【作者】 陆新良；

【导师】 姒健敏； 蔡建庭；

【作者基本信息】 浙江大学 ， 内科学， 2009， 博士

【摘要】 胰腺星状细胞（pancreatic stellate cells,PSCs）位于胰腺小叶间和腺泡周围区,围绕邻近腺细胞基底部,与慢性胰腺炎和胰腺癌密切相关的一类肌成纤维细胞样细胞。在未损伤的胰腺组织中,胰腺星状细胞呈静止状态,胞内富含Vit A脂滴和油脂酸视黄醇,具有摄取和酯化视黄醇的能力,结蛋白（desmin）和胶质纤维酸性蛋白（GFAP）染色阳性。当胰腺受损后,胰腺星状细胞活化,发生形态学和功能学的双重变化,表现为:细胞体积变大,增殖活跃;脂质丢失;仅α-平滑肌肌动蛋白（alpha-SMA）表达阳性;对细胞因子如血小板衍生生长因子（PDGF）、转化生长因子-β（TGF-β）反应性增加;分泌细胞外基质（ECM）,尤其是Ⅰ、Ⅲ型胶原。慢性胰腺炎（chronic pancreatitis,CP）是指由于各种不同病因引起的胰腺组织和功能的持续性损害,其特征为胰腺基本结构发生永久性改变,广泛纤维化,即使病因已去除仍常伴胰腺的功能性缺陷。临床上以反复发作的上腹部疼痛和胰腺外分泌功能不全为主要特征,可合并胰腺内分泌功能不全、胰腺实质钙化、胰管结石和胰腺假性囊肿形成。慢性胰腺炎病程迁延,临床表现多样,严重影响患者的日常生活。并且有报道认为慢性胰腺炎是胰腺癌的危险因素之一。但目前对慢性胰腺炎的病因、发病机制尚不甚清楚,慢性胰腺炎的治疗更为棘手。胰腺纤维化的形成是一个由复杂的信号网络通路所介导的、以胰腺星状细胞活化为中心、多种细胞因子与炎性介质参与的、最终以成纤维细胞增生和细胞外基质（ECM）沉积为特征的复杂的病理发展过程。胰腺星状细胞活化是胰腺纤维化发生的关键步骤,因此抑制胰腺星状细胞活化在抗胰腺纤维化过程中占有重要地位。体内外实验均证实细胞因子、乙醇及其代谢产物乙醛、氧应激等能促进胰腺星状细胞活化。活化的胰腺星状细胞分泌ECM成分,促进胰腺纤维化形成。在胰腺星状细胞活化通路中,丝裂原激活蛋白激酶（mitogen-activated protein kinase,MAPKs）信号传递途径是最为重要一条。ERK通路的主要功能是介导有丝分裂信号向胞核传递,调控细胞的生长。Jaster等在研究细胞外信号调节激酶1/2（ERK1/2）通路与PSC活化的关系时,发现不同转化阶段的PSC都能持续表达磷酸化的Smads;血小板衍生生长因子（PDGF）在促进PSC增殖的同时增加ERK通路成分的表达;应用Trapidil或PD98059（MAPK激酶的抑制剂）均可以抑制PSC的增殖。这也证实ERK通路在PSC的活化中起到了重要作用。丹酚酸B（Salvianolic acid B,Sal-B）,又称丹参酚酸B或丹酚酸乙,是唇形科植物丹参Salvia Miltiorrhiza Bge.的根及根茎提取而得,为三分子丹参素与一分子咖啡酸缩合而成。丹酚酸B具有活血化瘀,通经活络之功效,是目前研究较多的丹酚酸之一,对心、脑、肝、肾等器官均具有重要药理作用。药理学研究发现丹酚酸B具有很强的抗氧化、抗纤维化、抗肿瘤作用,抑制血小板聚集和抑制血栓形成作用,并能延长缺氧条件下动物的存活时间。实验证明,丹酚酸B能抑制TGF-β的肝星状细胞胞（HSC）内信号转导及其受体蛋白的表达,拮抗TGF-β的促HSC活化,还能抑制HSC增殖和胶原合成。我们试图通过丹酚酸B灌胃治疗大鼠慢性胰腺炎模型,观察血液生化指标、胰腺病理改变、胰腺星状细胞激活状态和胶原蛋白的表达,初步阐明丹酚酸B对慢性胰腺炎的保护作用及可能机制。并且通过丹酚酸B干预大鼠胰腺星状细胞,观察胰腺星状细胞活化,胶原蛋白和细胞外信号调节激酶（ERK1/2）表达水平,进一步阐明丹酚酸B对胰腺星状细胞的保护作用及机制。方法:1)动物模型建立与胰腺星状细胞评价雄性大鼠行胆胰管注射2%TNBS乙醇磷酸盐缓冲液诱导慢性胰腺炎模型,分别于术后2周4周和8周处死大鼠行胰腺病理学检查,采用文献报道的胰腺组织学评分方法,主要评价胰腺腺泡细胞萎缩,纤维化和炎症细胞浸润;免疫组化检测胰腺星状细胞活化的情况;RT-PCR检测Ⅰ胶原蛋白mRNA表达水平。同时设立相应的对照组。2)丹酚酸B动物试验雄性大鼠行胆胰管注射2%TNBS乙醇磷酸盐缓冲液诱导慢性胰腺炎模型,分为模型对照组（组1）和丹酚酸B治疗组（组2）,组3是胆胰管注射乙醇磷酸盐缓冲液的对照组（非模型对照组）,组4是假手术组。30只大鼠胆胰管注射2%TNBS乙醇磷酸盐缓冲液诱导慢性胰腺炎模型,12只大鼠胆胰管注射乙醇磷酸盐缓冲液,12只大鼠接受假手术。每组各取10只大鼠进入正式实验,第5周开始,组2接受每天10mg/kg体重的丹酚酸B灌胃治疗,组1、组3和组4接受等体积的生理盐水灌胃,共治疗8周。治疗结束时抽取血标本,分离血清,行血清生化和纤维化指标检测;同时分离胰腺称重,并取胰腺组织中性福尔马林固定用于形态学研究;免疫组化检测胰腺星状细胞活化的情况;取部分胰腺组织抽提总RNA检测Ⅰ胶原蛋白mRNA表达水平。3)细胞增殖抑制试验采用MTT方法,取对数生长期的细胞PSC（LTC-14,德国Robert Jaster教授涝?,用0.25%胰酶消化为单个细胞,作细胞计数,以培养液稀释为终浓度为2×10⁴/ml的单细胞悬液,每孔190ul接种于96孔培养板中,置37℃、5%CO₂培养箱中过夜,待细胞贴壁后进行试验。将稀释为不同浓度的丹酚酸B 10ul分别加入各孔中（终浓度为1μmol/L,10μmol/L,100μmol/L）,每个浓度设5个复孔,并设只加细胞不加药液的对照孔和只加培养液的调零孔。继续培养48小时后,进行MTT检测。选择570nm波长,在免疫酶标仪上比色测定OD值,记录结果。4）丹酚酸B细胞试验取对数生长期LTC-14细胞,消化为单个细胞,以培养液稀释为终浓度为1×10⁵/ml的单细胞悬液,每孔接种4×10⁴的单细胞悬液于6孔培养板中,置37℃、5%CO₂培养箱中过夜,待细胞贴壁后进行试验。将稀释为不同浓度的丹酚酸B加入各孔中（终浓度为1μmol/L,10μmol/L,100μmol/L）,以等体积的PBS作为对照。培养48小时后收集细胞抽提总RNA行RT-PCR检测alpha-平滑肌肌动蛋白和Ⅰ型胶原蛋白mRNA表达水平。免疫印迹检测Ⅰ型胶原蛋白和细胞外信号调节激酶（ERK1/2）表达水平。