激活素整合素内皮素在慢性胰腺炎胰腺纤维化机制中的作用及其对策的研究
|
摘要
胰腺纤维化是与慢性胰腺炎(chronic pancreatitis)相伴随的特殊的组织病理学特点,往往导致胰腺功能性组织丧失,富含连接组织的细胞外基质(extracellularmatrix ECM)在胰腺内沉积等后果。然迄今为止,胰腺纤维化过程精确的调控机制仍未明了。
胰腺星状细胞(pancreatic Stellate cell,PSC)是产生胰腺纤维化的关键细胞,并最终导致慢性胰腺炎的形成。在此过程中有多种细胞因子参与,而各种因子的产生途径、相互作用机制不尽相同。激活素(Activin)最早是在性腺发现的糖蛋白激素,后来发现其作用不仅限于性腺,具有多种生物学功能,属于TGF-β超家族的成员,其三种受体属于丝氨酸/苏氨酸蛋白激酶。激活素参与机体炎症反应和组织修复过程,一定水平激活素不但呈剂量依赖性的促进PSC增殖并表达a平滑肌肌动蛋白(a-SMA),而且可增强其胶原蛋白分泌。内皮素(endothelin ET)作为迄今所知最强的缩血管物质,其作用机制是通过增加PSC的胶原合成和分泌,以及提高PSC的摄取与合成能力有关。整合素(Integrin)是位于细胞表面的糖蛋白受体家族分子,为介导细胞—细胞以及细胞—细胞外基质,且作为介导信号传递的膜分子,在许多重要的病理生理过程:如细胞增殖、分化、伸展和迁移、凋亡、炎症反应、组织修复和肿瘤侵袭转移等中起着十分重要的作用。
细胞因子在促进PSC活化、增殖、迁徙过程中,都要通过细胞信号传导通路对PSC进行控制。c-Jun氨基端激酶(c-Jun-terminal kinase,JNK)是丝裂原活化蛋白激酶(mitogen-activaed proren kinase,MAPK)信号传导通路家族成员。MAPK广泛参与细胞的多种生命过程,包括生长、分化、细胞周期调节,肿瘤发生,细胞凋亡等。JNK主要参与ECM的合成。
汉丹肝乐是在临床辩证的基础上,以丹参、黄芪、汉防己碱、银杏、赤芍等五味中药组方而成。已有的研究证实,它可通过减轻肝细胞脂质过氧化、保护肝细胞、抑制HSC增殖及胶原合成、提高胶原酶活性、并促进胶原蛋白降解等多种机制,来发挥抗肝纤维化效应。
本课题采用细胞生物学和分子生物学技术(包括免疫组化、RT-PCR,westernblot等),检测激活素、整合素、内皮素在胰腺组织损伤修复及纤维化形成过程中的动态变化以及应用药物干预胰腺纤维化的形成。旨在探讨激活素、整合素、内皮素在慢性胰腺炎胰腺纤维化发生、发展中的作用和细胞内传导途径以及药物干预效果。本课题主要分为三部分:(1)慢性胰腺炎大鼠模型的建立;(2)激活素、整合素、内皮素在慢性胰腺炎胰腺组织中的表达;(3)汉丹肝乐对大鼠慢性胰腺炎胰腺纤维化干预的研究。
实验方法
1、实验动物模型的建立
通过向Wistar大鼠胆胰管输注含2%三硝基苯磺酸(TNBS)的10%乙醇PBS溶液复制慢性胰腺炎动物模型,在胆胰管中注射等量的10%乙醇PBS溶液复制对照组,干预组(模型组动物)术后第三天给予口服汉丹肝乐,大鼠术后分1、2、3、4、5、6、7周等观察时点。
2、实验标本的制备
抽取血清分别应用速率法、电化学发光免疫法测定血清淀粉酶及C肽;取胰腺组织部分即刻按透视电镜观察胰腺组织超微结构要求制片后行电镜观察,部分胰腺组织行组织学标本制备,部分胰腺组织-80℃保存。
3、免疫组化
组织学标本经脱蜡、脱苯,水化后采用免疫组化法检测FN胶原、ACT-A、ET-1、Integrinα_5β_1、JNK、a-SMA,结果判定:光镜下胰腺间质出现黄、棕色为阳性染色。免疫组化半定量分析表达程度:应用彩色图象分析系统,对各组大鼠胰腺切片随机选取10个视野测量,计算阳性染色面积占胰腺视野面积比,并进行定量分析,以染色面积在标本中所占百分比表示。
4、Masson胶原染色
组织学标本经脱蜡、脱苯,水化后采用采用Masson胶原染色法行胰腺组织胶原染色,结果判断:胰腺腺泡呈红色,胶原纤维呈蓝色或绿色。胶原面积半定量分析:选取每一例标本10个不同视野下相同大小的区域,测算出胶原面积占该区域的百分比,取其平均值作为胶原的相对含量值。
5、RT-PCR
RT-PCR技术检测模型组激活素mRNA、整合素mRNA、内皮素mRNA的表达。实验所用物品的去Rnase处理,组织总RNA的提取,逆转录(RT)反应,聚合酶链反应(PCR),基因扩增产物在1.5%琼脂糖凝胶上电泳,拍照。结果分析:将凝胶电泳图像输入,自动电泳凝胶成像分析系统,进行表达强度分析,按下公式计算相对系数:相对系数=细胞因子表达强度/β-actin表达强度。每张电泳图重复进行3次,计算均数。
6、western blot(蛋白印迹)
western blot法检测α-SMA、JNK表达。组织中总蛋白的提取,聚丙稀酰胺凝胶(SDS一PAGE)电泳,转膜,印迹(blotting),将胶片图像输入凝胶成像分析系统,进行表达强度分析,按下公式计算相对系数:相对系数=蛋白表达强度/β-actin表达强度,每张图像重复进行3次,计算均数。
结果
1、实验动物大体及形态学变化
模型组大鼠术后第一周内体重明显减轻,其后体重有所增加,但与对照组相比,体重仍较轻。模型组大鼠病理形态变化:自术后第二周胰腺小叶周围及间质内有纤维化形成,第4周达高峰,第5、6、7周胰腺组织变化基本同第4周。
2、透视电镜观察
模型组第1、2周胰腺间质内可见少量活化的胰腺星状细胞(PSC),其内维生素A脂滴消失,胰腺腺胞线粒体肿胀、有多种囊泡形成。第3周胰腺组织间质内、透明活化的PSC以及成纤维细胞明显增多,间质内纤维增多,腺胞内线粒体明显肿胀、透明。第4周时,胰腺组织间质内活化的PSC、成纤维细胞、巨噬细胞明显增多,间质内可见大量的纤维成分。胰腺腺胞内粗面内质网明显扩张,腺胞体积减少,第5、6、7周胰腺组织变化基本同第4周。
3、血清学检测
模型组各时点淀粉酶测定值较对照组均明显升高,模型组C肽测定值在术后第1、2周较对照组无差异,3～7周较对照组均明显降低
4、免疫组化检测
(1)FN胶原在模型组表达逐渐上调,于第四周达高峰,明显高于干预组及对照组,干预组较模型组有所下调。模型组第5、6、7周胰腺组织变化基本同第4周。
(2)对照组:激活素在腺泡细胞、胰管壁、胰腺间质内低表达;模型组与干预组:阳性细胞多位于胰腺间质、纤维间隔区、炎症细胞浸润区表达。模型组在1-7周呈逐渐上调趋势,明显高于对照组,尤以第4周后明显,干预组较模型组有所下调.
(3)对照组:内皮素在胰腺腺泡、胰管壁有弱表达;模型组与干预组:阳性细胞多位于纤维化区域及炎症细胞浸润区。模型组分别在1、4周出现两个高峰,明显高于对照组,干预组较模型组明显下调。
(4)对照组:整合素在胰腺腺泡、胰管壁有弱表达;模型组及干预组:阳性细胞多位于纤维化区域及炎症细胞浸润区。模型组表达逐渐上调,于3,4周达高峰,随后表达下调,明显高于对照组,干预组较模型组明显下调。
(5)对照组:JNK在胰腺腺泡、胰腺间质内少量表达;模型组及干预组:阳性细胞多位于胰腺间质及纤维化区细胞浆和细胞核中,两者明显高于对照组,干预组较模型组明显下调。
(6)对照组:α-SMA少量表达在血管壁;模型组及干预组:α-SMA表达在纤维化区域及胰管周围,两者明显高于对照组,干预组较模型组明显下调。
5、胰腺组织胶原分布情况
对照组于胰腺组织纤维间隔可见少量胶原纤维染色;模型组胰腺组织中可见大量呈蓝色的胶原纤维沉积,小叶间及胰管周边集中,小叶内可见散在的胶原纤维分布,干预组较模型组小叶间及胰管周围胶原纤维沉积均明显减少,小叶内仅见少量胶原纤维沉积.
6、RT-PCR
(1)激活素mRNA在模型组表达在第1-3周时测不到,4周后逐渐升高,6-7周为最高.
(2)内皮素mRNA在模型组表达在第1、4周出现两个高峰.
(3)整合素在mRNA在模型组表达逐渐升高,第4周达高峰,随后下调.
7、western blot
(1)α-SMA在胰腺组织中蛋白表达与对照组比较:模型组、干预组α-SMA表达明显上调;α-SMA在干预组表达较模型组明显下调。
(2)JNK在胰腺组织中蛋白表达与正常对照组比较:模型组、干预组JNK表达明显上调;JNK在干预组表达较模型组明显下调。
结论
1、胆胰管内注射2%TNBS乙醇PBS溶液的方法成功复制慢性胰腺炎的动物模型。
2、本方法可反映急性胰腺炎向慢性胰腺炎转化的动态过程,符合人类慢性胰腺炎病情演变的规律。本方法造模后4周,即可满足研究需要。
3、激活素、整合素、内皮素可促进慢性胰腺炎发生纤维化过程。
4、激活素促慢性胰腺炎胰腺纤维化的作用主要发生在病程的后期,整合素、内皮素促慢性胰腺炎胰腺纤维化的作用主要发生在病程的早、中期。
5、汉丹肝乐具有抑制大鼠慢性胰腺炎胰腺纤维化的作用。
6、JNK信号传导通路在慢性胰腺炎胰腺纤维化发生过程中起重要作用。
7、内皮素、整合素、激活素对PSC的活化、增殖作用可能与JNK信号传导通路的活化有关。
8、汉丹肝乐对胰腺纤维化的干预与其减少内皮素、整合素、激活素的生成、抑制JNK信号传导通路的活化有关。
preface
Pancreatic fibrosis is a special histopathological changes of the pancreas accompanying with chronic pancreatitis,which often leads to functional losing of organization and deposition of the cell-rich connective tissue of extracellular matrix. However,to date,the precise regulatory mechanism of pancreatic fibrosis remains unclear.
Pancreatic stellate cell(PSC) is a key cell in pancreatic fibrosis,which eventually led to the formation of chronic pancreatitis.A variety of cytokines involved in this process,and different cytokines have different origins as well as mechanism of interaction.Activin was first found in the gonadal glycoprotein hormone,but it has a variety of biological functions,it belongs to TGF-βsuperfamily members,and its three types of receptor belong to serine / threonine protein kinase.Activin organism involved in inflammatory response and tissue repair process,A certain level of activin not only promote the PSC proliferation in a dose-dependent manner to expressing a smooth muscle actin(a-SMA),but also enhance its secretion of collagen.Endothelin (endothelin ET) as the strongest known vasoconstrictor substances,its mechanism associates with increasing synthesis and secretion of PSC collagen as well as improving its ability of uptake and synthesis.Integrins are glycoprotein receptor family molecules located on cell surface,as mediating membrane signaling molecules and mediating cell -cell and cell-extracellular matrix molecules they play an important role in many pathophysiological processes:such as cell proliferation,differentiation,spreading and migration,apoptosis,inflammatory response,tissue repair and tumor invasion and metastasis.
