吡喹酮与γ-干扰素对肝纤维化胶原代谢影响的实验研究

目的：探讨吡喹酮杀虫治疗和IFN－γ抗肝纤维化的分子机制。方法：以日本血吸虫尾蚴感染新西兰兔制成肝纤维化动物模型,感染后第16周随机分成3组：感染组,吡喹酮治疗组和吡喹酮联合IFN－γ治疗组,另以10只正常兔为阴性对照组,感染后28周处死,取肝组织进行病理检测以及I型胶原,Ⅲ型胶原,Ⅳ型胶原,MMP－1,MMP－9,TGFβ1和IFN－γmRNA水平检测。结果：吡喹酮治疗后肝纤维化程度明显减轻,但仍显著高于正常组;I型胶原,Ⅲ型胶原和Ⅳ型胶原mRNA水平显著下降,尤其Ⅲ型胶原已接近正常水平,而MMP－1和MMP－9 mRNA水平变化不大;TGFβ1 mRNA水平下降不明显,而IFN-γ mRNA水平显著增高,TGFβ1/IFN-γmRNA比值明显减少,但仍高于正常。杀虫治疗后加IFN－γ后进一步降低I型胶原,Ⅳ型胶原mRNA水平,并使MMP－1和MMP－9 mRNA的表达显著增加,TGFβ1 mRNA水平明显下降。结论： 杀虫治疗后抗肝纤维化仍属必要,IFN－γ主要通过直接抑制胶原合成,促进胶原降解,抑制TGFβ1起抗肝纤维化作用。     

吡喹酮治疗血吸虫病肝纤维化的实验研究

目的观察吡喹酮对血吸虫病肝纤维化的治疗作用。方法将小鼠随机分为正常对照组、模型组、吡喹酮组。模型组、吡喹酮组小鼠经腹部皮肤感染日本血吸虫尾蚴诱导肝纤维化模型,吡喹酮组于感染8周后灌胃吡喹酮治疗2次。16周末测小鼠血清中ALT、AST;观察小鼠肝脏形态,HE染色观察肝纤维化病理组织变化,测定虫卵肉芽肿面积;免疫组化测定肝脏Ⅰ和Ⅲ型胶原表达。结果吡喹酮治疗可降低血吸虫病肝纤维化小鼠肝脏指数及血清ALT、AST,改善肝组织形态结构,降低肝脏Ⅰ和Ⅲ型胶原表达及虫卵肉芽肿面积。结论吡喹酮对血吸虫病肝纤维化有明显治疗作用。     

芍药甙对日本血吸虫病小鼠肝组织纤维化的影响

目的观察芍药甙对日本血吸虫感染小鼠肝组织纤维化的影响,探讨芍药甙预防血吸虫病肝虫卵肉芽肿及纤维化的作用机制。方法日本血吸虫尾蚴感染小鼠,制备肝虫卵肉芽肿和纤维化小鼠模型。随机分为5组:正常对照组(A组),给予芍药甙组(B组、C组、D组),3组分别为予芍药甙30mg/(kg·d)、60mg/(kg·d)、120 mg/(kg·d),及感染对照组(E组)。B、C、D和E 4组小鼠均在感染后6周灌胃吡喹酮(400mg/(kg·d)×2 d)。感染后14周处死小鼠取血及肝脏、用HE及Masson胶原纤维染色观察肝虫卵肉芽肿而积及纤维化程度,用免疫组化技术检测转化生长因子β1(TGF-β1)和Ⅰ、Ⅲ型胶原表达情况。结果 B、C、D组与阳性对照组E组相比.TGF-β1、Ⅰ和Ⅲ型胶原水平明显下降(均P0.05),而且随芍药甙给药浓度增加,B组、C组、D组TGF-β1、Ⅰ和Ⅲ型胶原表达水平逐渐减少。结论在吡喹酮杀虫处理的基础上联合应用芍药甙,可通过减少肝组织TGF-β1和Ⅰ、Ⅲ型胶原的表达,明显减少肝虫卵肉芽肿形成,减轻肝纤维化程度,对血吸虫病小鼠早期肝纤维化形成有阻抑作用。     