结果:1)动物模型建立与胰腺星状细胞评价胆胰管注射TNBS成功诱导慢性胰腺炎,术后第2周胰腺组织有炎症细胞浸润,未见明显萎缩和纤维化,免疫组化提示少量alpha-平滑肌肌动蛋白阳性细胞,第4周胰腺组织表面苍白、不规则、结节状,胆胰管明显扩张,内含淡黄色液体;HE染色光镜观察胰管周围、小叶间和小叶内广泛纤维化,伴炎症细胞浸润,局灶腺体萎缩,免疫组化明显alpha-平滑肌肌动蛋白阳性细胞,第8周胰腺组织改变与第4周相似,但病理该变更严重,alpha-平滑肌肌动蛋白阳性细胞更多。术后第2周胰腺组织Ⅰ型胶原蛋白mRNA水平已经升高,第4周胰腺组织Ⅰ型胶原蛋白mRNA水平明显升高,第8周升高幅度更大。2)丹酚酸B动物试验（1）丹酚酸B改善大鼠慢性胰腺炎模型病理形态:第12周末,模型对照组大鼠胰腺腺泡萎缩、间质水肿、炎症细胞浸润和纤维化明显。丹酚酸B可以明显改善胰腺组织病理损害。与模型对照组和丹酚酸B治疗组比较,非模型对照组和假手术组大鼠胰腺无或仅有轻微病理损害。模型对照组大鼠胰腺的组织病理学评分明显高于丹酚酸B治疗组（p＜0.01）。丹酚酸B治疗组大鼠胰腺的腺泡萎缩和纤维化评分明显低于模型对照组（p＜0.05）,而炎症细胞浸润评分没有差别（p＞0.05）。（2）丹酚酸B抑制大鼠慢性胰腺炎模型的胰腺星状细胞活化:模型对照组大鼠胰腺中有明显的alpha-SMA阳性细胞。与模型对照组和丹酚酸B治疗组比较,非模型对照组和假手术组大鼠胰腺仅有少数alpha-SMA阳性细胞。丹酚酸B治疗组大鼠胰腺的alpha-SMA阳性细胞明显少于模型对照组。（3）丹酚酸B抑制慢性胰腺炎模型胰腺组织Ⅰ胶原蛋白mRNA表达:与模型对照组比较,丹酚酸B可以明显抑制胰腺组织Ⅰ胶原蛋白mRNA表达（p＜0.01）。与非模型对照组和假手术组大鼠相比,模型对照组大鼠的胰腺组织Ⅰ胶原蛋白mRNA水平明显升高（p＜0.01）,而丹酚酸B治疗组大鼠的胰腺组织Ⅰ胶原蛋白mRNA升高水平没有统计学差异（p＞0.05）。3)胰腺星状细胞增殖抑制试验根据MTT结果,丹酚酸B1μmol/L组对PSC细胞没有明显的增殖抑制作用（p＞0.05）,而丹酚酸B10μmol/L和100μmol/L组与对照组相比,丹酚酸B对PSC细胞有明显的增殖抑制作用（p＜0.01）。4)丹酚酸B细胞试验（1）丹酚酸B抑制胰腺星状细胞alpha平滑肌肌动蛋白和Ⅰ型胶原蛋白mRNA表达:与对照组相比,丹酚酸B组胰腺星状细胞alpha平滑肌肌动蛋白和Ⅰ型胶原蛋白mRNA表达明显下调,而且随着丹酚酸B1μmol/L,10μmol/L和100μmol/L逐步升高,胰腺星状细胞alpha平滑肌肌动蛋白和Ⅰ型胶原蛋白mRNA表达逐步下调。（2）丹酚酸B抑制胰腺星状细胞Ⅰ型胶原蛋白表达:与对照组相比,丹酚酸B1μmol/L组胰腺星状细胞Ⅰ型胶原蛋白表达没有下调,但丹酚酸B10μmol/L和100μmol/L组胰腺星状细胞Ⅰ型胶原蛋白表达明显下调,且随着丹酚酸B浓度升高,胰腺星状细胞Ⅰ型胶原蛋白表达逐步下调。（3）丹酚酸B抑制胰腺星状细胞细胞外信号调节激酶（ERK1/2）表达:与对照组相比,丹酚酸B组胰腺星状细胞细胞外信号调节激酶（ERK1/2）表达明显下调,而且随着丹酚酸B1μmol/L,10μmol/L和100μmol/L逐步升高,胰腺星状细胞细胞外信号调节激酶（ERK1/2）表达逐步下调。结论:1)胆胰管注射法诱导的慢性胰腺炎模型基本符合人类慢性胰腺炎特征,胰腺星状细胞在慢性胰腺炎发生发展中起重要作用;2)丹酚酸B通过抑制胰腺星状细胞细胞外信号调节激酶活性,降低alpha平滑肌肌动蛋白mRNA表达水平和抑制胰腺星状细胞活化;3)丹酚酸B通过抑制胰腺星状细胞活化,减少Ⅰ胶原蛋白和改善慢性胰腺炎大鼠模型胰腺组织病理损害。更多还原

【Abstract】 Stellate cells derive their name from their shape and are also present in several organs,including the liver,kidney,pancreas and lung.Cells in the pancreas that were similar to hepatic stellate cells（HSCs） in that they were fat-storing cells were first observed with the use of autofluorescence and electron microscopy in 1982. Subsequently,two landmark reports described the isolation and initial characterization of what have henceforth been termed pancreatic stellate cells（PSCs）.PSCs express intermediate filament proteins that usually characterize several cell types-for example, desmin,which characterizes myocytes;GFAP,which characterizes astrocytes;vimentin, which characterizes cells such as leukocytes,fibroblasts,and endothelial cells;and nestin,which characterizes neuroepithelial stem cells.The expression of such a diversity of intermediate filament proteins highlights that PSCs have a broad range of potential properties,including contractility,the presence of cellular extensions to sense their environment,the potential to elaborate ECM components,and the potential to proliferate.However,it is important to note that