Periacinar stellate shaped cells in rat pancreas: identification, isolation, and culture

Background—The pathogenesis ofpancreatic fibrosis is unknown. In the liver, stellate cells (vitamin Astoring cells) play a significant role in the development of fibrosis.
Aims—To determine whether cellsresembling hepatic stellate cells are present in rat pancreas, and ifso, to compare their number with the number of stellate cells in theliver, and isolate and culture these cells from rat pancreas.
Methods—Liver and pancreaticsections from chow fed rats were immunostained for desmin, glialfibrillary acidic protein (GFAP), and α smooth muscle actin(α-SMA). Pancreatic stellate shaped cells were isolated using aNycodenz gradient, cultured on plastic, and examined by phase contrastand fluorescence microscopy, and by immunostaining for desmin, GFAP,and α-SMA.
Results—In both liver andpancreatic sections, stellate shaped cells were observed; these werepositive for desmin and GFAP and negative for α-SMA. Pancreaticstellate shaped cells had a periacinar distribution. They comprised3.99% of all pancreatic cells; hepatic stellate cells comprised 7.94%of all hepatic cells. The stellate shaped cells from rat pancreas grewreadily in culture. Cells cultured for 24 hours had an angularappearance, contained lipid droplets manifesting positive vitamin Aautofluorescence, and stained positively for desmin but negatively forα-SMA. At 48 hours, cells were positive for α-SMA.
Conclusions—Cells resemblinghepatic stellate cells are present in rat pancreas in a numbercomparable with that of stellate cells in the liver. These stellateshaped pancreatic cells can be isolated and cultured in vitro.

Keywords:pancreas; fibrosis; stellate cells

     

胰腺星状细胞在大鼠胰腺纤维化形成中的作用

目的 动态观察三硝基苯磺酸(TNBS)胰管内注射诱导大鼠胰腺纤维化过程中胰腺星状细胞(PSC)的活化情况，并研究PSC相关介质转化生长因子β1(TGF-β1)、基质金属蛋白酶-2(MMP-2)和Ⅰ型胶原蛋白表达，探讨PSC在胰腺纤维化形成过程中的作用。方法 通过胰管内注射含2％TNBS的乙醇磷酸盐缓冲液诱导大鼠胰腺纤维化模型，并分别于术后72h和3、4、5、6、7周处死大鼠，留取胰腺组织。胰腺组织切片经HE染色，光镜下观察病理学变化。分别用免疫组化、RT-PCR、Western blot检测胰腺组织α-平滑肌肌动蛋白(α-SMA)、TGF-β1、MMP-2 mRNA和Ⅰ型胶原蛋白表达；电镜观察不同时点胰腺组织超微结构变化。结果 2％TNBS胰管内注射后早期主要以胰腺组织炎症、水肿、坏死为主，3周后则以纤维组织增生、腺泡萎缩、胶原沉积等纤维化表现为主，以第4周为甚。第3周时α-SMA表达量较少，第4周时则显著增多，而后又渐减。第3周时胰腺组织TGF-β1蛋白表达显著增加，4周时达峰值。正常胰腺组织仅有极微量MMP-2 mRNA表达，而制模早期胰腺组织即有MMP-2 mRNA表达显著增加，随时间延长其表达量呈波动性变化，但仍明显高于正常。正常胰腺组织间质有少量I型胶原表达，而在胰腺纤维化区域则可见大量Ⅰ型胶原沉积。结论 PSC可能参与了TNBS诱导的大鼠胰腺纤维化的发生与发展过程；而此作用可能是PSC在促纤维化因子TGF-β1作用下活化，并合成Ⅰ型胶原等细胞外基质、分泌MMP-2等细胞外基质代谢相关酶而实现。     