抗独特型抗体NP30对血吸虫病虫卵肉芽肿及肝纤维化的调节作用

目的　研究单克隆抗独特型抗体NP30主动免疫对血吸虫病虫卵肉芽肿及肝纤维化的影响。方法　实验组ICR小鼠腹腔注射NP30 10 0 μg/次 ,连续免疫 3次 ,对照组腹腔注射SP2 / 0腹水。尾蚴攻击感染后第 4、8、12、16、2 0和 2 4周分别处死小鼠剖取肝脏 ,用VG(VanGieson)组织化学染色 ,Ⅰ型胶原、Ⅲ型胶原和纤维连接蛋白 (FN)免疫组织化学染色 ,应用计算机图像分析系统对肝脏虫卵肉芽肿体积和虫卵肉芽肿内的胶原沉积进行定量测定。结果　尾蚴攻击感染第 12周后 ,实验组虫卵肉芽肿的体积明显小于对照组 ,虫卵肉芽肿细胞组分与对照组明显不同 ,出现两种不典型肉芽肿。VG染色显示实验组虫卵肉芽肿内胶原的平均光密度值明显小于对照组。免疫组织化学显示实验组虫卵肉芽肿内Ⅰ型胶原、Ⅲ型胶原及FN含量均比对照组低。结论　NP30接种可能诱导体液和细胞两种保护性免疫 ,对血吸虫病虫卵肉芽肿具有负调节作用 ,对血吸虫性肝纤维化有明显的抑制作用     

布洛芬抗兔血吸虫病肝纤维化的实验研究

目的:观察布洛芬抗兔血吸虫病肝纤维化时对兔肝组织Ⅰ型胶原(Collagen Ⅰ)、Ⅲ型胶原(Collagen Ⅲ)、基质金属蛋白酶-1(MMP-1)、金属蛋白酶组织抑制因子-1(TIMP-1)mRAN表达的影响。方法:建立兔血吸虫病肝纤维化模型。应用布洛芬治疗8周后处死全部实验兔,取其肝组织作冰冻切片,应用原位杂交技术检测CollagenⅠ、CollagenⅢ、MMP-1、TIMP-1 mRNA,并用计算机彩色病理图像分析系统进行图像分析。结果:布洛芬组与模型对照组相比,CollagenⅠ、CollagenⅢ、TlMP-1 mRNA表达减少,MMP-1 mRNA表达增加(P<0.05)。结论:布洛芬能抑制兔血吸虫病肝纤维化肝组织CollagenⅠ、CollagenⅢ、TIMP-1 mRNA表达,增加MMP-1 mRNA表达,促进胶原降解,减少胶原沉积。     

人日本血吸虫性大肠纤维化胶原蛋白和细胞因子观察

目的观察日本血吸虫病传播阻断后人大肠纤维化胶原蛋白和细胞因子的改变。方法在日本血吸虫病传播阻断达标10年地区收集102例血吸虫感染患者大肠标本,在组织切片上进行Ⅰ-Ⅳ型胶原蛋白以及转化生长因子β1（TGFβ1）、碱性纤维母细胞生长因子（bFGF）、基质金属蛋白酶-1（MMP-1）和基质金属蛋白酶组织抑制剂-1（TIMP-1）的免疫组化染色和定量分析。收集同期51例无血吸虫感染者大肠标本作对照。结果102例血吸虫感染患者大肠肠壁内虫卵均已死亡和钙化。26份标本（25.5%）在死亡和钙化虫卵周围发现肉芽肿反应。虫卵高密度组（〉60个/mm^2）大肠的Ⅰ-Ⅲ型胶原平均吸光度（A）值均较对照组增高,而Ⅳ型胶原平均A值下降。Ⅰ-Ⅲ型胶原平均A值的增加均与肉芽肿反应呈正相关。血吸虫感染大肠肉芽肿组的TGFβ1、bFGF和MMP-1的平均A值分别高于对照组,而TIMP-1的A值低于对照组。结论参与日本血吸虫性大肠纤维化的胶原蛋白和细胞因子含量与虫卵密度、纤维化时期和有无肉芽肿反应有关。     