these markers have clear limitations and that there are species differences.Activation of quiescent PSCs,which occurs when primary PSCs are cultured and in the pancreas as a consequence of pancreatic injury,is associated with several morphologic changes,including nuclear enlargement and enhanced prominence of the endoplasmic reticulum network.Furthermore,in situ hybridization and immunohistochemical studies indicated that activated PSCs express alpha-SMA（also known as ACTA2） and collagen typeⅠ,therefore marking these cells as a source of fibrosis in chronic pancreatitis and pancreatic adenocarcinoma.Chronic pancreatitis is a progressive chronic inflammatory disease of the pancreas characterized by glandular atrophy,ductal changes,and extensive fibrosis.Although the clinical,morphological,and etiological characteristics of chronic pancreatitis are well known,the pathogenic mechanism has remained elusive.Pancreatic fibrosis is regulated by a balance between production and degradation of the ECM.ECM secreted by PSCs is degraded by matrix metalloproteinases（MMPs）,and their activity is blocked by tissue inhibitors of matrix metalloproteinases（TIMPs）.Multiple studies have identified several major signaling pathways involved in the regulation of PSC function. Mitogen-activated protein kinase（MAPKs） are key mediators of activating signals initiated by growth factors,angiotensinⅡ,and ethamol.Other signaling pathways regulating PSC activation include PI3K,RHO kinase,the JAK/STAT pathways,the activator protein-1 and NF-κB pathways,and the TGF-β/SMAD-related pathways.Salvia miltiorrhiza（also known as Danshen）,one of the well-known Chinese herbal medicines,is officially listed in the Chinese Pharmacopoeia and used as an important component of certain recipes for treatment of cardiovascular disorders as well as inflammatory diseases such as arthritis,and chronic hepatitis.Salvianolic acid B（Sal-B）, one of water soluble component from Danshen,its molecular formula C36H30O16 with MW718,is the major and most active radical scavenging and antioxidant from Danshen. Previous studies have showed that Sal-B can effectively reverse liver fibrosis induced by tetrachloride carbon or di-methylnitrosamine,ameliorate oxidative damage, eliminate ROS accumulation in hepatocytes,and attenuate hepatic stellate cells activation,potentially conferring hepatoprotective and anti-fibrogenic effects.As we all known,there is no effective therapy for chronic pancreatitis,and little progress has recently been made in the field of diagnosis and therapy of chronic pancreatitis.On the basis of the above-mentioned evidence,we reasoned that Sal-B might prevent or mitigate chronic pancreatitis.Materials