Cell factors control PSC through the cell signal-transduction pathways in the process of promoting activation,proliferation,migration of PSC.c-Jun N-terminal kinase(JNK) are signal transduction pathway members of the mitogen-activated protein kinase(MAPK) family.MAPK widely involved in variety of life processes of cells,including growth,differentiation,cell cycle regulation,tumorigenesis,apoptosis and so on.JNK mainly involved in the synthesis of ECM.
Han-Dan-Gan-Le is consisted by salvia,astragalus,Chinese Tetrandrine,gingko, flavors Chishao and other sides of the Chinese Medicines Board on the basis of the clinical differentiation.Research has confirmed that it can exert anti-liver fibrosis effect through many mechanisms,such as reducing lipid peroxidation in liver cells, protecting liver cells,inhibiting HSC proliferation and collagen synthesis,enhancing collagenase activity,promoting collagen degradation and so on.
This issue detected dynamic change of activin,integration and endothelin in the process of pancreatic tissue repair and fibrosis,and applied drug intervention of pancreatic fibrosis,by using cell biology and molecular biology techniques(including immunohistochemistry,RT-PCR,western blot,etc.).The purpose of this issue is to discuss the role of activin,integration and endothelin in the process of the occurrence and development of chronic pancreatic fibrosis,discuss the intracellular transduction pathway as well as the effect of drug intervention in this process.This topic is divided into three parts:(1) setting up rat model of chronic pancreatitis;(2),Expression activin, integration and endothelin in chronic pancreatitis tissues;(3) studying of the intervention of Khan-Dan-Gan-Le-Grand in rats with chronic pancreatitis.
methods
1、The establishment of experimental animal models
Injecting 10%ethanol solution PBS containing 2%trinitrobenzene sulfonic acid (TNBS) into pancreatic duct of Wistar rats in order to replicate animal model of chronic pancreatitis,and injecting equivalent 10%ethanol solution PBS as the control group, Three days later we give oral Han-Dan-Gan-Le to the intervention group(model group) and observe after the 1,2,3,4,5,6,7-week.
2、Preparation of experimental samples
we determinated serum amylase and C-peptide of serum samples by using rate method and electrochemiluminescence immunoassay respectively.Then,some of this pancreatic tissue was taken immediately to observe ultrastructure by the electron microscope lens,some was prepared to form histology specimens,and some was preserved in -80℃.
3、Immunohistochemistry
Dewaxing,from benzene,hydration Detecting FN collagen,ACT-A,ET-1, Integrin a5β1,JNK,a-SMA,after dewaxing,from benzene,and hydration of histology specimens through immunohistochemistry,we took this result as positive staining:the pancreas appeared interstitial yellow and brown under light microscopy.Expression degree of Immunohistochemical semi-quantitative analysis:By using color image analysis system,we selected 10 sections vision randomly to measure the pancreatic samples of rats in each group,calculated the area of positive staining area then the total pancreatic vision,and gave quantitative analysis expressed as stained area in percentage of specimens
4、Masson staining of collagen
The histological specimens of pancreatic tissue was stained by using of Masson staining of collagen method after dewaxing,froming benzene and hydration,resulting in judgments:acinar red,collagen fibers were blue or green.Semi-quantitative analysis of collagen area:selected 10 different visions of each specimen under the same size region,measured area of the collagen percentage in the region,and choose the average as the value of the relative content of collagen.
5、RT-PCR
Using RT-PCR technology we can detect the expression of.endothelin-mRNA, activin-mRNA and integrin-mRNA in the model group.After Rnase treatment of items used in the experiment,extraction of Organizational total RNA,reaction of reverse transcription(RT),polymerase chain reaction(PCR),as well as electrophoresis and photographed of gene amplificational products in 1.5%agarose gel we can give such analysis:entered the gel electrophoresis image,used automatic electrophoresis gel image analysis system to analysis the degree of expression,according to this formula to get the relative coefficient:relative coefficient = intensity of protein expression /β-actin expression.Each electrophoresis map repeated 3 times,and calculated the mean.
6、western blot
western blot detected the expression ofα-SMA,JNK.After extraction of organizational total protein,electrophoresis of gel polypropylene(SDS ONE PAGE), transferred membrane and blotting,the film image was entered to gel image analysis system for expression analysis,calculated the relative coefficient as this formula: relative coefficient = intensity of protein expression /β-actin expression.Each electrophoresis map repeated 3 times,and calculated the mean.
results
1、experimental animal general and morphology change
Model group rats body weight extremely decreased in the first week after operation,then increased later step by step,but still lighter than control group.Model group rats pathological change:fibrosis around the pancreatic foliole and the stoma,in the second week and reached pick at the forth weak,the changes of pancreatic tissue in the fifth,sixth,seventh week were similar with the forth week.
2、observation under electron microscope
model group appeared PBS cell in the pancreatic stroma in the first and second week and the Vitamin A lipid droplet disappeared in it,pancreatic cell mitochondrial swelling and multiple cyst formatted.Clearing activated PSC and fibroblasts increased markedly in pancreatic stroma at the third week,mitochondrial swelling and hyalinized apparently.In the forth week,activated PSC,fibroblasts and macrophages increased significantly in pancreatic stroma and generous fibers appeared in the stroma, pancreatic cell rough endoplasmic reticulum extended and cell volume decrease.The changes in the fifth,sixth,seventh week were the same with the forth week.
3、serum detect
model group every time point serum diastase was significantly higher than control group,C-peptide had no difference in the first and second week,buy model group became lower from the third week to the seventh week.
4、immunohistochemisty detect
(1) model group FN collagen expression increased steeply and reached pick in the forth week,significantly higher than intervention and control group.The changes of model group in the fifth,sixth,seventh week were the same with the forth week.
(2) Control group:Activin expressed low level in alveolus cell,pancreatic duct wall cell and pancreatic stroma.Model group and intervention group:positive cell located in pancreatic stroma,fibrosis septation area and inflammatory cell invasion area. Mode group increased gradually form the first to the seventh week,significantly higher than control group especially in the post 4 weeks,compared with model group the intervention group went down-regulation.
(3) Control group:endothelin slightly expressed at pancreatic alveolus cell and duct cell;model group and intervention group:positive cell located in fibrosis septation area and inflammatory cell invasion area.Model group appeared tow pick in the first and the forth week and significantly higher than control group,compared with model group the intervention group went down-regulation markedly.
(4) Control group:integrin slightly expressed at pancreatic alveolus cell and duct cell;model group and intervention group:positive cell located in fibrosis area and inflammatory cell invasion area.Model group went up-regulation step by step and appeared pick in the third or the forth week then went down and significantly higher than control group,compared with model group the intervention group went down-regulation markedly.
(5) Control group:JNK slightly expressed at pancreatic alveolus cell and duct cell;model group and intervention group:positive stain located in pancreatic storma and fibrosis area cell plasma and nucleus.Both were significantly higher than control group,compared with model group the intervention group went down-regulation markedly.
(6) Control group:a-SMA slightly expressed at blood vessel wall cell;model group and intervention group:a-SMA located in fibrosis area and around pancreatic duct.Both were significantly higher than control group,compared with model group the intervention group went down-regulation markedly.
5、pancreatic tissue collagen distribution
control group appeared slightly collagen fiber stain in the pancreatic fibrosis septation area;model group showed plenty of blue stained collagen fiber deposition, especially between foliole and parapancreatic duct,scattered distributed in the foliole, compared with model group,intervention group interfoliole and parapancreatic duct collagen fiber deposition significantly decreased,there were only a little collagen fiber deposition in the foliole.
6、RT-PCR
(1) activin mRNA could not be detected from the first to the third week in model group,progressive increased from the forth week and reached pick in the sixth or seventh week.
(2) Endothelin mRNA appeared tow pick in the first week and the forth week in model group.
(3) Integrin mRNA expression progressively increased and reached pick in the forth week,then went down.
7、western blot
(1) a-SMA protein expression in pancreatic tissue compared with control group: model and intervention group significantly up-regulation;compared with model group the intervention group went down-regulation markedly.
(2) JNK protein expression in pancreatic tissue compared with control group: model and intervention group significantly up-regulation;compared with model group the intervention group went down-regulation markedly.
Conclusion
1、Injecting 10%ethanol solution PBS containing 2%trinitrobenzene sulfonic acid (TNBS) into pancreatic duct of Wistar rats in order to replicate animal model of chronic pancreatitis witch was successful.
2、this methods can image the process of acute pancreatitis transformed into chronic pancreatitis and consistent with human chronic pancreatitis succession.It can be satisfied with any study after 4 weeks.
3、activin,Endothelin and Integrin can promote chronic pancreatitis fibrosis.
4、activin promote chronic pancreatitis fibrosis in the late stage of chronic pancreatitis,Endothelin and Integrin work in the early and middle stage.
5、Han-Dan-Gan-Le can inhibit rats chronic pancreatitis fibrosis.
6、JNK signal transport pathway does important work in the process of chronic pancreatitis fibrosis.
7、the effct of activin,Endothelin and Integrin witch can improve PSC activation and proliferation may have correlation with JNK signal transport pathway activity.
8、the effect of Han-Dan-Gan-Le witch can inhibit pancertitis fibrosis may have correlation with decrease activin,Endothelin and Integrin expresson and inhibit JNK signal transport pathway activity.
