肝星状细胞的抗原提呈特性

肝星状细胞(hepatic stellate cells,HSC),曾被称为Ito细胞、窦周细胞、脂细胞和贮脂细胞.由德国学者Kupffer在1876年首先用氯化金染色确认,称之为sternzellen,当时曾被误认为是一种肝内吞噬细胞.1951年,Ito用电镜观察将窦周HSC与窦内Kupffer细胞(肝吞噬细胞)予以清楚鉴别.1996年国际上将该细胞统一命名为HSC[1].     

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。     

Arg-gly-asp-mannose-6-phosphate inhibits activation and proliferation of hepatic stellate cells in vitro

AIM: To investigate the effect of arg-gly-asp-mannose-6 phosphate (RGD-M6P) on the activation and proliferation of primary hepatic stellate cells in vitro. METHODS: Hepatic stellate cells (HSCs) were isolated from rats by in situ collagenase perfusion of liver and 18% Nycodenz gradient centrifugation and cultured on uncoated plastic plates for 24 h with DMEM containing 10% fetal bovine serum (FBS/DMEM) before the culture medium was substituted with 2% FBS/DMEM for another 24 h. Then, HSCs were cultured in 2% FBS/DMEM with transforming growth factor beta1, M6P, RGD, or RGD-M6P, respectively. Cell morphology was observed under inverted microscope, smooth muscle alpha-actin (alpha-SMA) was detected by immunocytochemistry, type III procollagen (PC III) in supernatant was determined by radioimmunoassay, and the proliferation rate of HSCs was assessed by flow cytometry. RESULTS: RGD-M6P significantly inhibited the morphological transformation and the alpha-SMA and PC III expressions of HSCs in vitro and also dramatically prevented the proliferation of HSCs in vitro. Such effects were remarkably different from those of RGD or M6P. CONCLUSION: The new compound, RGD-M6P, which has a dramatic effect on primary cultured HSCs in vitro, can inhibit the transformation of HSCs in culture caused by TGFbeta1, suppresses the expression of PC III and decreases proliferation rate of HSC. RGD-M6P can be applied as a selective drug carrier targeting at HSCs, which may be a new approach to the prevention and treatment of liver fibrosis.     

致纤维化生长因子对肝星状细胞移行的影响

目的观察肝纤维化过程中Disse间隙生长因子微环境的改变对肝星状细胞（HSC）移行的影响，从细胞移行角度探讨肝纤维化病变的新机制。方法运用改良的Boyden腔系统，在体外条件下模拟体内正常Disse间隙的微环境及肝纤维化时的相关改变，以人HSC为研究对象，通过细胞迁移实验、细胞增殖实验等方法，观察肝纤维化时致纤维化生长因子对HSC移行的影响。结果肝纤维化时增高的血小板衍化生长因子（PDGF）-BB，转化生长因子β1（TGF-β1）及上皮细胞生长因子（EGF）均可导致活化的HSC移行能力增强，而肝纤维化时同样也增高的内皮细胞生长因子（VEGF）和碱性成纤维细胞生长因子（bFGF）则无此效果。PDGF—BB诱导的HSC移行能力的增强与其导致的HSC增殖有关，而由TGF-β1和EGF诱导的这种能力的增强与细胞增殖无关。结论肝纤维化时Disse间隙微环境的改变促进了HSC的移行，TGF-β1、PDGF—BB和EGF具有促进HSC移行的作用，而bFGF和VEGF则无。     

MicroRNAs在肝星状细胞活化过程中表达差异谱的研究

目的 应用MicroRNAs基因芯片技术筛选静止和活化肝星状细胞差异表达的MicroRNAs.方法 用链酶蛋白酶和胶原酶原位灌流,Nycodenz密度梯度离心分离大鼠HSCs,并进行体外培养,借助荧光倒置显微镜观察细胞形态,MicroRNAs基因芯片技术和荧光定量PCR检验MicroRNAs在HSCs静止期(2天)和活化期(14天)的表达.结果 HSCs的miRNAs表达谱中差异表达miRNAs共21个,其中上调12个,下调9个(P＜0.01);荧光定量RT-PCR证实其表达水平在静止和活化期肝星状细胞中均有差异(P＜0.01).结论 MicroRNAs基因芯片筛选差异表达miRNAs可望为肝纤维化的基因治疗提供全新的靶标和策略,为肝纤维化的发病机制和诊断治疗研究提供了新的思路.     

