胰腺星状细胞的调控及转归

活化的胰腺星状细胞(pancreatic stellate cells,PSCs)是胰腺炎致胰腺纤维化的主要效应细胞。近年来,学者普遍认为,胰腺纤维化早期阶段是动态可逆的,因此,若在胰腺损伤的早期阶段,抑制PSCs的增殖、迁移,减少损伤部位PSCs的数目,降低细胞外基质(extracellular matrix,ECM)的生成,将可能逆转胰腺纤维化。该文以PSCs为靶点阐述了抗胰腺纤维化的新策略。     

结缔组织生长因子在胰腺组织纤维化中的作用及机制探讨

目的 观察胰腺纤维化后结缔组织生长因子(Connective tissue growth factor,CTGF)在胰腺组织内的表达;进一步研究参与CTGF作用于胰腺星状细胞(pancreatic stellate cells,PSCs)的分子信号调控通路.方法 建立大鼠胰腺纤维化动物模型,HE染色、天狼猩红染色和免疫组织化学染色等方法观察胰腺纤维化后PSCs的活化情况及CTGF在胰腺组织的表达.Real-time RT PCR检测CTGF的基因表达.Western Blot检测PSCa内α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)及胶原蛋白Ⅰ(CollagenⅠ)水平.结果 胰腺组织纤维化后,PSCs大量活化,并显著表达CTGF.CTGF作用后,PSCs内CTGF mRNA、α-SMA和Collagen Ⅰ的合成均有显著增加,在给予不同的细胞信号通路阻断剂后,PSCs内α-SMA的合成有显著下降,而Collagen Ⅰ的降低没有表现出统计学差异.结论 CTGF参与了胰腺纤维化的调控,MAPK和PI3-K信号通路均参与了CTGF的调控作用.     

慢性胰腺炎胰腺纤维化发病机制研究新进展

慢性胰腺炎(chronic pancreatitis, CP)是由多种因素引起的胰腺内外分泌功能紊乱,可导致胰腺结构和功能发生不可逆性损伤,是临床常见的消化系统疾病。CP的病理特点是腺泡细胞损伤导致巨噬细胞等多种炎症细胞浸润,从而分泌大量促炎细胞因子,在胰腺组织微环境中引起胰腺星状细胞活化,进而产生大量的细胞外基质,表现为胰腺纤维化。而新近的研究提示:胰腺纤维化是一种动态病理现象,需要由多种自分泌和旁分泌的细胞因子组成复杂的网络,作用于相应的信号通路,最终导致纤维化形成。现以CP胰腺组织微环境中出现的主要细胞,如胰腺星状细胞、巨噬细胞、腺泡细胞及其在CP胰腺纤维化进展中的变化和作用为切入点,对CP胰腺纤维化的发病机制研究进展做一综述。     

胰腺星状细胞在胰腺内外分泌疾病中的作用

胰腺纤维化是胰腺炎、胰腺癌主要的病理学特征。活化的胰腺星状细胞(pancreatic stellate cell,PSC)是胰腺炎致胰腺纤维化的主要效应细胞,在胰腺炎与胰腺癌的纤维化进程中发挥重要作用。近期研究发现,PSC也存在于糖尿病动物胰岛中,可能参与糖尿病胰岛纤维化及β细胞衰竭的发展进程。现就PSC与胰腺炎、胰腺癌及糖尿病关系的研究进展作一综述。     

抗胰腺星状细胞药物研究进展

慢性胰腺炎传统治疗方法效果不确定,需要寻找新型有效药物。活化的胰腺星状细胞在胰腺纤维化过程中起了重要的作用,使之成为新的治疗靶点。活化PSC的物质分为三类,即炎症介质、氧化应激和毒素。本文就近年来针对以上不同活化PSC的介质和相关通路的抗星状细胞药物进行综述。     