胰腺星状细胞(pancreatic Stellate cell,PSC)是产生胰腺纤维化的关键细胞,并最终导致慢性胰腺炎的形成。在此过程中有多种细胞因子参与,而各种因子的产生途径、相互作用机制不尽相同。激活素(Activin)最早是在性腺发现的糖蛋白激素,后来发现其作用不仅限于性腺,具有多种生物学功能,属于TGF-β超家族的成员,其三种受体属于丝氨酸/苏氨酸蛋白激酶。激活素参与机体炎症反应和组织修复过程,一定水平激活素不但呈剂量依赖性的促进PSC增殖并表达a平滑肌肌动蛋白(a-SMA),而且可增强其胶原蛋白分泌。内皮素(endothelin ET)作为迄今所知最强的缩血管物质,其作用机制是通过增加PSC的胶原合成和分泌,以及提高PSC的摄取与合成能力有关。整合素(Integrin)是位于细胞表面的糖蛋白受体家族分子,为介导细胞—细胞以及细胞—细胞外基质,且作为介导信号传递的膜分子,在许多重要的病理生理过程:如细胞增殖、分化、伸展和迁移、凋亡、炎症反应、组织修复和肿瘤侵袭转移等中起着十分重要的作用。
细胞因子在促进PSC活化、增殖、迁徙过程中,都要通过细胞信号传导通路对PSC进行控制。c-Jun氨基端激酶(c-Jun-terminal kinase,JNK)是丝裂原活化蛋白激酶(mitogen-activaed proren kinase,MAPK)信号传导通路家族成员。MAPK广泛参与细胞的多种生命过程,包括生长、分化、细胞周期调节,肿瘤发生,细胞凋亡等。JNK主要参与ECM的合成。
汉丹肝乐是在临床辩证的基础上,以丹参、黄芪、汉防己碱、银杏、赤芍等五味中药组方而成。已有的研究证实,它可通过减轻肝细胞脂质过氧化、保护肝细胞、抑制HSC增殖及胶原合成、提高胶原酶活性、并促进胶原蛋白降解等多种机制,来发挥抗肝纤维化效应。
本课题采用细胞生物学和分子生物学技术(包括免疫组化、RT-PCR,westernblot等),检测激活素、整合素、内皮素在胰腺组织损伤修复及纤维化形成过程中的动态变化以及应用药物干预胰腺纤维化的形成。旨在探讨激活素、整合素、内皮素在慢性胰腺炎胰腺纤维化发生、发展中的作用和细胞内传导途径以及药物干预效果。本课题主要分为三部分:(1)慢性胰腺炎大鼠模型的建立;(2)激活素、整合素、内皮素在慢性胰腺炎胰腺组织中的表达;(3)汉丹肝乐对大鼠慢性胰腺炎胰腺纤维化干预的研究。
实验方法
1、实验动物模型的建立
通过向Wistar大鼠胆胰管输注含2%三硝基苯磺酸(TNBS)的10%乙醇PBS溶液复制慢性胰腺炎动物模型,在胆胰管中注射等量的10%乙醇PBS溶液复制对照组,干预组(模型组动物)术后第三天给予口服汉丹肝乐,大鼠术后分1、2、3、4、5、6、7周等观察时点。
2、实验标本的制备
抽取血清分别应用速率法、电化学发光免疫法测定血清淀粉酶及C肽;取胰腺组织部分即刻按透视电镜观察胰腺组织超微结构要求制片后行电镜观察,部分胰腺组织行组织学标本制备,部分胰腺组织-80℃保存。
3、免疫组化
组织学标本经脱蜡、脱苯,水化后采用免疫组化法检测FN胶原、ACT-A、ET-1、Integrinα_5β_1、JNK、a-SMA,结果判定:光镜下胰腺间质出现黄、棕色为阳性染色。免疫组化半定量分析表达程度:应用彩色图象分析系统,对各组大鼠胰腺切片随机选取10个视野测量,计算阳性染色面积占胰腺视野面积比,并进行定量分析,以染色面积在标本中所占百分比表示。
4、Masson胶原染色
组织学标本经脱蜡、脱苯,水化后采用采用Masson胶原染色法行胰腺组织胶原染色,结果判断:胰腺腺泡呈红色,胶原纤维呈蓝色或绿色。胶原面积半定量分析:选取每一例标本10个不同视野下相同大小的区域,测算出胶原面积占该区域的百分比,取其平均值作为胶原的相对含量值。
5、RT-PCR
RT-PCR技术检测模型组激活素mRNA、整合素mRNA、内皮素mRNA的表达。实验所用物品的去Rnase处理,组织总RNA的提取,逆转录(RT)反应,聚合酶链反应(PCR),基因扩增产物在1.5%琼脂糖凝胶上电泳,拍照。结果分析:将凝胶电泳图像输入,自动电泳凝胶成像分析系统,进行表达强度分析,按下公式计算相对系数:相对系数=细胞因子表达强度/β-actin表达强度。每张电泳图重复进行3次,计算均数。
6、western blot(蛋白印迹)
western blot法检测α-SMA、JNK表达。组织中总蛋白的提取,聚丙稀酰胺凝胶(SDS一PAGE)电泳,转膜,印迹(blotting),将胶片图像输入凝胶成像分析系统,进行表达强度分析,按下公式计算相对系数:相对系数=蛋白表达强度/β-actin表达强度,每张图像重复进行3次,计算均数。
结果
1、实验动物大体及形态学变化
模型组大鼠术后第一周内体重明显减轻,其后体重有所增加,但与对照组相比,体重仍较轻。模型组大鼠病理形态变化:自术后第二周胰腺小叶周围及间质内有纤维化形成,第4周达高峰,第5、6、7周胰腺组织变化基本同第4周。
2、透视电镜观察
模型组第1、2周胰腺间质内可见少量活化的胰腺星状细胞(PSC),其内维生素A脂滴消失,胰腺腺胞线粒体肿胀、有多种囊泡形成。第3周胰腺组织间质内、透明活化的PSC以及成纤维细胞明显增多,间质内纤维增多,腺胞内线粒体明显肿胀、透明。第4周时,胰腺组织间质内活化的PSC、成纤维细胞、巨噬细胞明显增多,间质内可见大量的纤维成分。胰腺腺胞内粗面内质网明显扩张,腺胞体积减少,第5、6、7周胰腺组织变化基本同第4周。
3、血清学检测
模型组各时点淀粉酶测定值较对照组均明显升高,模型组C肽测定值在术后第1、2周较对照组无差异,3～7周较对照组均明显降低
4、免疫组化检测
(1)FN胶原在模型组表达逐渐上调,于第四周达高峰,明显高于干预组及对照组,干预组较模型组有所下调。模型组第5、6、7周胰腺组织变化基本同第4周。
(2)对照组:激活素在腺泡细胞、胰管壁、胰腺间质内低表达;模型组与干预组:阳性细胞多位于胰腺间质、纤维间隔区、炎症细胞浸润区表达。模型组在1-7周呈逐渐上调趋势,明显高于对照组,尤以第4周后明显,干预组较模型组有所下调.
(3)对照组:内皮素在胰腺腺泡、胰管壁有弱表达;模型组与干预组:阳性细胞多位于纤维化区域及炎症细胞浸润区。模型组分别在1、4周出现两个高峰,明显高于对照组,干预组较模型组明显下调。
(4)对照组:整合素在胰腺腺泡、胰管壁有弱表达;模型组及干预组:阳性细胞多位于纤维化区域及炎症细胞浸润区。模型组表达逐渐上调,于3,4周达高峰,随后表达下调,明显高于对照组,干预组较模型组明显下调。
(5)对照组:JNK在胰腺腺泡、胰腺间质内少量表达;模型组及干预组:阳性细胞多位于胰腺间质及纤维化区细胞浆和细胞核中,两者明显高于对照组,干预组较模型组明显下调。
(6)对照组:α-SMA少量表达在血管壁;模型组及干预组:α-SMA表达在纤维化区域及胰管周围,两者明显高于对照组,干预组较模型组明显下调。
5、胰腺组织胶原分布情况
对照组于胰腺组织纤维间隔可见少量胶原纤维染色;模型组胰腺组织中可见大量呈蓝色的胶原纤维沉积,小叶间及胰管周边集中,小叶内可见散在的胶原纤维分布,干预组较模型组小叶间及胰管周围胶原纤维沉积均明显减少,小叶内仅见少量胶原纤维沉积.
6、RT-PCR
(1)激活素mRNA在模型组表达在第1-3周时测不到,4周后逐渐升高,6-7周为最高.
(2)内皮素mRNA在模型组表达在第1、4周出现两个高峰.
(3)整合素在mRNA在模型组表达逐渐升高,第4周达高峰,随后下调.
7、western blot
(1)α-SMA在胰腺组织中蛋白表达与对照组比较:模型组、干预组α-SMA表达明显上调;α-SMA在干预组表达较模型组明显下调。
(2)JNK在胰腺组织中蛋白表达与正常对照组比较:模型组、干预组JNK表达明显上调;JNK在干预组表达较模型组明显下调。
结论
1、胆胰管内注射2%TNBS乙醇PBS溶液的方法成功复制慢性胰腺炎的动物模型。
2、本方法可反映急性胰腺炎向慢性胰腺炎转化的动态过程,符合人类慢性胰腺炎病情演变的规律。本方法造模后4周,即可满足研究需要。
3、激活素、整合素、内皮素可促进慢性胰腺炎发生纤维化过程。
4、激活素促慢性胰腺炎胰腺纤维化的作用主要发生在病程的后期,整合素、内皮素促慢性胰腺炎胰腺纤维化的作用主要发生在病程的早、中期。
5、汉丹肝乐具有抑制大鼠慢性胰腺炎胰腺纤维化的作用。
6、JNK信号传导通路在慢性胰腺炎胰腺纤维化发生过程中起重要作用。
7、内皮素、整合素、激活素对PSC的活化、增殖作用可能与JNK信号传导通路的活化有关。
8、汉丹肝乐对胰腺纤维化的干预与其减少内皮素、整合素、激活素的生成、抑制JNK信号传导通路的活化有关。
preface
Pancreatic fibrosis is a special histopathological changes of the pancreas accompanying with chronic pancreatitis,which often leads to functional losing of organization and deposition of the cell-rich connective tissue of extracellular matrix. However,to date,the precise regulatory mechanism of pancreatic fibrosis remains unclear.
Pancreatic stellate cell(PSC) is a key cell in pancreatic fibrosis,which eventually led to the formation of chronic pancreatitis.A variety of cytokines involved in this process,and different cytokines have different origins as well as mechanism of interaction.Activin was first found in the gonadal glycoprotein hormone,but it has a variety of biological functions,it belongs to TGF-βsuperfamily members,and its three types of receptor belong to serine / threonine protein kinase.Activin organism involved in inflammatory response and tissue repair process,A certain level of activin not only promote the PSC proliferation in a dose-dependent manner to expressing a smooth muscle actin(a-SMA),but also enhance its secretion of collagen.Endothelin (endothelin ET) as the strongest known vasoconstrictor substances,its mechanism associates with increasing synthesis and secretion of PSC collagen as well as improving its ability of uptake and synthesis.Integrins are glycoprotein receptor family molecules located on cell surface,as mediating membrane signaling molecules and mediating cell -cell and cell-extracellular matrix molecules they play an important role in many pathophysiological processes:such as cell proliferation,differentiation,spreading and migration,apoptosis,inflammatory response,tissue repair and tumor invasion and metastasis.
Cell factors control PSC through the cell signal-transduction pathways in the process of promoting activation,proliferation,migration of PSC.c-Jun N-terminal kinase(JNK) are signal transduction pathway members of the mitogen-activated protein kinase(MAPK) family.MAPK widely involved in variety of life processes of cells,including growth,differentiation,cell cycle regulation,tumorigenesis,apoptosis and so on.JNK mainly involved in the synthesis of ECM.
Han-Dan-Gan-Le is consisted by salvia,astragalus,Chinese Tetrandrine,gingko, flavors Chishao and other sides of the Chinese Medicines Board on the basis of the clinical differentiation.Research has confirmed that it can exert anti-liver fibrosis effect through many mechanisms,such as reducing lipid peroxidation in liver cells, protecting liver cells,inhibiting HSC proliferation and collagen synthesis,enhancing collagenase activity,promoting collagen degradation and so on.
This issue detected dynamic change of activin,integration and endothelin in the process of pancreatic tissue repair and fibrosis,and applied drug intervention of pancreatic fibrosis,by using cell biology and molecular biology techniques(including immunohistochemistry,RT-PCR,western blot,etc.).The purpose of this issue is to discuss the role of activin,integration and endothelin in the process of the occurrence and development of chronic pancreatic fibrosis,discuss the intracellular transduction pathway as well as the effect of drug intervention in this process.This topic is divided into three parts:(1) setting up rat model of chronic pancreatitis;(2),Expression activin, integration and endothelin in chronic pancreatitis tissues;(3) studying of the intervention of Khan-Dan-Gan-Le-Grand in rats with chronic pancreatitis.
methods
1、The establishment of experimental animal models
Injecting 10%ethanol solution PBS containing 2%trinitrobenzene sulfonic acid (TNBS) into pancreatic duct of Wistar rats in order to replicate animal model of chronic pancreatitis,and injecting equivalent 10%ethanol solution PBS as the control group, Three days later we give oral Han-Dan-Gan-Le to the intervention group(model group) and observe after the 1,2,3,4,5,6,7-week.
2、Preparation of experimental samples
we determinated serum amylase and C-peptide of serum samples by using rate method and electrochemiluminescence immunoassay respectively.Then,some of this pancreatic tissue was taken immediately to observe ultrastructure by the electron microscope lens,some was prepared to form histology specimens,and some was preserved in -80℃.