and Methods:1) Induction of chronic pancreatitis and activation of pancreatic stellate cell in modelChronic pancreatitis was induced using the method described Puig-Divi et al. Animals were sacrificed at the end of two,four and eight weeks.Pancreas were quickly removed,portions immediately snap-frozen and stored at -80℃until the analyses were carried out.Collagen typeⅠmRNA was evaluated by reverse transcriptase-polymerase chain reaction（RT-PCR）.Other parts were fixed in 10%formalin and used for light microscopic using a previously described scoring system:（0=absent,1=a lesion slightly shown in the 2-3 lobules,2=a lesion widely shown in less than one haif lobules,and 3=a lesion shown in more than one half lobules or with destruction of lobular architecture;overall results are expressed as the total scores of rats assigned to each of the three histologic grades）.And the activation of PSCs was evaluated by immunohistochemical technique.The rats received 0.4 ml solvent（phosphate-buffered saline,PBS,pH 8.0 with 10%ethanol） were used as controls.2) Treatment of salvianolic acid B in modelChronic pancreatitis was induced using the same method.Thirty rats in the experimental chronic pancreatitis groups（including group 1 and group 2）,12 rats in the solvent treated group and 12 rats in the sham-operation group were used in order to reach 10 rats in each group.The rats in the group 3 received 0.4 ml solvent,and the rats in the group 4 were sham-operation without TNBS or solvent.Each group included 10 animals.Rats that died within four weeks after TNBS（group 1 and group 2） or solvent （group 3） administration were replaced with new ones to maintain 10 animals in each group until the end of four weeks.Mortality rate of the experimental groups（including group 1 and group 2） was 13%（4/30）,and there were no death in other groups.Because chronic pancreatitis develops two to three weeks after TNBS administration,treatment was started at the beginning of five weeks.The dose of Sal-B was daily 10mg/kg body weight for rats（i.e.10 times adult clinic therapeutic dose）.At the beginning of five weeks,the rats in the group 2 was treated with about 2 ml of Sal-B by garage,and the rats in other groups received the same volume of water for eight weeks.One rat from the group 1,which died between four and twelve weeks,was excluded from the study.Body weight was monitored weekly.Animals were sacrificed at the end of twelve weeks.Serum was kept at -20℃until the assays were carried out using an automatic biochemical analyzer according to the instructions of the manual（TBA-40FR,Tokyo,Japan）.Pancreas were quickly removed, freed from fat and lymph nodes and weighted.Portions immediately snap-frozen