胰腺微环境外泌体miRNA参与胰腺炎症和纤维化的发病机制研究进展

慢性胰腺炎(chronic pancreatitis, CP)发病率逐年上升，目前尚无明确根治性治疗方法且后期有进展为胰腺癌的风险。CP的典型病理学特征是胰腺慢性炎症和纤维化，CP进展与胰腺微环境中三种主要细胞(腺泡细胞、巨噬细胞以及胰腺星状细胞)间的相互作用密切相关，然而它们具体是如何进行细胞间联系的目前尚不清楚。新近研究表明外泌体作为细胞间重要的通讯介质，其携带的miRNA可通过调控主要细胞内基因表达和信号通路等影响CP的发生发展。本文围绕胰腺微环境中外泌体来源miRNA与三种主要细胞相互作用的机制，对其最新研究进展进行归纳和总结分析，以期为CP发病机制的深入认识提供参考。     

Notch3对促进胰腺星形细胞活化的基因表达及信号通路的影响

目的: 分离培养小鼠胰腺星形细胞(PSCs),检测Notch3 对促进PSCs活化的基因表达及信号通路的影响。方法: 对小鼠PSCs进行分离培养及传代。采用免疫荧光染色检测活化的小鼠PSCs中α-SMA, fibronectin及collagen I的表达;细胞分组为空白对照组(MOCK组),阴性对照组(转染Notch3 siRNA negative control,NC组),Notch3 siRNA组(转染Notch3 siRNA,N3 siRNA组)及Notch3 siRNA-1组(转染Notch3 siRNA-1,N3 siRNA-1组),提取各组总RNA,测定RNA浓度及纯度后,送至安诺优达基因科技(北京)有限公司进行转录组测序。结果: 免疫荧光结果显示,在活化的PSCs中α-SMA,fibronectin及collagen I都有明显的表达。测序结果分析表明,与NC组相比较,在N3 siRNA组与N3 siRNA-1组,α-SMA基因,collagen I基因,fibronectin基因及CTGF基因均表达下调,与胶原蛋白代谢过程相关的基因表达上调,正向调节胶原生物合成的基因表达下调,而负向调节胶原生物合成的基因表达上调,PCNA基因表达下调;在N3siRNA组与N3siRNA-1组,调节细胞聚集的基因表达下调;在细胞组分部分,细胞外基质的基因表达下调;抑制PSCs中Notch3的表达可对细胞粘附分子信号通路,MAPK信号通路及TGF-β信号通路的组成成员的基因表达产生影响。结论: 抑制Notch3的表达可抑制PSCs的活化,降低细胞增殖能力,降低迁移聚集能力及ECM合成的能力;抑制Notch3的表达可对其他的信号如细胞粘附分子信号通路,MAPK信号通路及TGF-β信号通路产生影响。     

白介素-17A在小鼠慢性胰腺炎中的作用研究

目的:研究白细胞介素-17A在小鼠慢性胰腺炎模型中的表达及其对小鼠星状细胞的影响。方法:建立雨蛙肽诱导的小鼠实验性慢性胰腺炎动物模型,利用实时荧光定量PCR、ELISA、免疫组化和Western-blot等手段检测白介素-17A在雨蛙肽诱导的小鼠慢性胰腺炎模型中的表达变化及免疫活性。用重组白介素-17A作用于小鼠胰腺星状细胞,检测其对星状细胞活化的作用,并进一步探究其对促胰腺纤维化炎症因子白介素-6、白介素-1β、TGF-β在mRNA水平的表达变化。结果:慢性胰腺炎胰腺组织中白介素-17A受体IL-17RA及IL-17RC mRNA水平的表达较正常胰腺明显升高,慢性胰腺炎小鼠胰腺组织中IL-17A蛋白水平较正常小鼠明显升高,CP小鼠血清中IL-17A蛋白水平(56.40±10.50 pg/L)较NC组(27.88±5.74pg/L)亦明显升高,IL-17A在正常胰腺组织中鲜有表达(8.9±2.72%),而在CP组织中呈强阳性表达(55.84±5.71%),其免疫活性主要定位于间质炎性细胞及导管样复合体中;重组白介素-17A可促进小鼠星状细胞活化,并直接诱导星状细胞表达白介素-6、白介素-1β以及TGF-β等促纤维化细胞因子。结论:白介素-17A在雨蛙肽诱导的小鼠慢性胰腺炎模型中表达上调,并可能通过诱导小鼠星状细胞表达促炎细胞因子白介素-6、白介素-1β和TGF-β,促进胰腺星状细胞活化以及胰腺纤维化。     