3、Immunohistochemistry
Dewaxing,from benzene,hydration Detecting FN collagen,ACT-A,ET-1, Integrin a5β1,JNK,a-SMA,after dewaxing,from benzene,and hydration of histology specimens through immunohistochemistry,we took this result as positive staining:the pancreas appeared interstitial yellow and brown under light microscopy.Expression degree of Immunohistochemical semi-quantitative analysis:By using color image analysis system,we selected 10 sections vision randomly to measure the pancreatic samples of rats in each group,calculated the area of positive staining area then the total pancreatic vision,and gave quantitative analysis expressed as stained area in percentage of specimens
4、Masson staining of collagen
The histological specimens of pancreatic tissue was stained by using of Masson staining of collagen method after dewaxing,froming benzene and hydration,resulting in judgments:acinar red,collagen fibers were blue or green.Semi-quantitative analysis of collagen area:selected 10 different visions of each specimen under the same size region,measured area of the collagen percentage in the region,and choose the average as the value of the relative content of collagen.
5、RT-PCR
Using RT-PCR technology we can detect the expression of.endothelin-mRNA, activin-mRNA and integrin-mRNA in the model group.After Rnase treatment of items used in the experiment,extraction of Organizational total RNA,reaction of reverse transcription(RT),polymerase chain reaction(PCR),as well as electrophoresis and photographed of gene amplificational products in 1.5%agarose gel we can give such analysis:entered the gel electrophoresis image,used automatic electrophoresis gel image analysis system to analysis the degree of expression,according to this formula to get the relative coefficient:relative coefficient = intensity of protein expression /β-actin expression.Each electrophoresis map repeated 3 times,and calculated the mean.
6、western blot
western blot detected the expression ofα-SMA,JNK.After extraction of organizational total protein,electrophoresis of gel polypropylene(SDS ONE PAGE), transferred membrane and blotting,the film image was entered to gel image analysis system for expression analysis,calculated the relative coefficient as this formula: relative coefficient = intensity of protein expression /β-actin expression.Each electrophoresis map repeated 3 times,and calculated the mean.
results
1、experimental animal general and morphology change
Model group rats body weight extremely decreased in the first week after operation,then increased later step by step,but still lighter than control group.Model group rats pathological change:fibrosis around the pancreatic foliole and the stoma,in the second week and reached pick at the forth weak,the changes of pancreatic tissue in the fifth,sixth,seventh week were similar with the forth week.
2、observation under electron microscope
model group appeared PBS cell in the pancreatic stroma in the first and second week and the Vitamin A lipid droplet disappeared in it,pancreatic cell mitochondrial swelling and multiple cyst formatted.Clearing activated PSC and fibroblasts increased markedly in pancreatic stroma at the third week,mitochondrial swelling and hyalinized apparently.In the forth week,activated PSC,fibroblasts and macrophages increased significantly in pancreatic stroma and generous fibers appeared in the stroma, pancreatic cell rough endoplasmic reticulum extended and cell volume decrease.The changes in the fifth,sixth,seventh week were the same with the forth week.
3、serum detect
model group every time point serum diastase was significantly higher than control group,C-peptide had no difference in the first and second week,buy model group became lower from the third week to the seventh week.
4、immunohistochemisty detect
(1) model group FN collagen expression increased steeply and reached pick in the forth week,significantly higher than intervention and control group.The changes of model group in the fifth,sixth,seventh week were the same with the forth week.
(2) Control group:Activin expressed low level in alveolus cell,pancreatic duct wall cell and pancreatic stroma.Model group and intervention group:positive cell located in pancreatic stroma,fibrosis septation area and inflammatory cell invasion area. Mode group increased gradually form the first to the seventh week,significantly higher than control group especially in the post 4 weeks,compared with model group the intervention group went down-regulation.
(3) Control group:endothelin slightly expressed at pancreatic alveolus cell and duct cell;model group and intervention group:positive cell located in fibrosis septation area and inflammatory cell invasion area.Model group appeared tow pick in the first and the forth week and significantly higher than control group,compared with model group the intervention group went down-regulation markedly.
(4) Control group:integrin slightly expressed at pancreatic alveolus cell and duct cell;model group and intervention group:positive cell located in fibrosis area and inflammatory cell invasion area.Model group went up-regulation step by step and appeared pick in the third or the forth week then went down and significantly higher than control group,compared with model group the intervention group went down-regulation markedly.
(5) Control group:JNK slightly expressed at pancreatic alveolus cell and duct cell;model group and intervention group:positive stain located in pancreatic storma and fibrosis area cell plasma and nucleus.Both were significantly higher than control group,compared with model group the intervention group went down-regulation markedly.
(6) Control group:a-SMA slightly expressed at blood vessel wall cell;model group and intervention group:a-SMA located in fibrosis area and around pancreatic duct.Both were significantly higher than control group,compared with model group the intervention group went down-regulation markedly.
5、pancreatic tissue collagen distribution
control group appeared slightly collagen fiber stain in the pancreatic fibrosis septation area;model group showed plenty of blue stained collagen fiber deposition, especially between foliole and parapancreatic duct,scattered distributed in the foliole, compared with model group,intervention group interfoliole and parapancreatic duct collagen fiber deposition significantly decreased,there were only a little collagen fiber deposition in the foliole.
6、RT-PCR
(1) activin mRNA could not be detected from the first to the third week in model group,progressive increased from the forth week and reached pick in the sixth or seventh week.
(2) Endothelin mRNA appeared tow pick in the first week and the forth week in model group.
(3) Integrin mRNA expression progressively increased and reached pick in the forth week,then went down.
7、western blot
(1) a-SMA protein expression in pancreatic tissue compared with control group: model and intervention group significantly up-regulation;compared with model group the intervention group went down-regulation markedly.
(2) JNK protein expression in pancreatic tissue compared with control group: model and intervention group significantly up-regulation;compared with model group the intervention group went down-regulation markedly.
Conclusion
1、Injecting 10%ethanol solution PBS containing 2%trinitrobenzene sulfonic acid (TNBS) into pancreatic duct of Wistar rats in order to replicate animal model of chronic pancreatitis witch was successful.
2、this methods can image the process of acute pancreatitis transformed into chronic pancreatitis and consistent with human chronic pancreatitis succession.It can be satisfied with any study after 4 weeks.
3、activin,Endothelin and Integrin can promote chronic pancreatitis fibrosis.
4、activin promote chronic pancreatitis fibrosis in the late stage of chronic pancreatitis,Endothelin and Integrin work in the early and middle stage.
5、Han-Dan-Gan-Le can inhibit rats chronic pancreatitis fibrosis.
6、JNK signal transport pathway does important work in the process of chronic pancreatitis fibrosis.
7、the effct of activin,Endothelin and Integrin witch can improve PSC activation and proliferation may have correlation with JNK signal transport pathway activity.
8、the effect of Han-Dan-Gan-Le witch can inhibit pancertitis fibrosis may have correlation with decrease activin,Endothelin and Integrin expresson and inhibit JNK signal transport pathway activity.
引文
11 Khokhar AS,Seidner DL.The pathophysiolgy of Pancreatilis[J].Nutr Clin Pract,2004,19(1):5.
22张汝玲,王兴鹏,慢性胰腺炎胰腺纤维化发生机制及抗纤维化治疗进展,胰腺病学,.2001,1:56-58.
3Kim CD.Pancreatilis -etiology and pathogenesis[J].Korean J Gastroenterol,2005,46(5):321.
4Buchholz M,Kestler HA,Holzmann K,et al.Transcriptome analysis of hmanhepatic and pancreatic stellate cells:organ-specific variations of a common transcriptional phenotype[J].J Mol Med,2005,83:795.
5Etemad B,Whitcom DC.Chronic pathogenesis:diagnosis,classification,and new genetic deveiopment[J].Gastroenteroly,2001;120(3):6822.
6Meili S,Graf R,Perren A et al.Secretory apparatus assessed by analysis of pancreatic secretory stress protein expression in a rat model of chronic pancreatitis[J].Cell Tissue Res,2003,312(3):291.
7Graf R,Schiesser M,Bimmler D.Increased secretion of the pancreatic secretory trypisn inhibitor,(PSTI -I,monitor peptide)during development of chronic pancreatitis in the WBN/Kob rat[J].Pancreatology,2002,2(2):108.
8Widdison AL,Alvarez C,Schwarz M,et al.The influence of ethanol on pancreatic blood flow in cats with chronic pancreatitis[J].Surgery,1992,112(2):202
9Boerma D,Straatsburg IH,Offerhaus GJ,et al.Experimental model of obstructive,chronic pancreatitis in bigs[J].Dig Surg,2003,20(6):520
10Kishi S,Takeyama Y,Ueda T,et al.pancreatic duct obstruction itself induces expression of alpha smooth muscle actin in pancreatic stellate cells[J].J Surg Res,2003,114(1):6
11Neuschwander-Tetri BA,Talkad V,et al.Otis Stephen F.Induced thrombospondin expression in the mouse pancreas during pancreatic injury[J].Int J Biochem Cell Biol,2006,38(1):102
12Westerloo DJ,Florquin S,de Boer AM,et al.Therapeutic effects of troglitazone in experimental chronic pancreatitis in mice[J].Am J Pathol,2005,166(3):721
13 Elsasser HP,Haake T,Grimming M,et al.Repeptitive caerule induced pancreatitis and pancreatic fibrosis in the rat.pancreas,1992,7(3):385-90
14 Inoue M,Ino Y,Gibo J,et al.The role of monocyte chemoattractant protein-1 in Experimental chronic pancreatitis model induced by dibutyltin dichloride in rats[J].pancreas,2002,25(4):64
15 Sparmann G,Sass S,Bohme HJ,et al.Persistence of memory type lymphocytes in an experimental model of chronic pancreatitis in rats[J].Cell Mol Biol(Noisy-le-grand),2002,48(3):309
16 Otte JM,Schwenger M,Brunke G,et al.Expression of hepatocyte growth factor,keratinocy growth factor and their receptors in experimental chronic pancreatitis[J].Eur J Clin Invest,2001,31(10):865
17 Watari N,Hotta Y,et al.Mabuchi,et al.Morphological studies on avitamin A-storing cell and its complex with macrophage observed in mouse pancreatic tissues following excess vitamin A administration.Okajimas Folia Anat jpn.1982,58:837-858
18 Lkejiri N,The vitamin A-storing rells in human and rat pancreas.kurume Med J.1990,37:67-81
19 Saotome T,Inoue H,Fujimiya A,et al.Morphological and immunocytochemical odentification of periacinar fibroblast-like cells derived from human pancreatic acini.pancreas.1997,14:373-382
20 Bachem MG,Schnneider E,Gross H,et al.Identification,culture,and characterization of pancreatic stellate cells in rats and humans.Gastroenterology.1998,115:421-432