Pancreatic stellate cells express Toll-like receptors

BACKGROUND: Toll-like receptors (TLRs) are proteins involved in recognition of foreign pathogen-associated molecular patterns (PAMPs) and activation of innate immunity. This study aimed to clarify whether pancreatic stellate cells (PSCs), a major profibrogenic cell type in the pancreas, expressed TLRs and responded to PAMPs. METHODS: PSCs were isolated from rat pancreas tissue, and expression of TLRs was examined. PSCs were treated with lipoteichoic acid (a ligand for TLR2), polyinosinic-polycytidylic acid (a ligand for TLR3), lipopolysaccharide (a ligand for TLR4), or flagellin (a ligand for TLR5). The effects of the TLR ligands on key cell functions and activation of signaling pathways were examined. The ability of PSCs to perform endocytosis and phagocytosis was also examined. RESULTS: PSCs expressed TLR2, 3, 4, and 5, as well as the associated molecules CD14 and MD2. All of the TLR ligands activated nuclear factor-kappaB, and three classes of mitogen-activated protein kinases (extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase). TLR ligands induced expression of monocyte chemoattractant protein 1, cytokine-induced neutrophil chemoattractant 1 (a rat homolog of interleukin-8), and inducible nitric oxide synthase, but not proliferation or type I collagen production. PSCs could perform fluid-phase and receptor-mediated endocytosis, as well as phagocytosis of Escherichia coli. CONCLUSIONS: PSCs expressed a variety of TLRs and responded to TLR ligands, leading to the activation of signaling pathways and proinflammatory responses. PSCs could process exogenous antigens by endocytosis and phagocytosis. PSCs might play a role in the immune functions of the pancreas through the recognition of PAMPs.     

肝星状细胞在逆转肝纤维化中的作用

肝纤维化是一种能够导致门静脉高压、肝硬化、肝衰竭的严重疾病。已经发现肝星状细胞的活化是引起肝纤维化的中心环节,因此抑制肝星状细胞的活化、加速肝星状细胞的清除有望逆转肝纤维化。本文将对活化的肝星状细胞的凋亡、衰老以及清除的研究进展作综述,阐明肝星状细胞在肝纤维化过程中所起的作用及相关机制。     

过氧化物酶体增殖物激活受体γ与肝星状细胞在肝纤维化中的关系

肝纤维化是慢性肝损伤后常见的形态学表现，大多数是由慢性肝脏疾病发展而来。而肝损伤（肝实质炎症、坏死）激活肝星状细胞（HSC）引起大量细胞外基质沉积，是肝纤维化发生机制的中心环节。在正常肝脏中，HSC处于静息状态，细胞质中脂滴丰富，具有合成和分泌少量细胞外基质和胶原酶的能力。在肝损伤及各种慢性肝病时，HSC被激活转化为肌成纤维母样细胞，发生明显的形态和结构变化：细胞质中脂滴减少或消失，增殖迁移活性明显增强，分泌多种细胞因子和黏附分子，合成各种细胞外基质（ECM）的能力明显增强，抑制基质金属蛋白酶（MMPs）的合成和分泌，而上调基质金属蛋白酶抑制剂（TIMPs）的表达，同时发生多种基因表达的改变。     

Xanthine oxidase-derived free radicals directly activate rat pancreatic stellate cells

BACKGROUND AND AIM: Free radicals are reported to be associated with fibrosis in the pancreas. It is generally accepted that pancreatic stellate cells (PSC) play an important role in pancreatic fibrosis. However, the exact role of free radicals in activation of PSC has not been fully elucidated. In the present study, using a superoxide dismutase (SOD) inhibitor, diethyldithiocarbamate (DDC) with cultured PSC, we investigated how free radicals act on the activation of PSC. METHODS: PSC were isolated from male Wister rats. Cultured rat PSC were incubated with DDC for 48 h. Intracellular SOD activity and lipid peroxidation were examined in DDC-treated PSC. Activation of PSC was examined by determining the expression of alpha-smooth muscle actin (alpha-SMA) by immunocytochemistry. The number of PSC using a hemocytometer, type I collagen secretion with ELISA and matrix metalloproteinases (MMP) activities with gelatin zymography were also examined. Secretion of transforming growth factor-beta1 (TGF-beta1) was evaluated by ELISA. The effects of the allopurinol, a xanthine oxidase (XOD) inhibitor, on PSC were also examined. RESULTS: DDC decreased SOD activity and increased lipid peroxidation products in PSC. DDC activated PSC, increasing the number of alpha-SMA positive cells, enhancing secretion of type I collagen and MMP, inhibiting PSC proliferation. Secretion of TGF-beta1, which is known to activate PSC, was increased by DDC treatment. These alterations were prevented by allopurinol. CONCLUSION: These results suggest that free radicals generated by XOD might directly activate PSC.     