芍药苷对感染血吸虫小鼠肝纤维化的影响

目的探讨小鼠感染日本血吸虫后经吡喹酮治疗的不同时期,给予芍药苷对肝组织虫卵肉芽肿和纤维化的影响。方法以日本血吸虫尾蚴感染BALB/c小鼠构建肝纤维化模型,随机分为(Ⅰ)杀虫前给药组、(Ⅱ)杀虫同时给药组及(Ⅲ)杀虫后给药组。除正常组外,杀虫治疗、芍药苷治疗组和对照组小鼠分别于感染后12d、42d和72d给予芍药苷和对照,并于102d、132d和162d处死。检测透明质酸(HA)、Ⅲ型前胶原氨基端肽(PⅢP)、羟脯氨酸(Hyp)、虫卵肉芽肿大小、肝纤维化分级以及胶原Ⅰ的表达。结果在组Ⅰ和组Ⅲ,芍药苷明显降低血清中HA、PⅢP和肝组织中Hyp的含量,减小虫卵结节并降低肝纤维化严重程度分级,降低胶原Ⅰ的表达(P<0.05或P<0.01);在组Ⅱ,大部分指标没有明显差别(P>0.05)。结论芍药苷无论是早期或延后给药,都具有抑制虫卵肉芽肿,减少胶原的生成从而对抗小鼠血吸虫性肝纤维化的作用。     

安络化纤丸联合干扰素γ干预鼠血吸虫性肝纤维化及对肝色素沉积的影响

目的 评价安络化纤丸联合干扰素γ治疗鼠血吸虫肝纤维化的效应机制及其对肝色素沉积的影响.方法 将30只昆明小鼠分为健康对照组、感染对照组及干扰素γ+安络化纤丸治疗组.采用日本血吸虫尾蚴(40条/只)攻击感染小鼠,建立血吸虫性肝纤维化模型,连续干预8周,观察肝色素沉积及血吸虫卵肉芽肿改变;免疫组织化学检测肝组织Ⅰ型和Ⅲ型胶原的表达;荧光定量法检测肝组织TGF-β1 mRNA、组织病理学评价及电子计算机图像定量分析.对数据进行正态性检验、方差齐性检验及单因素方差分析.结果 肝色素沉积百分比与TGF-β1 mRNA量呈相关性,相关系数=0.8;安络化纤丸联合干扰素γ治疗后明显减轻鼠血吸虫肝组织纤维化、减少色素沉着、使虫卵肉芽肿变小、下调Ⅰ及Ⅲ型胶原的表达及减少TGF-β1 mRNA量表达,与感染对照组比较,差异均有统计学意义(P<0.05).结论 肝色素沉积量与TGF-β1 mRNA量有一定相关性,安络化纤丸联合干扰素γ明显减轻鼠血吸虫性肝纤维化.下调Ⅰ型及Ⅲ型胶原及TGF-β1 mRNA表达、减少色素沉积是其作用机制之一.     

中药对日本血吸虫肝纤维化形成的影响

用免疫组化SABC技术结合计算机彩色病理图文分析系统，定量检测并分析复方中药制剂治疗对日本血吸虫鼠肝内Ⅰ、Ⅲ型胶原及转化生长因子β1（TGF—β1）的影响。结果：感染组鼠肝内Ⅰ、Ⅲ型胶原及TGF—β1。均主要分布在虫卵肉芽肿内，呈密集片状着色。经中药治疗后着色均明显减少；治疗组Ⅰ、Ⅲ型胶原及TGF-β。的含量均显著低于感染组。提示中药对日本血吸虫病肝纤维化的形成有抑制作用。     

pHGF对日本血吸虫病宿主肝内TNF及肝纤维化的影响

本文用免疫组化SABC技术，动态观察感染日本血吸虫小鼠不同时期肝内TNF－α、FN、LN、Ⅰ及Ⅲ型胶原的分布及含量变化。结果显示感染后第8－12wk，肝内TNF的量即明显增加，第16wk时达到高峰，主要分布在虫卵肉芽肿内及其周围。炎症反应越重，TNF量越多。在慢性感染期，肝内FN、LN、Ⅰ及Ⅲ型胶原的量逐渐增加，使得感染初期呈细线、环状结构分布的Ⅰ及Ⅲ型胶原，逐步增宽变厚，呈网状连结分布，虫卵肉芽钟内开始有较多的胶原纤维沉积。但TNF的含量变化与肝内胶原的增加不成正比。注射pHGF后．对肝内肉芽肿的大小及炎症反应、TNF、FN及Ⅲ型胶原的量影响不大（P＞0．05），但LN及Ⅰ型胶原的含量明显低于同期非注射组（P＜0.05）。提示早期应用足量pHGF治疗日本血吸虫病，对于减轻虫卵引起的肝损害及肝纤维化可能具有一定的作用。     

