间质性胶原酶(MMP 1 3)及其抑制因子(TIMP-1)表达水平的动态变化规律及相互间的关系.方法将大鼠随机分成正常对照组与模型组.模型组以二甲基亚硝胺腹腔内注射,第1周注射3次,随后每周2次,共注射6周,停用后再观察2周;正常对照组以等渗盐水代替腹腔内注射.分别于第1、4、10、17、28、42、56天分批处死大鼠.留取的肝脏组织做HE与Masson染色,按0～4期标准判定肝纤维化程度,测定羟脯氨酸含量,应用半定量RTPCR检测Ⅰ、Ⅲ型胶原、MMp-13和TIMP-1 mRNA.结果在肝纤维化形成过程中,胶原持续增高,其中Ⅰ型胶原mRNA在肝组织受损后10 d开始较正常对照明显增高(正常组0.468±0.015,模型组0.603±0.031,t=2.8 5,P＜0.05),并保持不断增高的表达水平直至实验结束;Ⅲ型胶原mRNA从28d左右开始显著增高(正常组0.774±0.043,模型组0.922±0.079,t=4.16,P＜0.01),直至实验结束;TIMP-1 mRNA从第4天即开始明显增高(正常组0.618±0.030,模型组0.728±0.013,t=2.60,P＜0.05),并保持不断增高的趋势直至实验结束;MMP-13 mRNA从10 d后到28 d有显著增高(17d左右达到高峰,正常组0.987±0.048,模型组1.141±0.033,t=4.08,P＜0.01),此后便逐渐下降至实验结束.结论在肝纤维化形成过程中MMP-13的表达虽有增高,但由于TIMP-1的表达在肝受损后早期即开始持续不断增高,从而MMP-13降解胶原的能力受到抑制,致使过度产生与沉积的胶原得不到有效降解,促进了肝纤维化的发展.     

Glycoproteomics analysis of plasma proteins associated with Opisthorchis viverrini infection-induced cholangiocarcinoma in hamster model

Objective: To apply lectin affinity chromatography and glycoproteomics-based LC-MS/MS to preliminarily investigate the possible potential plasma biomarkers of Opisthorchis viverrini(OV)-associated CCA in OV/dimethylnitrosamine(DMN)-induced CCA hamster model. Methods: Nine Syrian hamsters were divided into 3 groups as follows(n=3 each): normal(healthy control group); OV group; and OV/DMN group(CCA group). Pooled plasma samples collected from animals in each group at the 6th month post-infection with OV metacercarae were subjected to glycoproteomics analysis. Glycoproteins in the pooled sample from each group were initially isolated by concanavilin A(Con A)-based affinity chromatography. The expression of glycoproteins isolated by both enrichment methods were determined using LCMS/MS. Results: Among the 24 Con A-binding glycoproteins isolated, two proteins, N-myc downstream regulated gene 1(NDRG1) and fetuin-B(FETUB) were found up-regulated only in the samples from the OV and control groups, but not in the OV/DMN(CCA) groups. On the other hand, one protein, i.e., NSFL1 cofactor p47 isoform x3(NSFL1C) was found only in the samples from OV/DMN(CCA) and control groups, but not in the OV group. The remaining 21 proteins were upregulated in the samples from all groups. Conclusions: NDRG1, FETUB and NSFL1 C glycoproteins isolated by Con A-based affinity chromatography could be potential biomarkers for CCA. Plasma samples with negative for NDRG1 and FETUB proteins but positive for NSFL1 C are likely to be OV-associated CCA. Nevertheless, this conclusion remains to be confirmed whether this battery test can discriminate OV-associated CCA from other risk factors.     