Effects of interleukin-10 on activation and apoptosis of hepatic stellate cells in fibrotic rat liver

AIM:To study the effects of interleukin-10(IL-10)on the expression of α-smooth muscle actin(α-SMA),nuclear factor-κB(NF-κB)and Fas/Fas ligand(FasL)inhepatic stellate cells of experimental rats with hepaticfibrosis.METHODS:Sixty clean SD rats were randomly dividedinto control group(group N),liver fibrotic group(groupC)and IL-10 treatment group(group I).Control groupreceived intraperitoneal injection of saline(2ml·kg~(-1)),twicea week.Fibrotic group was injected intraperitoneallywith 50% carbon tetrachloride(CCl_4)(2 ml·kg~(-1)),twicea week.IL-10 treatment group was given IL-10 at adose of 4 μg·kg~(-1)20 minutes before CCl_4 administrationfrom the third week.Hepatic stellate cells(HSCs)wereisolated from these rats at the seventh and eleventhweeks during the course of liver fibrosis,respectively.The expression of α-SMA and NF-κB in HSCs wasmeasured by S-P immunohistochemistry.The expressionof Fas and FasL mRNA was measured by RT-PCR.Furthermore,liver tissues were harvested from threegroups at the same time.RESULTS:The CCl_4- induced experimental rat hepaticfibrosis model was established successfully.The purityof extracted hepatic stellate cells was about 95% andthe yield of hepatic stellate cells was 1.2-2.3×10~6/g livertissue averagely.The positive expression of α-SMA andNF-κB was 36.5% and 28.5% respectively in group N.The positive levels of α-SMA and NF-κB were increasedsignificantly in group C compared to group N(P<0.01).The positive signals decreased significantly(P<0.05)ingroup I.In the 11~(th)week,the HSCs of group I becameround with visible pyknotic nuclei.The expression ofNF-μB in group C was significantly increased in a time-dependentmanner(P<0.01),but there was no difference in the α-SMA expression(P>0.05).The mRNA of Fasand FasL in group C was significantly increased in a time-dependent manner compared to that in control group.After treated with IL-10,the expression level of Fas andFasL was higher in group I than in group C.CONCLUSION:The positive expression of α-SMA andNF-κB in hepatic stellate cells is decreased by ectogenicIL-10 in liver fibrosis induced by CCl_4.The expression ofFas and FasL is increased in the course of liver fibrosis,and is further increased by IL-10.IL-10 could inhibitthe activation of HSCs and cause apoptosis of activatedHSCs.     

洛伐他丁对肝星状细胞增殖及细胞外基质分泌的影响

目的 观察洛伐他丁对肝星状细胞增殖及细胞外基质分泌的影响,并探讨其作用机制。方法 用不同浓度的洛伐他丁和胆固醇合成过程中产生的非脂性中间产物香叶基香叶基焦磷酸处理大鼠肝星状细胞株;用MTT法检测细胞增殖,流式细胞仪检测细胞周期,ELISA检测细胞外基质IV型胶原和层粘连蛋白,免疫细胞化学结合计算机图文分析系统检测c—jun、c-fos基因表达。结果 洛伐他丁可剂量依赖性地抑制肝星状细胞增殖（对照组A值24 h和48 h分别为0.736±0.090和0.972±0.097,洛伐他丁浓度在10μmol/L时24h和48h分别为0.602±0.049和0.785±0.028,两组比较差异有显著性）,影响其细胞周期,使G0/G1期细胞增多,S期细胞减少,并明显抑制c-jun、c—fos表达（洛伐他丁浓度在 50μmol/L时分别较对照组降低51.5％和 54.5％）,抑制IV型胶原和层粘连蛋白分泌（P<0.01）。而香叶基香叶基焦磷酸可部分拮抗洛伐他丁的上述抑制作用。结论 洛伐他丁可显著抑制肝星状细胞增殖及细胞外基质分泌,其机制可能与抑制香叶基香叶基焦磷酸产生而阻止信号转导有关。     