and stored at -80℃until the analyses were carried out.Collagen typeⅠmRNA was evaluated by reverse transcriptase-polymerase chain reaction（RT-PCR）.Other parts were fixed in 10%formalin and used for light microscopic using the same scoring system.And the activation of PSCs was evaluated by immunohistochemical technique.3) Cell proliferation assayCell growth experiments were performed using the 3-（4,5-methylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide（MTT） assay.LTC-14（A present from Professor Robert Jaster） were counted and seeded at a density of 3800 cells/well onto 96-well plates containing Iscoves modified Dulbeccos medium（IMDM） supplement with 10%fetal bovine serum,non-essential amino acid,and cultured at 37℃in a humidified environment containing 5%CO2.After treatment with various concentrations of salvianolic acid B for 48 hours,twenty microliter of MTT（5mg/ml） was added to each well for an additional 4 hours.Formazan products were solubilized with dimethyl sulphoxide（DMSO）,and the optical density was measured at 570 nm.4) Treatment of salvianolic acid B in PSCLTC-14 cells were counted and seeded at a density of 4×10⁴ cells/well onto 6-well plates containing IMDM supplement with 10%fetal bovine serum,non-essential amino acid,and cultured at 37℃in a humidified environment containing 5%CO₂.After treatment with three concentrations（1μmol/L,10μmol/L,100μmol/L） of salvianolic acid B for 48 hours,alpha smooth muscle actin and collagen typeⅠmRNA was evaluated by RT-PCR,and extracellular signal-regulated kinase-1/2（ERK1/2） was evaluated using western blot technique.Results:1) Induction of chronic pancreatitis and activation of pancreatic stellate cell in modelIn pancreatic tissue after two weeks after surgical procedure,there were inflammatory cell infiltration,few alpha-smooth muscle actin positive cells except for vascular smooth muscle cells and higher level of collagen typeⅠmRNA compared with the control,but without atrophy and fibrosis.In pancreatic tissue after four weeks after surgical procedure,there were inflammatory cell infiltration,prominent fibrosis and atrophy,many alpha-smooth muscle actin positive cells and higher level of collagen typeⅠmRNA compared with the control.In pancreatic tissue after eight weeks after surgical procedure,the changes were similar with that of four weeks,but more obvious.2) Treatment of salvianolic acid B in modelHistopathologic analysesAt the end of twelve weeks,segmental glandular atrophy was prominent in the group 1.Interstitial edema,mononuclear inflammatory cell infiltration,destruction of acini,and intralobular or interlobular and periductal fibrosis were also observed.Sal-B clearly improved pancreatic