21 Potts J R,Campbell I D.Structure and function of fibronectin modules.Matrix Biol.l996,15:313～320.
22 Magnusson M K,Mosher D F.Fibronectin:structure,assembly,and cardiovascular implication.Arterioscler Thromb Vase Biol,1998,18:1363-1370
23 Beagiey Kw,Black CA,Elson CO.Strain differentces in susceptibility to TNBS-induced colitis(abstract).Gastroenterology.1991,100.A560
24Grisham MB,Volkmer C,Tso P,et al.Metabolism of trinityorbenzene sulfonic acid by the rat colon produces reactive oxygen specis.Gastroenterology.1991,101:540-547
25Alder G,and Schmid RM.Chronic pancreatitis:still puzzling?Gastroenterology.1997,112(5):1762-1765
26Koto Y,et al.Gastroenterol.1996,31(4):565-571
27SaotomeT,et al.Pancreas.1997,14(4):373-382
28Apte MV,et al.Gut.1998,13(1):128-133
29Lau AL,KumarTR,NishimorK,et al.ActivinbetaE genesare notessential formouse liver growth,differentiation,and regeneration[J].MolCellBiol,2000,20(18):6127-6137
30OhnishiN,MiyataT,OhnishiH,et al.Activin Aisanautocrine activattorof rat pancreatic stellate cells otential the rapeuticrole offollis statin for pancreatic fibrosis[J].Gut,2003,52(10):1487-1493
31Liu ZH,ShintaniY,WakatsukiM,et al.Regulation of immunoreative activin Asecretion from cultured rat anter iorpituitary cells[J].EndocrineJ.1996,43(1):39
32黄新,李定国,陆汉明,等.激活素和卵泡抑素mRNA在肝纤维化形成过程中的作用[J].中华肝脏病杂志,2002,10(2):85-87
33Shing O,et al.Integrin function:Molecular hiearchies of cytoskeletal and signaling molecules.J-cell-Biol,1995,131(3):791-805
34Longhursc CM,et al.Cell Mol Life Sci,1998,54:514-526
35Tuckwell D,Humphries M Integrin collagen binding.Semin Cell Develop Biol,1996,7:649-657
36ejjari M,Couvelard A,Mosnier JF,et al.Integrin up-regulation in chronic liver disease:relationship with inflammation and fibrosis in chronic hepatitis C.J Pathol,2001,195:473-481
37Pinzani M,Marra F.Cytokine receptors and signaling in hepatic stellate cells.Semin Liver Dis,2001,21:397-416
38Mcnamee.H.P,et al.Adhesion to fibronectin stimulates inositol lipid synthesis and enhances PDGF-induced inositol lipid breakdown.J-cell-Biol,1995,121:673-8
39Mould A.P.Regulation of integrin by a_5β_1-fibronectin interaction divalent cations.J Biol Chern,1995,270(44):26270-77
40Zhu M,et al.Studies on the mechanism of the selenite-induced decrease in cell attachment:effect of selenite on the levels of fibronection receptor mRNAs.Biol Trace Elem Res,1998;62(3):123-34
41Gopalakrishna-p,et al.Modulation of alpha5betal integrin functions by the phospholipid and cholesterol contents of cell membrances.J cell Biochem,2000,77(4):517-28
42Miranti CK et al.Nat Cell Biol,2002,4(4):E83-90
43Yanagisawa M,Kurihara H,Kimura S,et al.A novel potent vasoconstrictor peptide produced by vascular endothelial cells[J].Nature,1988,332(6163):411-415
44Shi-Wen X,Denton CP,Dashwood MR,et al.Fibroblast matrix gene expression and connective tissue remodeling:role of endothelin-1 J Invest Dermatol,2001,116:417-425
45Koracs EJ,Dipietro LA.Fibrogenic cytokines and connective tissue production.FASEB J,1994,8:854
46Batlistini B,Chailler P,Dorleans-Juste P,et al.Growth regulatory properties of endothelins.Peptides,1993,14:385
47黄志芳,朱妙珍,等.内皮素1对人肾小球基质金属蛋白酶3及其组织抑制剂1表达的影响[J].中华肾脏病杂志,2003,19(3):182-183
48Reinehr RM,Kubitz R,Peters-Regehr T,Bode JG,Haussinger D.Activation of rat heptic stellate cells in culture is associated with increased sensitivity to endothelin.Hepatology,1998,28:1566-1577
49Cho JJ,Hocher B,Herbst H,Jia JD,Ruehl M,Hahn EG,Riecken EO,Schuppan D.An oral endothelin-A receptor antagonist blocks collagen synihesis and deposition in advanced rat liver fibrosis.Gastroenterotogy.2000,118:1169-1176
50Plusczyk T,Bersal B,Menger M D,et al.Differential effects of ET-1,ET-2 and ET-3 on pancreatic microcirculation,tissue integrity,and inflammation[J].Dig Dis Sci,2001,46(6):1343
51陆萌英、陆彤、程明亮等.汉丹肝乐对猪血清免疫性纤维化人鼠胶原酶活性的影响.中华 肝脏病杂志,2000,8(2):108-109
52张惠娜,李诚秀,黄能慧,等.汉丹肝乐对免疫性肝纤维化的预防作用[J].贵州医药,2001,25(5):415
53程明亮,陆彤.a_3型基在工程干扰素和汉丹冲剂治疗早期肝硬化的组织学研究[J].中国中西医结合杂志,1995,15(5):300-303
54Apte MV,Haber PS,Applegate TL,et al.Periacinar stellate shaped cells in rat pancreas-Identification,isolation,and culture.Gut 1998,43:128-133
55Schneider E,Schmid-Kotsas A,Zhao J,et al.Identification,of mediators stimulating proliferation and matrix synthesis of rat pancreatic stellate cells.Am J Physiol Cell Physiol 2001,281:C532-543
56Svegliati BG,Ridolfi F,Di Sario A,et al.Intracellular signaling pathways involved in acetaldehyde-induced collagen and fibronectin gene-expression in human hepetic stellate cells.Hepatology 2001,33:1130-1140
57Schnabl B,Kweon YO,Frederick JP,Wang XF,RippeRA,Brermer DA.The role of Smad3 in mediating mouse hepatic stellate cellactivation.Hepatology 2001,34:89-100
58Lcotado R,Arthur MJ,Iredate JP.Pathogenesis of liver fibrosis.Clin Sci 1997,92:103-112
59Robino G,Parola M,Marra F,et al.Interaction between 4-hydroxy-2,3-alkenals and the platelet-derived growth factor-beta receptor.Reduced tyrosine phosphorylation and downstream signaling in hepatic stellate cells.J Biol Chem 2000;275:40561-40567
60Matsuda N,Horikawa M,Wang LH,et al.Differential activation of ERK1/2 and JNK in normal human fibroblast-like cells in response to UVC radiation under different oxygen tensions.Photochem Photobiol 2000;72:334-339
61Shimohashi N,Nakamuta M,Uchimura K,et al.Selenoorganic compound,ebselen,inhibits nitric oxide and tumor necrosis factor-alpha phoduction by the modulation of Jun-N-terminal knase and the NF-kappab signaling pathway in rat Kupffer cells.J Cell Biochem 2000;78:595-596
62Cano E,Mahadevan LC.Parallel signal procesing among mammalian MAPKs.TIBS 1995;20:117-122
63Seger,EG.The MAPK signal cascde.FASEB J 1995;9:726
64Abe J,Kusuhara M,Ulevitch RJ,et al.Big mitogen activated protein kinase(BMK1) is a redox-sensitive kinase.J Biol Chem 1996;271:16586
65Lee M,Yea SS.Hydrogen peroxide inhibits the immune response to lipopolysac charide by attenuating signaling through c-jun N-terminal kinase and p38 associated with protein kinase C.Immunopharmacology 2000;48:165-172
66Tournier C,Whitmarsh AJ,Cavanagh J,et al.Mitogen-activated protein kinase7 isan activator of the c-jun NH2-terminal kinase.Proc Natl Acad Sci USA,1997,94(14):7337
67Derijard B,Hibi M,Wu IH,et al.JNK1:a protein kinases timulated by UV lightand Ha-rasthat binds and phosphory lates the c-jun activation domain.Cell,1994,76(6):1025
68Gupta S,Campbell D,Derijard B,et al.Transcription factor ATF2 regulation by the JNK signal transduction pathway.Science,1995,267(5196):389
69Svegliati-Baroni G,Ridolfi F,et al.Regulation of ERK/JNK/P70S6K in two rat models of liver injury and fibrosis.J Hepatol.2003 Oct;39(4):528-37
70Schnabl B,Bradham CA,Bennett BL,et aI.TAK1/JNK and p38 have opposite effects on rat hepatic stellate cells.Hepatology 2001 Nov;34(5):953-63
71Anania FA,Womack L,Jiang MD,et al.Aldehydes potentiate alpha(2)-1 collagen gene activity by JNK in hepatic stellate cells.Free Radic Bial Med 2001;30(8):451
72马学惠,韩德伍,赵元吕等.丹参对肝纤维化重吸收的作用.中西医结合杂志,1998,8:23-25
73张文胜,程明亮,陆萌英.中药复方对肝纤维化大鼠细胞因子的影响.中华肝脏病杂志,2003,11(5):285-288
74李诚秀,罗俊,李玲等.汉丹肝乐治疗肝纤维的作用研究.中国医药学报,1998,13:199
75Bataller R,Gabele E,Schoonhoven R,et al.Prolonged infusion of arrgiotensin Ⅱ into normal rats induces stellate cell activation and proinflammatory events in liver[J].Am J Physiol Gastrointest Liver Physiol,2003,285(3):642-651
1 Stevens T,Conwell DL,Zueearo G.Pathogenesis of chronic pancreatitis:an evidence-based review of pasttheories and recent developments.Am J Gastroenterol 2004;99:2256-2270
2 Apte MV,Haber PS,Applegate TL,Norton ID,McCaughan GW,Korsten MA,Pirola RC,Winlson JS.Periacinar stellate shaped cell in rat pancreas:identification isolation,and culture.Gut 1998;43:128-133
3 Bachem M,Schneider H,Weidenbach H,Scmid RM,Menke A,Beger H,Grunert A,Adler G.Identification,culture and characteri-zation of characterization of pancreatic stellate cells in rate and humans.Gastroenterology 1998;115:421-432
4 Haber PA,Keogh GW,Apte MV.Moran CS,Stewart NL,Crawford DH,Pirola RC,McCaughan GW,Ramm GA,Wilson IS.Activation of pancreatic stellate cells I human and experimental pancertic fibrosis.Am J Pathol 1999;155:1087-1095
5 Apte MV,Haber PS,Darby SJ,Rodgers SC,McCaughan GW,Korsten MA,Piorla RC,Wilson JS.Pancreatic stellate cell are activated by proinflammatory cytokines:implications for pancreatic fibrogenesis.Gut 1999;44:534-541
6 MasamuneA,Satoh M,Kikuta K,Suzuki N,Shimosegawa T.Establishment and charaeterization of a rat Pancreatic stel-Late cell line by spontaneous immortalization.World J Gastroenterol2003;9:2751-2758
7 Kikuta K,Masamune A.Satoh M,Suzuki N,Shimosegawa T.4-hydroxy-2,3-nonenal activates activator protein-1 and mi-togen-activated protein kinases in rat pancreatic stellate cells.World J Gastroenterol,2004;10:2344-2351
8 watari N,Hotta Y,Mabuehi Y.Morphological studies on a Vitamin A-storing cell and its complex with macrophage ob-Served in mouse pancreatic tissues following excess vitamin A administration.Okaiimas Folia Anat Jpn 1982;58:837-858
9 Ikejire N.The vitamin A-storing cells in the huaman and rat pancreas.Kurume Med J 1990;37:67-81
10 Wells RG,Crawford JM.Pancreatic stellate cells:the new stars Of chronic pancreatitis? Gastroenterology!998;115:491-493