细胞外基质金属蛋白酶诱导分子（CD147）在胰腺癌细胞及胰腺星状细胞中的表达

目的：探讨细胞外基质金属蛋白酶诱导分子（CD147）在胰腺癌细胞（Panc-1）及胰腺星状细胞（PSCs）的表达。方法：应用QRT—PCR,免疫细胞化学和免疫印迹分析方法检测Panc-1和PSCs细胞中EMMRPIN的表达,应用脱糖基化试剂N—glycosidase F及Endoglycosidase H鉴定CD147糖基化形式。结果：CD147在Panc-1和PSCs细胞质膜及细胞质中高表达,通过脱糖基化法首次鉴定出胰腺癌细胞及胰腺星状细胞中CD147不同的糖基化修饰。结论：CD147的糖基化修饰具有细胞特异性,可能与细胞恶性程度相关。     

PI3K、p-AKT及HIF—1α在胰腺癌中的表达及意义

目的检测PI3K、p-AKT及HIF-1α三种蛋白在胰腺癌组织中的表达并探讨其与临床病理因素的关系及三者之间的相关性。方法采用免疫组织化学SP法检测43例胰腺癌组织、9例胰腺炎组织和8例正常胰腺组织中PI3K、p-AKT及HIF-1α三种蛋白的表达。结果PI3K、p-AKT的阳性表达主要位于肿瘤细胞胞浆,HIF-1α的阳性表达主要位于细胞核或细胞浆。PI3K、p-AKT及HIF-1α在胰腺癌组织中的阳性表达率分别为62．79％、67．44％和69．77％,三者均显著高于在正常胰腺组织和慢性胰腺炎组织中的表达,差异有统计学意义（P〈0．01）。PI3K、p-AKT及HIF-1α的异常表达均与胰腺癌的淋巴结转移和TNM分期有关（P〈0．05）,与患者的性别、年龄、部位、分化程度和病理分型无关（P〉0．05）。PI3K、p-AKT及HIF-1α三者间的表达呈正相关。结论PI3K、p-AKT及HIF-1α的表达在胰腺癌的生长、浸润转移中起重要作用,PI3K-AKT信号通路激活可能促进HIF-1α的表达。     

Heat shock protein 90 inhibitor ameliorates pancreatic fibrosis by degradation of transforming growth factor-β receptor

Background and aimPancreatic fibrosis increases pancreatic cancer risk in chronic pancreatitis (CP). Pancreatic stellate cells (PSCs) play a critical role in pancreatic fibrosis by transforming growth factor-β (TGFβ) has been shown to inhibit transforming growth factor-β receptor (TGFβR)-mediated Smad and no-Smad signaling pathways. Thus, the effects of Hsp90 inhibitor on pancreatic fibrosis are evaluated in CP mice, and the association between Hsp90 and biological functions of PSCs is further investigated in vitro.MethodsThe effects of Hsp90 inhibitor 17AAG on pancreatic fibrosis were assessed in caerulein-induced CP mice, and primary PSCs were used to determine the role of Hsp90 inhibitor 17AAG in vitro.ResultsWe observed increased expression of Hsp90 in pancreatic tissues of caerulein-induced CP mice. Hsp90 inhibitor 17AAG ameliorated pancreatic inflammation and fibrosis in caerulein-induced CP mice. In vitro, Hsp90 inhibitor 17AAG inhibited TGFβ1-induced activation and extracellular matrix accumulation of PSCs by blocking TGFβR-mediated Smad2/3 and PI3K /Akt/GSK-3β signaling pathways.Hsp90 inhibitor 17AAG degraded TGFβRII by a ubiquitin-proteasome pathway, co-immunoprecipitation showed an interaction between Hsp90 and TGFβRII in PSCs.ConclusionsThe study suggests that an Hsp90 inhibitor 17AAG remarkable prevents the development of pancreatic fibrosis in caerulein-induced CP mice, and suppresses activation and extracellular matrix accumulation of PSCs in vitro. The current results provide a potential treatment strategy based on Hsp90 inhibition for pancreatic fibrosis in CP.     