histological findings in experimental pancreatitis rats,as demonstrated by light microscopy inspection.The animals in the group 3 and the group 4 had significantly less pancreatic injury and fibrosis when compared with other groups. The histopathologic scores were higher in the TNBS treated group than in the TNBS+ Sal-B treated group（p＜0.01）.Subgroup analysis of pathological scores revealed that the rats in the TNBS+Sal-B treated group had significantly lower scores than the rats in the TNBS treated group（p＜0.05）,except inflammation（p＞0.05）.Activation of pancreatic stellate cellsImmunostaining for alpha-SMA was performed to identify the activated PSCs.In the TNBS treated group,it revealed a marked proliferation of alpha-SMA positive cells in the pancreas.However,there were few alpha-SMA positive cells in the TNBS+ Sal-B treated group at the end of twelve weeks except for vascular smooth muscle cells. There were also few alpha-SMA positive cells in the pancreas of group 3 and group 4 rats.Expression of collagen typeⅠmRNASal-B suppressed the expression of collagen typeⅠmRNA in pancreatic tissue compared with group 1（p＜0.01）.Compared with group 3 and group 4,pancreatic collagen typeⅠmRNA obviously increased in group 1（p＜0.01）,but not in group 2 （p＞0.05）.3) Proliferation of pancreatic stellate cellsAccording to MTT assay,1μmol/L Sal-B had no effect on the proliferation of PSCs （p＞0.05）.10μmol/L and 100μmol/L Sal-B significantly suppressed PSCs proliferation compared with the control（p＜0.01）.4) Treatment of salvianolic acid B in PSCExpression of alpha-SMA and collagen typeⅠmRNASal-B suppressed expression of collagen typeⅠand alpha smooth muscle actin in pancreatic stellate cells compared with control（p＜0.01）.Expression of alpha-SMA and collagen typeⅠmRNA decreased gradually with increasing the concentration of Sal-B. Suppression of ERK 1/2 activity Sal-B suppressed activity of ERK1/2 in pancreatic stellate cells,and decreased collagen typeⅠprotein expression compared with control（p＜0.01）.The activity of ERK1/2 decreased with increasing increasing the concentration of Sal-B.Conclusion:1) Chronic pancreatitis model induced by trinitrobenzene sulfonic acid infusion into rat pancreatic ducts had the similar character of human chronic pancreatitis. Pancreatic stellate cell had an important role in chronic pancreatitis.2) Salvianolic acid B suppressed the expression of alpha-smooth muscle actin and the activation of pancreatic stellate cell by decreasing the activity of extracellular signal-regulated kinase 1/2.3) Salvianolic acid B decreased collagen typeⅠand attenuated the morphological pancreatic damage by suppressing the activation of pancreatic stellate cells.更多还原