11 Sehuppan D,Koda M,Bauer M,Hahn EG.Fibrosis of liver,pancreas and intestinercommon mechanisms and clear targets? Acta Gastroenterol Belg 2000;63:366-370
12 Mews P,phillips P,Fahmy R,Korsten M,pirola R,Wilson J,Apte M.Pancreatic stellate cells respond to inflammatory cytokines:potential role in chronie panereatitis.Gut 2002;50:
13 535-541
14 BerberatPO,FriessH,BuehierMW.Chronic pancreatitis-new Pathophysiological concepts.Swiss Surg 2000;6:227-230
15 Menke A,Geerling I,Giehi K,Vogelmann R,Reinshagen M,
16 Adler G.Transforming growth faetor-beta-indueed upregulation of transforming growth factor-beta receptor expression in pancreatic regeneration.Biochim Biopys Acta 1999;1449:178-185
17 Ebert M,KasPer HU,Hernberg S,Friess H,Buchler MW,Roessner A,Korc M,Malfertheiner P.Overexpression of platelet-derived growth factor(PDGF)B chain and type beta PDGF receptor in human chronic pancretitis.Dig Dis Scil998;43:567-574
18 Xie MJ,Motoo Y,Su SB,Sawabu N.Expression of tumor necrosis factor-alpha,interleukin-6 and interferon-gamma in spont-aneous chronic pancreatitis in the WBN/Kob rat.Pancreas 2001;22:400-408
19 Hanck C,Rossol S,Hartmann A,Singer MV.Cytokine gene expression in peripheral blood mononuclear cells reflects a systemic immune response in alcoholic chronic pancreatitis.Int J Pancreatol 1999;26:137-145
20 Marra F,Gentillini A,Pinzani M,Choudhury GG,Parola M,Herbst H,Dianzani MU,Laffi G,Abboud HE,Gentilini P.Phosphatidylinositol 3-kinase is required for platelet-derived growth factor's aetions on hepatic stellate cells.Gastroenterology 1997;112:1297-1306
21 Slater SD,Williamson RC,Foster CS.Expression of transform-ing growth factor beta 1 in chronic pancreatitis.Digestion 1995;56:237-241
22 Vogelmann R,Ruf D,Wagner M,Adler G,Menke A.Effects of fibrogenic mediators on the development of pancreatic fibrosis in a TGF-pi transgenic mouse model.Am J Physiol Gastrointest Liver Physiol 2001;280:G164-G 172
23 Kruse ML,Hildebrand PB,Tilnke C,Folseh UR,Sehmidt WE.TGFpl autocrine growth control in isolated pancreatic fibro-blastoid cells/stellate cells in vitro.Regul Pept 2000;90:47-52
24 Schneider E,Schmid-Kotsas A,Zhao J,Weidenbach H,Schmid RM,Menke A,Adler G,Waltenberger J,Grunert A,Baehem MG.Identiflcation of mediators stimulating proliferation and matrix synthesis of rat pancreatic stellate cells.Am J Physiol Cell Physiol 2001;281:C532-C543
25 Claesson-WelshL.Platelet—derived growth factor receptor signals.J Biol Chem 1994;269:32023-32026
26 Carloni V,Pinzani M,Giusti S,Romanelh RG,Parola M,Bellomo G,Faiili P,Hamilton AD,Sebti SM,Laffi G,Gentilini P.Tyrosine phosphorylation of focal adhesion kinase by PDGF is dependent on ras in human hepatic stellate cells.Hepatology 2000;31:131-140
27 Lewis TS,Shapiro PS,Ahn SG.Signal transduction through MAP kinase cascades.Adu Cancer Res 1998;74:49-139
28 26.Luttenberger T,Schmld-Kotasis A,Menke A,Siech M,Beger H,Adler G,Grunert A,Baehem MG.Platelet-derived growth factors stimulate proliferation and extracellular matrix synthesis of pancreatic stellate cells implications in pathogenesis of pancreas fibrosis.Lab Invset 2000;80:47-55
29 Di Sebastiano P,Di Mola FF,Di Febbo C,Baccante G,Porreca E,Innocenti P,Friess H,BuehierMV.Expression of interleukin
30 8(IL-8)and substance P in human chronic pancreatitis.Gut 2000;47:423-428
31 Madro A,Cellnski K,Slomka M.The role of pancreatic stellate cells and cytokines in the development of chronic pancreatitis.Med Sci Monit 2004;10:RA166-RA 170
32 Cook JW,Karakozis S,Kim D,Provido H,Gongora E,KirkPatriek JR.Interleukin—10 attenuates proinflammatory cytokine production and improves survival inlethal pancreatitis.Am Surg2001;67:237 241
33 WangSC,OhataM,Sehrum L,RippeRA,Tsukamoto H.Expression of interleukin 10 by in vitro and in vivo activated hepatic stellate cells.J Biol Chem 1998;273:302-308
34 Demols A,Van Laethem JL,Quertinmont E,Degraef C,Delhaye M,Geerts A,Deviere J.Endogenous interleukin-10 modulates fibrosis and regeneration in experimental chronic pancreatitis.Am J Physiol Gastrointest Liver Physiol 2002;282:G1105-G1112
35 Saxena NK,Ikeda K,Roekey DC,Friedman SL,Anania FA.Leptin in hepatic fibrosisrevidence for increased collagen production in stellate cells and lean littermates of ob/ob mice.Hepatology 2002;35:762-771
36 Ikejima K,Takei Y,Honda H,Hirose M,Yoshikawa M,Zhang YJ,Lang T,Fukuda T,Yamashina S,Kitamura T,Sato N.Leptin receptor-mediated signaling regulates hepatic fibrogenesis and remodeling of extracellular matrix in the rat.Gastro-enterology 2002;122:1399-1410
37 Potter JJ,Mezey E.Leptin deficiency reduces but does not eliminate the development of hepatic fibrosis in mice infected with schistosoma mansoni.Liver 2002;22:173-177
38 Lang T,Dcejima K,Yoshikawa M,Enomoto N,Iijima K,Kitamura T,Takei Y,Sato N.Leptin facilitates proliferation of hepatic stellate cells through up-regulation of platelet-derived growth factor receptor.Biochem Biophys Res Commun 2004;323:1091—1095
39 Baldwin AS Jr.The NF-KB and 1 KB Proteins:new discoveries and insights.Annu Rev Immunol 1996;14:469-481
40 Ghosh S,May MJ,Kopp EB.NF-KB and Rel proteins:evolu-tionary conserved mediators of immune responses.Annu Reu Immunol 1998;16:225—260
41 Karin M,Ben-eriah Y.Phosphorylation meets ubiquitination:The control of NF-KB aetivity.Annu Rev Immunol 2000;18:621-663
42 Ohmori Y,Fukumoto S,Hamilton TA.Two structurally distinct KB sequence motifs cooperatively control LpS-induced KC gene transcription in mouse macrophages.J Immunol 1995;155:3593-3600
43 Wulczyn FG,Krappmann D,Scheidereit C.The NF—KB /Rel and I KB gene families:mediators of immune response and in-flammation.J Mol Med 1996;74:749-769
44 Hellerbrand C,Jobin C,Licato LL,Sartor RB,Brenner DA.Cytokines induec NF-KB in activated but not in quiescent rat hepatic stellate cells.Am J Physiol1998;275(2Ptl):G269-G278 42.HellerbrandC,Jobin C,limuro Y,Licato L,Sartor RB,Brenner DA.Inhibition of NF-KB in activated rat hepatic stellate cells by proteasome inhibitors and an I KB super-repressor.Hepa-tology 1998;27:1285-1295
45 43.Elsharkawy AM,Wright MC,Hay RT,Arthur MJ,Hughes T,Bahr MJ,Degitz K,Mann DA.Persistent activation of nuclearetor-kappa B in cultured rat hepatic stellate cells involves the induction of potentially novel Rel-like factors and prolonged changes in the expression of 1KB family proteins.Hepatology 1999;30:761-769
46 44.Gukovsky I,Gukovskaya AS,Blinman TA,Zaninovic V,Pandol SJ.Early NF-KB activation is associated with hormone-induced pancreatitis.Am J Physiol 1998;275(6Pt1):G 1402-G1414
47 45.Evans RM.The steroid and thyroid hormone receptor super-family.Science 1988;240:889-895
48 46.Tontonoz P.Hu E,Spiegelman BM.Stimulation of adipogenesis in fibroblasts by PPAR gamma2,a lipid-activated transcription factor.Cell 1994;79:1147-1156
49 47.Forman BM,Tontonoz P,Chen J,Bran RP,Spiegelman BM,Evans RM.15-Deoxy-deltal2,14-prostaglandin J2 is a ligand for the adipocyte determination faetor PPAR gamma.Cell 995;83:803-812 48.Masamune A,Kikuta K,Satoh M,Sakai Y,Satoh A,Shirnosegawa T.Ligands of peroxisome proliferator-activated receptor-ybock activation of pancreatic stellate cells.J Biol Chem 2002;277:141-147
50 49.Ellenrieder V,Sehneiderhan W,Bachm M,Adler G.Fibrogenesis in the pancreas.Roez Akad Med Bialymst 2004;49:40-46
51 50.Jaster R.Molecular regulation of panereatic stellate cell function.Mol Cancer 2004;3:26
52 51.Shimizu K,Shiratori K,Kobayashi M,Kawamata H.Troglitazone inhibits the progression of chronic pancreatitis and the pro-fibrogenic activity of pancreatic stellate cells via a PPAR gamma-independent mechanism.Pancreas 2004;29:67-74
53 52.Masamune A,Kikuta K,Satoh M,Suzuki N,Shimosegawa T.Pro-tease-activated receptor-2-mediated proliferation and colla-gen production of rat pancreatic stellate cells.J Pharmacol ExP Ther 2005;312:651-658
54 53.McCarroll JA,Phillips PA,Kumar RK,Park S,Pirola RC,Wilson JS,Apte MV.Pancreatic stellate cell migration:role of the Phosphatidylinositol 3-kinase(PI3-kinase)pathway.Biochem Pharmacol 2004;67:1215-1225
55 54,Ohnishi H,Miyata T,Yasuda H,Satoh Y,Hanatsuka K,Kita H,Ohashi A,TamadaK,Makita N,Iiri T,Ueda N,Mashima H Sugano K.Distinct roles of Smad2-,Smad3-,and ERK-dependent pathways in transforming growth factor-betal regulation of pancreatic stellate cellular functions.J Biol Chem 2004;279:8873-8878
22张汝玲,王兴鹏,慢性胰腺炎胰腺纤维化发生机制及抗纤维化治疗进展,胰腺病学,.2001,1:56-58.
3Kim CD.Pancreatilis -etiology and pathogenesis[J].Korean J Gastroenterol,2005,46(5):321.
4Buchholz M,Kestler HA,Holzmann K,et al.Transcriptome analysis of hmanhepatic and pancreatic stellate cells:organ-specific variations of a common transcriptional phenotype[J].J Mol Med,2005,83:795.
5Etemad B,Whitcom DC.Chronic pathogenesis:diagnosis,classification,and new genetic deveiopment[J].Gastroenteroly,2001;120(3):6822.
6Meili S,Graf R,Perren A et al.Secretory apparatus assessed by analysis of pancreatic secretory stress protein expression in a rat model of chronic pancreatitis[J].Cell Tissue Res,2003,312(3):291.
7Graf R,Schiesser M,Bimmler D.Increased secretion of the pancreatic secretory trypisn inhibitor,(PSTI -I,monitor peptide)during development of chronic pancreatitis in the WBN/Kob rat[J].Pancreatology,2002,2(2):108.