Peroxisome proliferator-activated receptor gamma overexpression inhibits pro-fibrogenic activities of immortalised rat pancreatic stellate cells

Pancreatic stellate cells (PSCs) play a key role in the development of pancreatic fibrosis, a constant feature of chronic pancreatitis and pancreatic cancer. In response to pro-fibrogenic mediators, PSCs undergo an activation process that involves proliferation, enhanced production of extracellular matrix proteins and a phenotypic transition towards myofibroblasts. Ligands of the peroxisome proliferator-activated receptor gamma (PPARgamma), such as thiazolidinediones, are potent inhibitors of stellate cell activation and fibrogenesis in pancreas and liver. The effects of PPARgamma ligands, however, are at least in part mediated through PPARgamma-independent pathways. Here, we have chosen a different approach to study regulatory functions of PPARgamma in PSCs. Using immortalised rat PSCs, we have established a model of tetracycline (tet)-regulated PPARgamma overexpression. Induction of PPARgamma expression strongly inhibited proliferation and enhanced the rate of apoptotic cell death. Furthermore, PPARgamma-overexpressing cells synthesised less collagen than controls. To monitor effects of PPARgamma on PSC gene expression, we employed Affymetrix microarray technology. Using stringent selection criteria, we identified 21 up- and 19 down-regulated genes in PPARgamma-overexpressing cells. Most of the corresponding gene products are either involved in lipid metabolism, play a role in signal transduction, or are secreted molecules that regulate cell growth and differentiation. In conclusion, our data suggest an active role of PPARgamma in the induction of a quiescent PSC phenotype. PPARgamma-regulated genes in PSCs may serve as novel targets for the development of antifibrotic therapies.     

Curcumin blocks activation of pancreatic stellate cells

Activated pancreatic stellate cells (PSCs) play a pivotal role in the pathogenesis of pancreatic fibrosis and inflammation. Inhibition of activation and cell functions of PSCs is a potential target for the treatment of pancreatic fibrosis and inflammation. The polyphenol compound curcumin is the yellow pigment in curry, and has anti-inflammatory and anti-fibrotic properties. We here evaluated the effects of curcumin on the activation and cell functions of PSCs. PSCs were isolated from rat pancreas tissue and used in their culture-activated, myofibroblast-like phenotype unless otherwise stated. The effects of curcumin on proliferation, alpha-smooth muscle actin gene expression, monocyte chemoattractant protein (MCP)-1 production, and collagen expression were examined. The effect of curcumin on the activation of freshly isolated cells in culture was also assessed. Curcumin inhibited platelet-derived growth factor (PDGF)-induced proliferation, alpha-smooth muscle actin gene expression, interleukin-1beta- and tumor necrosis factor (TNF)-alpha-induced MCP-1 production, type I collagen production, and expression of type I and type III collagen genes. Curcumin inhibited PDGF-BB-induced cyclin D1 expression and activation of extracellular signal-regulated kinase (ERK). Curcumin inhibited interleukin-1beta- and TNF-alpha-induced activation of activator protein-1 (AP-1) and mitogen-activated protein (MAP) kinases (ERK, c-Jun N-terminal kinase (JNK), and p38 MAP kinase), but not of nuclear factor-kappaB (NF-kappaB). In addition, curcumin inhibited transformation of freshly isolated cells to myofibroblast-like phenotype. In conclusion, curcumin inhibited key cell functions and activation of PSCs.     