Fasudil prevents liver fibrosis via activating natural killer cells and suppressing hepatic stellate cells

BACKGROUNDFasudil, as a Ras homology family member A (RhoA) kinase inhibitor, is used to improve brain microcirculation and promote nerve regeneration clinically. Increasing evidence shows that Rho-kinase inhibition could improve liver fibrosis.AIMTo evaluate the anti-fibrotic effects of Fasudil in a mouse model of liver fibrosis induced by thioacetamide (TAA). METHODSC57BL/6 mice were administered TAA once every 3 d for 12 times. At 1 wk after induction with TAA, Fasudil was intraperitoneally injected once a day for 3 wk, followed by hematoxylin and eosin staining, sirius red staining, western blotting, and quantitative polymerase chain reaction (qPCR), and immune cell activation was assayed by fluorescence-activated cell sorting. Furthermore, the effects of Fasudil on hepatic stellate cells and natural killer (NK) cells were assayed in vitro.RESULTSFirst, we found that TAA-induced liver injury was protected, and the positive area of sirius red staining and type I collagen deposition were significantly decreased by Fasudil treatment. Furthermore, western blot and qPCR assays showed that the levels of alpha smooth muscle actin (α-SMA), matrix metalloproteinase 2 (MMP-2), MMP-9, and transforming growth factor beta 1 (TGF-β1) were inhibited by Fasudil. Moreover, flow cytometry analysis revealed that NK cells were activated by Fasudil treatment in vivo and in vitro. Furthermore, Fasudil directly promoted the apoptosis and inhibited the proliferation of hepatic stellate cells by decreasing α-SMA and TGF-β1. CONCLUSIONFasudil inhibits liver fibrosis by activating NK cells and blocking hepatic stellate cell activation, thereby providing a feasible solution for the clinical treatment of liver fibrosis.     

肝星状细胞的可塑性及其对肝纤维化的意义

在肝纤维化形成过程中,肝星状细胞(hepatic stellate cells,HSC)发挥着重要的作用.基础和临床研究结果显示,在特殊的内环境因素的影响下HSC具有朝多方向分化的潜能.对HSC命运的干预能够在一定程度上预防肝纤维化的发生,甚至逆转肝纤维化,因此HSC的可塑性研究可能为慢性肝病治疗开辟一条新途径.本文就HSC的起源、结构、可塑性及其对肝纤维化的潜在治疗意义作一综述.     

肥大细胞在大鼠胰腺组织纤维化形成中的作用及其机制

目的探讨肥大细胞（MC）在大鼠胰腺组织纤维化形成中的作用和机制。方法建立经逆行胆胰管注射2％三硝基苯磺酸（TNBS）诱导大鼠慢性胰腺炎模型，将大鼠分为三组，每组40只，分别用肥大细胞膜稳定剂色甘酸钠和MC激动剂48／80化合物及生理盐水进行干预，并于第3、7、14、21和28天处死动物。H—E染色观察胰腺组织病理学改变；Van Gieson染色观察胰腺组织纤维化情况；硫堇蓝染色观察大鼠慢性胰腺炎过程中MC分布、形态和数量的改变；免疫组化染色观察大鼠慢性胰腺炎α-平滑肌肌动蛋白（α-SMA）、转化生长因子（TGF）β1的表达；逆转录-多聚酶链反应（RT—PCR）观察血管紧张素Ⅱ1型（AT1）和2型（AT2）受体蛋白的表达。结果2％TNBS胰管内注射后可于第4周引起典型大鼠胰腺组织纤维化，在胰腺纤维化区域可见大量Ⅰ型胶原沉积。在此过程中MC存在着活化及脱颗粒。胰腺组织α—SMA、TGFβ1、AT1和AT2 mRNA蛋白制模早期表达即为阳性，至第4周时最强。与对照（生理盐水）组比较，色甘酸钠组MC数量及脱颗粒现象明显减少，α-SMA、TGFβ1蛋白表达和AT1、AT2受体mRNA表达明显减少；48／80化合物组MC数量及脱颗粒现象明显增多，上述各指标的表达均有不同程度的增加。结论MC参与TNBS诱导的大鼠慢性胰腺炎的炎症和纤维化的发生及发展，其机制可能与MC促进胰腺星状细胞活化，上调血管紧张素Ⅱ受体表达等介导了胰腺纤维化的形成。     