8Widdison AL,Alvarez C,Schwarz M,et al.The influence of ethanol on pancreatic blood flow in cats with chronic pancreatitis[J].Surgery,1992,112(2):202
9Boerma D,Straatsburg IH,Offerhaus GJ,et al.Experimental model of obstructive,chronic pancreatitis in bigs[J].Dig Surg,2003,20(6):520
10Kishi S,Takeyama Y,Ueda T,et al.pancreatic duct obstruction itself induces expression of alpha smooth muscle actin in pancreatic stellate cells[J].J Surg Res,2003,114(1):6
11Neuschwander-Tetri BA,Talkad V,et al.Otis Stephen F.Induced thrombospondin expression in the mouse pancreas during pancreatic injury[J].Int J Biochem Cell Biol,2006,38(1):102
12Westerloo DJ,Florquin S,de Boer AM,et al.Therapeutic effects of troglitazone in experimental chronic pancreatitis in mice[J].Am J Pathol,2005,166(3):721
13 Elsasser HP,Haake T,Grimming M,et al.Repeptitive caerule induced pancreatitis and pancreatic fibrosis in the rat.pancreas,1992,7(3):385-90
14 Inoue M,Ino Y,Gibo J,et al.The role of monocyte chemoattractant protein-1 in Experimental chronic pancreatitis model induced by dibutyltin dichloride in rats[J].pancreas,2002,25(4):64
15 Sparmann G,Sass S,Bohme HJ,et al.Persistence of memory type lymphocytes in an experimental model of chronic pancreatitis in rats[J].Cell Mol Biol(Noisy-le-grand),2002,48(3):309
16 Otte JM,Schwenger M,Brunke G,et al.Expression of hepatocyte growth factor,keratinocy growth factor and their receptors in experimental chronic pancreatitis[J].Eur J Clin Invest,2001,31(10):865
17 Watari N,Hotta Y,et al.Mabuchi,et al.Morphological studies on avitamin A-storing cell and its complex with macrophage observed in mouse pancreatic tissues following excess vitamin A administration.Okajimas Folia Anat jpn.1982,58:837-858
18 Lkejiri N,The vitamin A-storing rells in human and rat pancreas.kurume Med J.1990,37:67-81
19 Saotome T,Inoue H,Fujimiya A,et al.Morphological and immunocytochemical odentification of periacinar fibroblast-like cells derived from human pancreatic acini.pancreas.1997,14:373-382
20 Bachem MG,Schnneider E,Gross H,et al.Identification,culture,and characterization of pancreatic stellate cells in rats and humans.Gastroenterology.1998,115:421-432
21 Potts J R,Campbell I D.Structure and function of fibronectin modules.Matrix Biol.l996,15:313～320.
22 Magnusson M K,Mosher D F.Fibronectin:structure,assembly,and cardiovascular implication.Arterioscler Thromb Vase Biol,1998,18:1363-1370
23 Beagiey Kw,Black CA,Elson CO.Strain differentces in susceptibility to TNBS-induced colitis(abstract).Gastroenterology.1991,100.A560
24Grisham MB,Volkmer C,Tso P,et al.Metabolism of trinityorbenzene sulfonic acid by the rat colon produces reactive oxygen specis.Gastroenterology.1991,101:540-547
25Alder G,and Schmid RM.Chronic pancreatitis:still puzzling?Gastroenterology.1997,112(5):1762-1765
26Koto Y,et al.Gastroenterol.1996,31(4):565-571
27SaotomeT,et al.Pancreas.1997,14(4):373-382
28Apte MV,et al.Gut.1998,13(1):128-133
29Lau AL,KumarTR,NishimorK,et al.ActivinbetaE genesare notessential formouse liver growth,differentiation,and regeneration[J].MolCellBiol,2000,20(18):6127-6137
30OhnishiN,MiyataT,OhnishiH,et al.Activin Aisanautocrine activattorof rat pancreatic stellate cells otential the rapeuticrole offollis statin for pancreatic fibrosis[J].Gut,2003,52(10):1487-1493
31Liu ZH,ShintaniY,WakatsukiM,et al.Regulation of immunoreative activin Asecretion from cultured rat anter iorpituitary cells[J].EndocrineJ.1996,43(1):39
32黄新,李定国,陆汉明,等.激活素和卵泡抑素mRNA在肝纤维化形成过程中的作用[J].中华肝脏病杂志,2002,10(2):85-87
33Shing O,et al.Integrin function:Molecular hiearchies of cytoskeletal and signaling molecules.J-cell-Biol,1995,131(3):791-805
34Longhursc CM,et al.Cell Mol Life Sci,1998,54:514-526
35Tuckwell D,Humphries M Integrin collagen binding.Semin Cell Develop Biol,1996,7:649-657
36ejjari M,Couvelard A,Mosnier JF,et al.Integrin up-regulation in chronic liver disease:relationship with inflammation and fibrosis in chronic hepatitis C.J Pathol,2001,195:473-481
37Pinzani M,Marra F.Cytokine receptors and signaling in hepatic stellate cells.Semin Liver Dis,2001,21:397-416
38Mcnamee.H.P,et al.Adhesion to fibronectin stimulates inositol lipid synthesis and enhances PDGF-induced inositol lipid breakdown.J-cell-Biol,1995,121:673-8
39Mould A.P.Regulation of integrin by a_5β_1-fibronectin interaction divalent cations.J Biol Chern,1995,270(44):26270-77
40Zhu M,et al.Studies on the mechanism of the selenite-induced decrease in cell attachment:effect of selenite on the levels of fibronection receptor mRNAs.Biol Trace Elem Res,1998;62(3):123-34
41Gopalakrishna-p,et al.Modulation of alpha5betal integrin functions by the phospholipid and cholesterol contents of cell membrances.J cell Biochem,2000,77(4):517-28
42Miranti CK et al.Nat Cell Biol,2002,4(4):E83-90
43Yanagisawa M,Kurihara H,Kimura S,et al.A novel potent vasoconstrictor peptide produced by vascular endothelial cells[J].Nature,1988,332(6163):411-415
44Shi-Wen X,Denton CP,Dashwood MR,et al.Fibroblast matrix gene expression and connective tissue remodeling:role of endothelin-1 J Invest Dermatol,2001,116:417-425
45Koracs EJ,Dipietro LA.Fibrogenic cytokines and connective tissue production.FASEB J,1994,8:854
46Batlistini B,Chailler P,Dorleans-Juste P,et al.Growth regulatory properties of endothelins.Peptides,1993,14:385
47黄志芳,朱妙珍,等.内皮素1对人肾小球基质金属蛋白酶3及其组织抑制剂1表达的影响[J].中华肾脏病杂志,2003,19(3):182-183
48Reinehr RM,Kubitz R,Peters-Regehr T,Bode JG,Haussinger D.Activation of rat heptic stellate cells in culture is associated with increased sensitivity to endothelin.Hepatology,1998,28:1566-1577
49Cho JJ,Hocher B,Herbst H,Jia JD,Ruehl M,Hahn EG,Riecken EO,Schuppan D.An oral endothelin-A receptor antagonist blocks collagen synihesis and deposition in advanced rat liver fibrosis.Gastroenterotogy.2000,118:1169-1176
50Plusczyk T,Bersal B,Menger M D,et al.Differential effects of ET-1,ET-2 and ET-3 on pancreatic microcirculation,tissue integrity,and inflammation[J].Dig Dis Sci,2001,46(6):1343
51陆萌英、陆彤、程明亮等.汉丹肝乐对猪血清免疫性纤维化人鼠胶原酶活性的影响.中华 肝脏病杂志,2000,8(2):108-109
52张惠娜,李诚秀,黄能慧,等.汉丹肝乐对免疫性肝纤维化的预防作用[J].贵州医药,2001,25(5):415
53程明亮,陆彤.a_3型基在工程干扰素和汉丹冲剂治疗早期肝硬化的组织学研究[J].中国中西医结合杂志,1995,15(5):300-303
54Apte MV,Haber PS,Applegate TL,et al.Periacinar stellate shaped cells in rat pancreas-Identification,isolation,and culture.Gut 1998,43:128-133
55Schneider E,Schmid-Kotsas A,Zhao J,et al.Identification,of mediators stimulating proliferation and matrix synthesis of rat pancreatic stellate cells.Am J Physiol Cell Physiol 2001,281:C532-543
56Svegliati BG,Ridolfi F,Di Sario A,et al.Intracellular signaling pathways involved in acetaldehyde-induced collagen and fibronectin gene-expression in human hepetic stellate cells.Hepatology 2001,33:1130-1140
57Schnabl B,Kweon YO,Frederick JP,Wang XF,RippeRA,Brermer DA.The role of Smad3 in mediating mouse hepatic stellate cellactivation.Hepatology 2001,34:89-100
58Lcotado R,Arthur MJ,Iredate JP.Pathogenesis of liver fibrosis.Clin Sci 1997,92:103-112
59Robino G,Parola M,Marra F,et al.Interaction between 4-hydroxy-2,3-alkenals and the platelet-derived growth factor-beta receptor.Reduced tyrosine phosphorylation and downstream signaling in hepatic stellate cells.J Biol Chem 2000;275:40561-40567
60Matsuda N,Horikawa M,Wang LH,et al.Differential activation of ERK1/2 and JNK in normal human fibroblast-like cells in response to UVC radiation under different oxygen tensions.Photochem Photobiol 2000;72:334-339
61Shimohashi N,Nakamuta M,Uchimura K,et al.Selenoorganic compound,ebselen,inhibits nitric oxide and tumor necrosis factor-alpha phoduction by the modulation of Jun-N-terminal knase and the NF-kappab signaling pathway in rat Kupffer cells.J Cell Biochem 2000;78:595-596
62Cano E,Mahadevan LC.Parallel signal procesing among mammalian MAPKs.TIBS 1995;20:117-122
63Seger,EG.The MAPK signal cascde.FASEB J 1995;9:726
64Abe J,Kusuhara M,Ulevitch RJ,et al.Big mitogen activated protein kinase(BMK1) is a redox-sensitive kinase.J Biol Chem 1996;271:16586
65Lee M,Yea SS.Hydrogen peroxide inhibits the immune response to lipopolysac charide by attenuating signaling through c-jun N-terminal kinase and p38 associated with protein kinase C.Immunopharmacology 2000;48:165-172
66Tournier C,Whitmarsh AJ,Cavanagh J,et al.Mitogen-activated protein kinase7 isan activator of the c-jun NH2-terminal kinase.Proc Natl Acad Sci USA,1997,94(14):7337
67Derijard B,Hibi M,Wu IH,et al.JNK1:a protein kinases timulated by UV lightand Ha-rasthat binds and phosphory lates the c-jun activation domain.Cell,1994,76(6):1025
68Gupta S,Campbell D,Derijard B,et al.Transcription factor ATF2 regulation by the JNK signal transduction pathway.Science,1995,267(5196):389
69Svegliati-Baroni G,Ridolfi F,et al.Regulation of ERK/JNK/P70S6K in two rat models of liver injury and fibrosis.J Hepatol.2003 Oct;39(4):528-37
70Schnabl B,Bradham CA,Bennett BL,et aI.TAK1/JNK and p38 have opposite effects on rat hepatic stellate cells.Hepatology 2001 Nov;34(5):953-63
71Anania FA,Womack L,Jiang MD,et al.Aldehydes potentiate alpha(2)-1 collagen gene activity by JNK in hepatic stellate cells.Free Radic Bial Med 2001;30(8):451
72马学惠,韩德伍,赵元吕等.丹参对肝纤维化重吸收的作用.中西医结合杂志,1998,8:23-25
73张文胜,程明亮,陆萌英.中药复方对肝纤维化大鼠细胞因子的影响.中华肝脏病杂志,2003,11(5):285-288
74李诚秀,罗俊,李玲等.汉丹肝乐治疗肝纤维的作用研究.中国医药学报,1998,13:199
75Bataller R,Gabele E,Schoonhoven R,et al.Prolonged infusion of arrgiotensin Ⅱ into normal rats induces stellate cell activation and proinflammatory events in liver[J].Am J Physiol Gastrointest Liver Physiol,2003,285(3):642-651
1 Stevens T,Conwell DL,Zueearo G.Pathogenesis of chronic pancreatitis:an evidence-based review of pasttheories and recent developments.Am J Gastroenterol 2004;99:2256-2270
2 Apte MV,Haber PS,Applegate TL,Norton ID,McCaughan GW,Korsten MA,Pirola RC,Winlson JS.Periacinar stellate shaped cell in rat pancreas:identification isolation,and culture.Gut 1998;43:128-133