TGF-beta signaling preserves RECK expression in activated pancreatic stellate cells

Activated pancreatic stellate cells (PSCs) play a pivotal role in the pathogenesis of pancreatic fibrosis, but the detailed mechanism for dysregulated accumulation of extracellular matrix (ECM) remains unclear. Cultured rat PSCs become activated by profibrogenic mediators, but these mediators failed to alter the expression levels of matrix metalloproteinases (MMPs) to the endogenous tissue inhibitors of metalloproteinases (TIMPs). Here, we examined the expression of RECK, a novel membrane-anchored MMP inhibitor, in PSCs. Although RECK mRNA levels were largely unchanged, RECK protein expression was barely detected at 2, 5 days after plating PSCs, but appeared following continued in vitro culture and cell passage which result in PSC activation. When PSCs at 5 days after plating (PSCs-5d) were treated with pepstatin A, an aspartic protease inhibitor, or TGF-beta1, a profibrogenic mediator, RECK protein was detected in whole cell lysates. Conversely, Smad7 overexpression or suppression of Smad3 expression in PSCs after passage 2 (PSCs-P2) led to the loss of RECK protein expression. These findings suggest that RECK is post-translationally processed in pre-activated PSCs but protected from proteolytic degradation by TGF-beta signaling. Furthermore, collagenolytic activity of PSCs-5d was greatly reduced by TGF-beta1, whereas that of PSCs-P2 was increased by anti-RECK antibody. Increased RECK levels were also observed in cerulein-induced acute pancreatitis. Therefore, our results suggest for the first time proteolytic processing of RECK as a mechanism regulating RECK activity, and demonstrate that TGF-beta signaling in activated PSCs may promote ECM accumulation via a mechanism that preserves the protease inhibitory activity of RECK.     

Distinct roles of Smad2-, Smad3-, and ERK-dependent pathways in transforming growth factor-beta1 regulation of pancreatic stellate cellular functions

Pancreatic stellate cells (PSCs) play a major role in promoting pancreatic fibrosis. Transforming growth factor-beta(1) (TGF-beta(1)) regulates PSC activation and proliferation in an autocrine manner. The intracellular signaling pathways of the regulation were examined in this study. Immunoprecipitation and immunocytochemistry revealed that Smad2, Smad3, and Smad4 were functionally expressed in PSCs. Adenovirus-mediated expression of Smad2, Smad3, or dominant-negative Smad2/3 did not alter TGF-beta(1) mRNA expression level or the amount of autocrine TGF-beta(1) peptide. However, expression of dominant-negative Smad2/3 inhibited PSC activation and enhanced their proliferation. Co-expression of Smad2 with dominant-negative Smad2/3 restored PSC activation inhibited by dominant-negative Smad2/3 expression without changing their proliferation. By contrast, co-expression of Smad3 with dominant-negative Smad2/3 attenuated PSC proliferation enhanced by dominant-negative Smad2/3 expression without altering their activation. Exogenous TGF-beta(1) increased TGFbeta(1) mRNA expression in PSCs. However, PD98059, a specific inhibitor of mitogen-activated protein kinase kinase (MEK1), inhibited ERK activation by TGF-beta(1), and consequently attenuated TGF-beta(1) enhancement of its own mRNA expression in PSCs. We propose that TGF-beta(1) differentially regulates PSC activation, proliferation, and TGF-beta(1) mRNA expression through Smad2-, Smad3-, and ERK-dependent pathways, respectively.     

The effects of nuclear factor-kappa B in pancreatic stellate cells on inflammation and fibrosis of chronic pancreatitis