大鼠胰星状细胞的分离与培养

目的　建立大鼠胰星状细胞 (pancreatic stellate cells,PSCs)的分离培养方法。方法　大鼠胰腺组织经胶原酶和链霉蛋白酶 E消化后 ,用 Nycodenz不连续密度梯度离心法分离 PSC,在 32 8nm紫外光激发下观察细胞的自发荧光现象 ,并以免疫组化技术检测结蛋白 (desmin)、神经胶质原纤维酸性蛋白 (glial fibrillary acidic protein,GFAP)和α-平滑肌动蛋白 (α- sm ooth m uscle actin,α- SMA )的表达 ,同时观察培养细胞的形态和生长特性。结果　新鲜分离的大鼠 PSCs产率、活力和纯度分别约为 2 .5 ×10 6 /g胰腺、95 %和 90 %。培养的 PSCs可自发活化 ,表达 α- SMA,细胞由静止型转化为肌成纤维样细胞表型。原代培养 10天后细胞纯度 >95 % ,传代培养后细胞纯度可达 99%以上。结论　利用 Nycodenz密度梯度离心方法可成功分离大鼠 PSCs,其细胞产率、活力和纯度均可满足体外研究需要。     

肝星状细胞凋亡调控因素的研究进展

诱导HSC凋亡成为阻止肝纤维化进程的途径之一.生长因子、死亡受体配体(TRAIL、FAS)、细胞外基质(胶原、整合素)、信号转导蛋白和转录因子(NF-κB、IKKJNK)等多种因素参与调控HSC凋亡.     

MicroRNA-194 inactivates hepatic stellate cells and alleviates liver fibrosis by inhibiting AKT2

BACKGROUND Activation of hepatic stellate cells(HSCs)is a pivotal event in the onset and progression of liver fibrosis.Loss of microRNA-194(miR-194)has been reported in activated HSCs,but the actual role of miR-194 in liver fibrosis remains uncertain.AIM To explore the role and potential mechanism of miR-194-mediated regulation of liver fibrosis in vitro and in vivo.METHODS The expression of miR-194 was examined in human fibrotic liver tissues,activated HSCs,and a carbon tetrachloride(CCl4)mouse model by qPCR.The effects of AKT2 regulation by miR-194 on the activation and proliferation of HSCs were assessed in vitro.For in vivo experiments,we reintroduced miR-194 in mice using a miR-194 agomir to investigate the functions of miR-194 in liver fibrosis.RESULTS MiR-194 expression was notably lacking in activated HSCs from both humans and mice.Overexpression of miR-194(OV-miR-194)inhibitedα-smooth muscle actin(α-SMA)and type I collagen(Col I)expression and suppressed cell proliferation in HSCs by causing cell cycle arrest in G0/G1 phase.AKT2 was predicted to be a target of miR-194.Notably,the effects of miR-194 knockdown in HSCs were almost blocked by AKT2 deletion,indicating that miR-194 plays a role in HSCs via regulation of AKT2.Finally,miR-194 agomir treatment dramatically ameliorated liver fibrosis in CCl4-treated mice.CONCLUSION We revealed that miR-194 plays a protective role by inhibiting the activation and proliferation of HSCs via AKT2 suppression.Our results further propose miR-194 as a potential therapeutic target for liver fibrosis.     

肝星状细胞活化相关信号通路在肝纤维化中的研究进展

肝纤维化是多种慢性肝损伤造成的细胞外基质(extracellular matrix, ECM)过度累积及降解不足的病理结果,如不加以干预会逐渐进展为肝硬化,甚至肝细胞癌。肝星状细胞(hepatic stellate cell, HSC)是ECM的主要来源,并且HSC在肝纤维化的起始、发展和消退过程中发挥关键作用。近年来,HSC活化涉及的信号传导通路成为研究热点,本文总结了HSC活化过程中的重要信号通路。     

肝星状细胞的活化与肝纤维化

肝纤维化是机体对损伤的一种修复作用，即使可以应用基因或其他疗法彻底消除纤维化，但机体对抑制或消除纤维化后将产生何种反应及后果尚难预测。肝星状细胞（hepatic stellate cells，HSC）是引起肝纤维化的主要细胞，对HSC与其活化型一肌成纤维细胞（myofibroblast，MF）在肝损伤中作用的研究已颇为深入，而HSC激活在肝纤维化发生、发展中的作用甚为重要。