High serum laminin and type IV collagen levels in schistosomiasis mansoni

BACKGROUND: Fibrosis is the process of excessive deposition of collagen and other extra cellular matrix components and large amounts of these components have been shown in periovular schistosomal granulomas, especially in the liver. Laminin and type IV collagen have been investigated in various hepatic disorders but their accuracy in fibrosis detection and in the evaluation of its progression in schistosomiasis have not been fully explained. AIM: To measure the serum levels of two markers of fibrosis, laminin and type IV collagen in schistosomiasis. PATIENTS AND METHODS: Sixty-four patients with different clinical forms of schistosomiasis mansoni: intestinal (group I), hepatointestinal (group II), compensated (group III) and decompensated hepatosplenic (group IV) and 18 healthy volunteers were included. RESULTS: Serum type IV collagen and laminin levels were significantly increased in patients compared to controls. At about clinical forms, serum type IV collagen was increased in groups II and IV, compared to controls and was significantly higher in group IV than in group I. Serum laminin was significantly increased in groups II, III and IV and was significantly higher in group IV than in group II. Serum type IV collagen was closely correlated with serum laminin in groups II and IV. CONCLUSIONS: Connective tissue marker levels did not correlate with periportal thickness. In schistosomiasis mansoni there is an increase of type IV collagen and laminin levels at the initial stage of the disease, as well as in advanced forms. We also suggest that these markers may be a useful predictor of disease progression.     

日本血吸虫感染兔Ⅰ型和Ⅲ型胶原动态变化及干扰素-γ对其的作用

目的　观察感染日本血吸虫的新西兰兔肝脏Ⅰ型和Ⅲ型胶原的动态变化以及γ 干扰素 (IFN γ)对Ⅰ型和Ⅲ型胶原的降解作用。方法　日本血吸虫感染兔在感染后不同时期取 8只病兔肝脏 ,作常规石蜡切片 ,分别进行免疫组织化学α SMA染色、伊红染色和天狼红染色 ,并在偏光显微镜下观察Ⅰ型和Ⅲ型胶原分布情况 ,计算机图像分析计算胶原含量。感染 16wk后 ,给予吡喹酮治疗 ,IFN γ治疗 8wk ,停药观察 4wk。观察IFN γ对Ⅰ型和Ⅲ型胶原沉积的降解作用。结果　感染日本血吸虫的病兔Ⅰ型胶原在第 8周时占总面积百分比的 5 73± 3 40 ,至第2 8周时达总面积百分比的 40 14± 17 0 0 ,约增加了 7倍 ;Ⅲ型胶原则由第 8周时总面积百分比的 1 15± 1 34增加到6 80± 5 19。α SMA阳性细胞表达数则由 2 8± 1 0增加至 7 3± 1 5。自血吸虫感染 16wk开始采用IFN γ治疗 ,8wk后 ,IFN γ治疗组的Ⅰ型和Ⅲ型胶原所占面积百分比分别由原来的 18 5 1± 7 5 2和 4 63± 3 64下降为 2 4wk时的3 0 9± 1 5 4和 0 40± 0 37(P <0 0 1) ,模型对照组和吡喹酮治疗组比较差异具有显著性 (P <0 0 1)。停药 4wk后IFN γ治疗组的Ⅰ型和Ⅲ型胶原所占面积百分比均有所回升 (P <0 0 5 )。结论　IFN γ对日本血吸虫感染的新西兰兔肝纤     

Effects of pentoxifylline on the hepatic content of TGF-β1 and collagen in Schistosomiasis japonica mice with liver fibrosis

To study the effects of pentoxif-ylline (PTX) on the content of hepatic TGF-β, type Ⅰand type Ⅲ collagen in schistosomiasis japonica mice with liver fibrosis and its mechanism of anti-fibrosis.     

Collagen isotypes, laminin, and fibronectin in granulomas of the liver and intestines of Schistosoma mansoni-infected mice