The activation of pancreatic stellate cells (PSCs) plays a critical role in the progression of pancreatic fibrosis. Nuclear factor-kappa B ( NF-κB) is associated with chronic pancreatitis (CP). Previous evidence indicated that NF-κB in acinar cells played a double-edged role upon pancreatic injury, whereas NF-κB in inflammatory cells promoted the progression of CP. However, the effects of NF-κB in PSCs have not been studied. In the present study, using two CP models and RNAi strategy of p65 in cultured PSCs, we found that the macrophage infiltration and MCP-1 expression were increased, and the NF-κBp65 protein level was elevated. NF-κBp65 was co-expressed with PSCs. In vitro, TGF-β1 induced overexpression of the TGF-β receptor 1, phosphorylated TGF-β1–activated kinase 1 (p-TAK1) and NF-κB in the PSCs. Moreover, the concentration of MCP-1 in the supernatant of activated PSCs was elevated. The migration of BMDMs was promoted by the supernatant of activated PSCs. Further knockdown of NF-κBp65 in PSCs resulted in a decline of BMDM migration, accompanied by a lower production of MCP-1. These findings indicate that TGF-β1 can induce the activation of NF-κB pathway in PSCs by regulating p-TAK1, and the NF-κB pathway in PSCs may be a target of chronic inflammation and fibrosis.     

Hic‐5 deficiency protects cerulein‐induced chronic pancreatitis via down‐regulation of the NF‐κB (p65)/IL‐6 signalling pathway

Chronic pancreatitis (CP), characterized by pancreatic fibrosis, is a recurrent, progressive and irreversible disease. Activation of the pancreatic stellate cells (PSCs) is considered a core event in pancreatic fibrosis. In this study, we investigated the role of hydrogen peroxide‐inducible clone‐5 (Hic‐5) in CP. Analysis of the human pancreatic tissue samples revealed that Hic‐5 was overexpressed in patients with CP and was extremely low in healthy pancreas. Hic‐5 was significant up‐regulated in the activated primary PSCs independently from transforming growth factor beta stimulation. CP induced by cerulein injection was ameliorated in Hic‐5 knockout (KO) mice, as shown by staining of tissue level. Simultaneously, the activation ability of the primary PSCs from Hic‐5 KO mice was significantly attenuated. We also found that the Hic‐5 up‐regulation by cerulein activated the NF‐κB (p65)/IL‐6 signalling pathway and regulated the downstream extracellular matrix (ECM) genes such as α‐SMA and Col1a1. Therefore, we determined whether suppressing NF‐κB/p65 alleviated CP by treating mice with the NF‐κB/p65 inhibitor triptolide in the cerulein‐induced CP model and found that pancreatic fibrosis was alleviated by NF‐κB/p65 inhibition. These findings provide evidence for Hic‐5 as a therapeutic target that plays a crucial role in regulating PSCs activation and pancreatic fibrosis.     

High glucose increases extracellular matrix production in pancreatic stellate cells by activating the renin-angiotensin system

Pancreatic stellate cells (PSCs) are involved in pancreatic inflammation and fibrosis. Recent studies have shown that blocking the renin-angiotensin system (RAS) attenuates pancreatic inflammation and fibrosis. However, there are few data about the direct effects of high glucose on extracellular matrix (ECM) protein synthesis and angiotensin II (Ang II) induction in PSCs. PSCs were isolated from male Sprague-Dawley rats and cultured in medium containing 5.5 mM (LG group) or 27 mM D-glucose (HG group). Levels of Ang II and transforming growth factor-beta (TGF-beta) in culture media were measured and Ang II-positive cells were counted. We used real-time polymerase chain reaction (PCR) to detect Ang II receptor expression and Western blot analysis for the expression of ECM proteins such as connective-tissue growth factor (CTGF) and collagen type IV. Cells were also treated with an Ang II-receptor antagonist (candesartan, 10 microM) or angiotensin-converting enzyme (ACE) inhibitor (ramiprilat, 100 nM). Thymidine uptake by PSCs increased fourfold with high glucose treatment. Ang II levels and the proportion of Ang II-positive PSCs were significantly increased after 6 h under high-glucose conditions. TGF-beta concentrations also increased significantly with high glucose. After 72 h, the expression of CTGF and collagen type IV proteins in high-glucose cultures increased significantly and this increase was effectively attenuated by the candesartan or the ramiprilat. All together, high glucose induced PSCs proliferation and ECM protein synthesis, and these effects were attenuated by an Ang II-receptor antagonist. The data suggest that pancreatic inflammation and fibrosis aggravated by hyperglycemia, and Ang II play an important role in this pathogenesis.