Incomplete septal cirrhosis associated with Wegener's granulomatosis

Incomplete septal cirrhosis, which is included in the spectrum of hepatoportal sclerosis, is characterized by parenchymal nodularity, incomplete fibrous septa, clustered or dispersed portal tract remnants, and abnormal spacing of portal tracts and hepatic veins. Hepatoportal sclerosis is known to be associated with collagen vascular diseases. Here, we describe a 73 year-old-female with incomplete septal cirrhosis. At 57 years, she presented with respiratory symptoms, and lung biopsy disclosed active arteritis with granuloma. Perinuclear antineutrophilic cytoplasmic antibody was also positive. Immunosuppressive therapy was done under the diagnosis of Wegener's granulomatosis. At 63 years, liver dysfunction was noted, and laparoscopy revealed uneven surface of the liver and dilatation of the umbilical vein. Liver dysfunction progressed, and she developed encephalopathy and massive ascites. She died of sepsis at 73 years. At autopsy the liver (700 g) was macronodular with several deep depressions. The parenchyma showed fine and diffuse nodularity. Grossly visible portal and hepatic veins were patent. The above-mentioned histologic features characterizing incomplete septal cirrhosis were found. This is the first report of incomplete septal cirrhosis associated with Wegener's granulomatosis implying that vascular and extravascular lesions of Wegener's granulomatosis might have been related to the pathogenesis of incomplete septal cirrhosis.     

二甲基亚硝胺致大鼠肝纤维化的造模研究

采用二甲基亚硝胺对大鼠进行急性小剂量间歇性腹腔注射,制作实验性肝纤维化模型。结果发现,在染毒期间大鼠肝内小叶中央出血性变性坏死的炎性浸润持续存在,肝细胞外基质进行性增加,染毒4周末在肝内形成中心—中心性和/或中心—门脉性纤维间隔。停止染毒后4周末肝内出血性病变基本消失,炎性浸润明显减轻,但纤维化仍然明显。血清和肝脏生化检测等结果与肝内病理改变相一致.本模型造模周期短,大鼠死亡率低,肝纤维化形成相对稳定,值得在中医药抗肝纤维化研究中使用。     

大鼠肝纤维化病理过程中Smads锚着蛋白表达变化

目的观察肝纤维化病理过程中肝脏Smads锚着蛋白(SARA)的表达变化及其与肝纤维化的关系。方法大鼠每kg体重10μl二甲基亚硝胺腹腔注射,1次/d,每周连续3 d,共4周,复制大鼠肝纤维化模型。模型大鼠分别设首次造模后1、3 d、1,2、3、4周末,与造模停止后1、2、4周,共9个时间段为观察组。每组5～8只,另设正常大鼠10只为对照组。天狼猩红染色观察肝组织胶原沉积,盐酸水解法测定肝组织羟脯氨酸(HYP)含量,免疫组织化学染色观察肝组织SARA蛋白表达,Western blot法分析肝组织转化生长因子(TGF)β1、α-平滑肌肌动蛋白(α-SMA)和SARA蛋白的表达,并进行以上指标的相关性分析。结果随二甲基亚硝胺染毒持续,模型大鼠肝脏胶原增生(Hyp含量)与沉积增加,4周末时达到高峰,可见宽大纤维间隔与假小叶；而后随染毒停止,肝脏胶原沉积与纤维间隔有所减轻。模型组4、5、6、8周肝组织Hyp平均含量(μg/g)分别为193．0±39．2、188．5±39．9．174．4±21．2、163．6±31．5,对照组分别为125．6±19．5,t值在3．43～4．9,P值均＜0．01。免疫组织化学染色发现,SARA主要表达于正常与纤维化肝脏的肝窦周围间质细胞,随肝纤维化发展SARA阳性染色细胞数量减少,随染毒停止与肝纤维化恢复,SARA渐恢复至正常组水平。Western blot发现,随肝纤维化形成,模型大鼠肝组织TGFβ1、α-SMA蛋白表达逐渐增加,而SARA蛋白表达逐渐减少；肝纤维化恢复过程中,SARA渐恢复接近正常水平,TGFβ1与α-SMA蛋白表达有所下降。在肝纤维化形成与恢复过程中,SARA蛋白与Hyp含量、TGFβ1与α-SMA表达均呈明显负相关。结论SARA蛋白主要表达于肝脏间质细胞；随大鼠肝纤维化发展,SARA表达减少,SARA蛋白与肝纤维化形成呈负相关关系。