Patterns of fibrosis within hepatic and intestinal granulomas of Schistosoma mansoni-infected mice were analyzed by indirect immunofluorescence. Deposition of collagen isotypes, laminin, and fibronectin was evaluated semiquantitatively between 8 and 20 weeks of the infection. Liver granulomas were the largest at 8 weeks and contained low amounts of type I and higher amounts of type III collagen and fibronectin. Collagen deposition became pronounced as infection progressed. The relative amounts of type I collagen deposits rose and equalled that of type III. In the smaller immunomodulated granulomas at 20 weeks both types I and III were high, and type IV collagen deposition was observed. Fibronectin and laminin deposits were also detected. The small ileal granulomas did not change their size during the course of the infection. At 8 weeks, connective tissue matrix deposition was barely detectable within these lesions. Gradually, small deposits of types I and III appeared in equal amounts and attained highest levels by 20 weeks of the infection. Fibronectin deposits at that time were very prominent but laminin and type IV collagen were absent. Colon granulomas at 8 weeks of the infection were only somewhat smaller than those of the liver, yet contained very sparse deposits of types I and III collagen. During the ensuing weeks collagen deposits rose only slightly. By 20 weeks the granulomas diminished in size and within those lesions type III collagen was predominant. Whereas the presence of fibronectin was pronounced, type IV collagen and laminin were detectable only in trace amounts. These observations indicate the existence of important organ-related differences in the intragranulomatous deposition of connective tissue matrix.     

Potential use of collagen and elastin degradation markers for monitoring liver fibrosis in schistosomiasis

Liver fibrosis is a serious complication of schistosomiasis infection, is associated with increased amounts of collagen and the collagen cross-link, pyridinoline. Non-invasive markers of liver fibrosis have been developed. Serum and urinary markers of collagen synthesis and degradation have been studied to assess the balance between collagen synthesis, measured with markers of collagen synthesis such as amino-terminal propeptide of type III procollagen (PIIINP), and markers of degradation such as pyridinoline or pyridinoline cross-linked carboxyterminal telopeptide of type I collagen (ICTP). It has been shown that mice infected with Schistosomiasis mansoni excrete excess pyridinoline cross links in urine and this was correlated with the collagen content of granulomas from the liver. Treatment of infected mice with an anti-parasitic drug, praziquantel, decreased the collagen content of parenchyma and excretion of pyridinoline in the urine. Although the connective tissue protein, elastin, is present in the liver, the role of elastin in liver fibrosis has not been investigated. However, it has been shown that the urinary concentration of elastin specific crosslinks, desmosine and isodesmosine, as well as the urinary concentration of the collagen crosslink, pyridinoline, correlated well with liver fibrosis score in biopsy specimens from patients with liver disease secondary to hepatitis C virus and alcohol. Each biopsy specimen was reviewed by two pathologists who were blinded as to the clinical data. The pathological evaluation generated scores for both inflammation and fibrosis. No correlation was seen between the urinary markers and inflammation scores. The measurement of non-invasive markers of collagen synthesis and degradation may be useful in monitoring the reversal of fibrosis following therapeutic intervention in schistosome infections.     

Expression of type I and type III collagens during the course of dimethylnitrosamine-induced hepatic fibrosis in rats

ABSTRACT— Aims/Background: We wished to clarify the mechanisms that account for the increase in hepatic collagen accumulation during hepatic fibrosis. Methods: The gene expression of type I and type III procollagens and matrix metalloproteinase-1 (MMP-1) was measured by Northern blot analysis; immunolocalization of both types of collagen was estimated by indirect immunohistochemical assay; and the hepatic content of collagen and malondialdehyde (MDA), a product of lipid peroxidation, were assayed in hepatic fibrosis induced in rats with a single dose of dimethylnitrosamine (DMN). Results: During the experimental period, more type I procollagen mRNA was found than type III procollagen mRNA. The immunoreactive intensity of type I collagen was greater in necrotic areas near central veins 3 days after DMN treatment than it was on day 9, whereas the type III collagen immunodeposition for the latter period of the hepatic fibrosis was stronger than it was on day 3. As compared with controls, hepatic collagen content increased significantly after 3 days and continued, increasing gradually, as did type I and III procollagen mRNA levels. On day 14, fibrosis was greatest and both types of procollagen gene expression were at their highest, and type I and III procollagen mRNA levels and hepatic collagen content increased as the dosage of DMN was raised. MMP-1 mRNA levels increased early in hepatic fibrogenesis, and increased on day 14 when DMN dosages were low. Hepatic MDA levels increased rapidly for 3 days after DMN treatment, remaining significantly higher than control values and showing a significant increase even in response to low DMN doses on day 14. Conclusions: Our results suggested that fibrotic liver collagen content may make its first notable increase due in part to the balance between type I collagen and MMP-1 expression rates. Also, lipid peroxidation may be important in the mechanism of hepatofibrogenesis.     

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.