3 Bachem M,Schneider H,Weidenbach H,Scmid RM,Menke A,Beger H,Grunert A,Adler G.Identification,culture and characteri-zation of characterization of pancreatic stellate cells in rate and humans.Gastroenterology 1998;115:421-432
4 Haber PA,Keogh GW,Apte MV.Moran CS,Stewart NL,Crawford DH,Pirola RC,McCaughan GW,Ramm GA,Wilson IS.Activation of pancreatic stellate cells I human and experimental pancertic fibrosis.Am J Pathol 1999;155:1087-1095
5 Apte MV,Haber PS,Darby SJ,Rodgers SC,McCaughan GW,Korsten MA,Piorla RC,Wilson JS.Pancreatic stellate cell are activated by proinflammatory cytokines:implications for pancreatic fibrogenesis.Gut 1999;44:534-541
6 MasamuneA,Satoh M,Kikuta K,Suzuki N,Shimosegawa T.Establishment and charaeterization of a rat Pancreatic stel-Late cell line by spontaneous immortalization.World J Gastroenterol2003;9:2751-2758
7 Kikuta K,Masamune A.Satoh M,Suzuki N,Shimosegawa T.4-hydroxy-2,3-nonenal activates activator protein-1 and mi-togen-activated protein kinases in rat pancreatic stellate cells.World J Gastroenterol,2004;10:2344-2351
8 watari N,Hotta Y,Mabuehi Y.Morphological studies on a Vitamin A-storing cell and its complex with macrophage ob-Served in mouse pancreatic tissues following excess vitamin A administration.Okaiimas Folia Anat Jpn 1982;58:837-858
9 Ikejire N.The vitamin A-storing cells in the huaman and rat pancreas.Kurume Med J 1990;37:67-81
10 Wells RG,Crawford JM.Pancreatic stellate cells:the new stars Of chronic pancreatitis? Gastroenterology!998;115:491-493
11 Sehuppan D,Koda M,Bauer M,Hahn EG.Fibrosis of liver,pancreas and intestinercommon mechanisms and clear targets? Acta Gastroenterol Belg 2000;63:366-370
12 Mews P,phillips P,Fahmy R,Korsten M,pirola R,Wilson J,Apte M.Pancreatic stellate cells respond to inflammatory cytokines:potential role in chronie panereatitis.Gut 2002;50:
13 535-541
14 BerberatPO,FriessH,BuehierMW.Chronic pancreatitis-new Pathophysiological concepts.Swiss Surg 2000;6:227-230
15 Menke A,Geerling I,Giehi K,Vogelmann R,Reinshagen M,
16 Adler G.Transforming growth faetor-beta-indueed upregulation of transforming growth factor-beta receptor expression in pancreatic regeneration.Biochim Biopys Acta 1999;1449:178-185
17 Ebert M,KasPer HU,Hernberg S,Friess H,Buchler MW,Roessner A,Korc M,Malfertheiner P.Overexpression of platelet-derived growth factor(PDGF)B chain and type beta PDGF receptor in human chronic pancretitis.Dig Dis Scil998;43:567-574
18 Xie MJ,Motoo Y,Su SB,Sawabu N.Expression of tumor necrosis factor-alpha,interleukin-6 and interferon-gamma in spont-aneous chronic pancreatitis in the WBN/Kob rat.Pancreas 2001;22:400-408
19 Hanck C,Rossol S,Hartmann A,Singer MV.Cytokine gene expression in peripheral blood mononuclear cells reflects a systemic immune response in alcoholic chronic pancreatitis.Int J Pancreatol 1999;26:137-145
20 Marra F,Gentillini A,Pinzani M,Choudhury GG,Parola M,Herbst H,Dianzani MU,Laffi G,Abboud HE,Gentilini P.Phosphatidylinositol 3-kinase is required for platelet-derived growth factor's aetions on hepatic stellate cells.Gastroenterology 1997;112:1297-1306
21 Slater SD,Williamson RC,Foster CS.Expression of transform-ing growth factor beta 1 in chronic pancreatitis.Digestion 1995;56:237-241
22 Vogelmann R,Ruf D,Wagner M,Adler G,Menke A.Effects of fibrogenic mediators on the development of pancreatic fibrosis in a TGF-pi transgenic mouse model.Am J Physiol Gastrointest Liver Physiol 2001;280:G164-G 172
23 Kruse ML,Hildebrand PB,Tilnke C,Folseh UR,Sehmidt WE.TGFpl autocrine growth control in isolated pancreatic fibro-blastoid cells/stellate cells in vitro.Regul Pept 2000;90:47-52
24 Schneider E,Schmid-Kotsas A,Zhao J,Weidenbach H,Schmid RM,Menke A,Adler G,Waltenberger J,Grunert A,Baehem MG.Identiflcation of mediators stimulating proliferation and matrix synthesis of rat pancreatic stellate cells.Am J Physiol Cell Physiol 2001;281:C532-C543
25 Claesson-WelshL.Platelet—derived growth factor receptor signals.J Biol Chem 1994;269:32023-32026
26 Carloni V,Pinzani M,Giusti S,Romanelh RG,Parola M,Bellomo G,Faiili P,Hamilton AD,Sebti SM,Laffi G,Gentilini P.Tyrosine phosphorylation of focal adhesion kinase by PDGF is dependent on ras in human hepatic stellate cells.Hepatology 2000;31:131-140
27 Lewis TS,Shapiro PS,Ahn SG.Signal transduction through MAP kinase cascades.Adu Cancer Res 1998;74:49-139
28 26.Luttenberger T,Schmld-Kotasis A,Menke A,Siech M,Beger H,Adler G,Grunert A,Baehem MG.Platelet-derived growth factors stimulate proliferation and extracellular matrix synthesis of pancreatic stellate cells implications in pathogenesis of pancreas fibrosis.Lab Invset 2000;80:47-55
29 Di Sebastiano P,Di Mola FF,Di Febbo C,Baccante G,Porreca E,Innocenti P,Friess H,BuehierMV.Expression of interleukin
30 8(IL-8)and substance P in human chronic pancreatitis.Gut 2000;47:423-428
31 Madro A,Cellnski K,Slomka M.The role of pancreatic stellate cells and cytokines in the development of chronic pancreatitis.Med Sci Monit 2004;10:RA166-RA 170
32 Cook JW,Karakozis S,Kim D,Provido H,Gongora E,KirkPatriek JR.Interleukin—10 attenuates proinflammatory cytokine production and improves survival inlethal pancreatitis.Am Surg2001;67:237 241
33 WangSC,OhataM,Sehrum L,RippeRA,Tsukamoto H.Expression of interleukin 10 by in vitro and in vivo activated hepatic stellate cells.J Biol Chem 1998;273:302-308
34 Demols A,Van Laethem JL,Quertinmont E,Degraef C,Delhaye M,Geerts A,Deviere J.Endogenous interleukin-10 modulates fibrosis and regeneration in experimental chronic pancreatitis.Am J Physiol Gastrointest Liver Physiol 2002;282:G1105-G1112
35 Saxena NK,Ikeda K,Roekey DC,Friedman SL,Anania FA.Leptin in hepatic fibrosisrevidence for increased collagen production in stellate cells and lean littermates of ob/ob mice.Hepatology 2002;35:762-771
36 Ikejima K,Takei Y,Honda H,Hirose M,Yoshikawa M,Zhang YJ,Lang T,Fukuda T,Yamashina S,Kitamura T,Sato N.Leptin receptor-mediated signaling regulates hepatic fibrogenesis and remodeling of extracellular matrix in the rat.Gastro-enterology 2002;122:1399-1410
37 Potter JJ,Mezey E.Leptin deficiency reduces but does not eliminate the development of hepatic fibrosis in mice infected with schistosoma mansoni.Liver 2002;22:173-177
38 Lang T,Dcejima K,Yoshikawa M,Enomoto N,Iijima K,Kitamura T,Takei Y,Sato N.Leptin facilitates proliferation of hepatic stellate cells through up-regulation of platelet-derived growth factor receptor.Biochem Biophys Res Commun 2004;323:1091—1095
39 Baldwin AS Jr.The NF-KB and 1 KB Proteins:new discoveries and insights.Annu Rev Immunol 1996;14:469-481
40 Ghosh S,May MJ,Kopp EB.NF-KB and Rel proteins:evolu-tionary conserved mediators of immune responses.Annu Reu Immunol 1998;16:225—260
41 Karin M,Ben-eriah Y.Phosphorylation meets ubiquitination:The control of NF-KB aetivity.Annu Rev Immunol 2000;18:621-663
42 Ohmori Y,Fukumoto S,Hamilton TA.Two structurally distinct KB sequence motifs cooperatively control LpS-induced KC gene transcription in mouse macrophages.J Immunol 1995;155:3593-3600
43 Wulczyn FG,Krappmann D,Scheidereit C.The NF—KB /Rel and I KB gene families:mediators of immune response and in-flammation.J Mol Med 1996;74:749-769
44 Hellerbrand C,Jobin C,Licato LL,Sartor RB,Brenner DA.Cytokines induec NF-KB in activated but not in quiescent rat hepatic stellate cells.Am J Physiol1998;275(2Ptl):G269-G278 42.HellerbrandC,Jobin C,limuro Y,Licato L,Sartor RB,Brenner DA.Inhibition of NF-KB in activated rat hepatic stellate cells by proteasome inhibitors and an I KB super-repressor.Hepa-tology 1998;27:1285-1295
45 43.Elsharkawy AM,Wright MC,Hay RT,Arthur MJ,Hughes T,Bahr MJ,Degitz K,Mann DA.Persistent activation of nuclearetor-kappa B in cultured rat hepatic stellate cells involves the induction of potentially novel Rel-like factors and prolonged changes in the expression of 1KB family proteins.Hepatology 1999;30:761-769
46 44.Gukovsky I,Gukovskaya AS,Blinman TA,Zaninovic V,Pandol SJ.Early NF-KB activation is associated with hormone-induced pancreatitis.Am J Physiol 1998;275(6Pt1):G 1402-G1414
47 45.Evans RM.The steroid and thyroid hormone receptor super-family.Science 1988;240:889-895
48 46.Tontonoz P.Hu E,Spiegelman BM.Stimulation of adipogenesis in fibroblasts by PPAR gamma2,a lipid-activated transcription factor.Cell 1994;79:1147-1156
49 47.Forman BM,Tontonoz P,Chen J,Bran RP,Spiegelman BM,Evans RM.15-Deoxy-deltal2,14-prostaglandin J2 is a ligand for the adipocyte determination faetor PPAR gamma.Cell 995;83:803-812 48.Masamune A,Kikuta K,Satoh M,Sakai Y,Satoh A,Shirnosegawa T.Ligands of peroxisome proliferator-activated receptor-ybock activation of pancreatic stellate cells.J Biol Chem 2002;277:141-147
50 49.Ellenrieder V,Sehneiderhan W,Bachm M,Adler G.Fibrogenesis in the pancreas.Roez Akad Med Bialymst 2004;49:40-46
51 50.Jaster R.Molecular regulation of panereatic stellate cell function.Mol Cancer 2004;3:26
52 51.Shimizu K,Shiratori K,Kobayashi M,Kawamata H.Troglitazone inhibits the progression of chronic pancreatitis and the pro-fibrogenic activity of pancreatic stellate cells via a PPAR gamma-independent mechanism.Pancreas 2004;29:67-74
53 52.Masamune A,Kikuta K,Satoh M,Suzuki N,Shimosegawa T.Pro-tease-activated receptor-2-mediated proliferation and colla-gen production of rat pancreatic stellate cells.J Pharmacol ExP Ther 2005;312:651-658
54 53.McCarroll JA,Phillips PA,Kumar RK,Park S,Pirola RC,Wilson JS,Apte MV.Pancreatic stellate cell migration:role of the Phosphatidylinositol 3-kinase(PI3-kinase)pathway.Biochem Pharmacol 2004;67:1215-1225
55 54,Ohnishi H,Miyata T,Yasuda H,Satoh Y,Hanatsuka K,Kita H,Ohashi A,TamadaK,Makita N,Iiri T,Ueda N,Mashima H Sugano K.Distinct roles of Smad2-,Smad3-,and ERK-dependent pathways in transforming growth factor-betal regulation of pancreatic stellate cellular functions.J Biol Chem 2004;279:8873-8878