大麻素受体2介导的N-花生四烯酸氨基乙醇对肝星状细胞增殖和活化的影响

目的 观察内源性大麻素N-花生四烯酸氨基乙醇(AEA)及大麻素受体(CBR)2对肝星状细胞(HSC)增殖活化的影响,以探讨内源性大麻素及其受体系统在肝纤维化发展中的作用.方法 采用免疫荧光观察血小板衍生生长因子(PDGF)刺激前后HSC中CBR1和CBR2的表达.Western blot、PCR法观察不同浓度AEA及CBR2拮抗剂AM630对PDGF刺激下HSC增殖及活化的影响,同时用四甲基偶氮唑盐、流式细胞仪分析AEA对HSC活力及凋亡的影响.结果 HSC中CBR2的表达较CBR1高(F=116.797,P<0.01),且PDGF刺激后CBR2的表达明显增强(F=7.878,P<0.05).AEA可剂量依赖地抑制HSC的增殖,在浓度为10,20、50μmol/L时抑制率分别为7.12%±0.34%、12.52%±0.78%、80.13%±1.57%,差异有统计学意义(F=533.41,P<0.01);但对HSC凋亡的影响不明显.同时AEA可抑制HSC的活化指标α-平滑肌肌动蛋白、转化生长因子β1、Ⅰ型胶原、Ⅲ型胶原及基质金属蛋白酶抑制因子等的表达,但这种抑制作用在给予CBR2拮抗剂AM630后明显减弱,差异有统计学意义(P<0.05).结论 CBR2在AEA引起的HSC增殖及活化抑制中起关键作用,AEA和CBR2可望成为肝纤维治疗的新靶点.     

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.     

木犀草素抑制肝星状细胞增殖及其胶原合成

目的 研究木犀草素对体外培养的肝星状细胞(hepatic stellate cells,HSC)增殖及其胶原表达、合成的影响。方法 从Wistar大鼠肝脏分离培养HSC,并用~3H-TdR和~3H-pro同位素掺入实验,基因探针原位杂交等技术研究了木犀草素对HSC增殖、胶原基因表达合成的影响。结果 当木犀草素的浓度分别达到10 μmol/L和20 μmol/L 时抑制HSC增殖(t=2.542,P＜0.05)和胶原合成(t=3.650,P＜0.01),其作用具有剂量依赖关系;25 μmol/L木犀草素使Ⅰ、Ⅲ型前胶原mRNA的表达降低,其中Ⅰ型前胶原基因表达降低具有统计学差异(x~2=6.850,P＜0.01)。结论 木犀草素在体外抑制HSC增殖和胶原表达合成,在体内可能会具有预防或冶疗肝纤维化的作用。     

sp600125对乙醛刺激的大鼠肝星状细胞增殖及bcl-2、c-myc蛋白表达的影响

肝星状细胞（HSC）的活化与增殖是肝纤维化发生的关键环节。乙醛刺激HSC活化与增殖，是导致酒精性肝纤维化发生的关键因素。研究表明，丝裂原活化蛋白激酶（MAPK），包括细胞外信号调节激酶（ERK）、JNK、P38是HSC活化与增殖导致肝纤维化发生的主要信号传导通路之一。乙醛刺激HSC中JNK磷酸化水平随sp600125（JNK信号传导通路特异性阻断剂）浓度增加而减少。本实验用sp600125处理乙醛刺激的HSC，观察sp600125对HSC增殖以及bcl-2、c-myc蛋白表达的影响，以探讨酒精性肝纤维化的发生机制。     

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.     

活化肝星状细胞的信号传导

肝纤维化是肝脏对各种急慢性肝损伤的疤痕修复反应的结果,是各种慢性肝病向肝硬化进展的共同环节,其最主要的特征是以胶原为主的细胞外基质(ECM)的生成与降解失衡,在肝脏中的过度沉积.活化的肝星状细胞(HSC)是肝纤维化发生时ECM的主要来源.HSC持续激活后增殖、迁移和表型转化,成为肌成纤维样细胞,是肝纤维化发生和发展的核心环节.活化HSC具有如下特点:(1)合成和分泌间质胶原等各种ECM;(2)自分泌产生致纤维化细胞因子如转化生长因子(TGF)β I;(3)释放胶原酶抑制物组织金属蛋白酶抑制剂(TIMP);(4)分泌趋化因子及其他炎症性细胞因子;(5)具有细胞收缩特性;(6)合成基质金属蛋白酶(MMP),使ECM降解异常;(7)对凋亡刺激的耐受性等.     

一种改良的肝星状细胞分离方法

目的改良肝星状细胞（HSC）的分离方法,为深入研究肝纤维化的发生机制奠定基础。方法采用Ⅳ型胶原酶和链酶蛋白酶原位消化肝脏,在1．040～1．060g／mL范围内多重多次密度梯度离心分离HSC,台盼蓝染色测定细胞存活率,328nm紫外光激发HSC自发蓝绿色荧光法和Desmin细胞免疫荧光法鉴定HSC及检测HSC纯度。Desmin和n—SMA细胞免疫荧光方法鉴定HSC静息和活化状态。结果每只大鼠肝脏HSC得率为（3～5）×10^7个,HSC存活率在95％以上,纯度在90％以上,体外培养14d后活化的HSC纯度几乎达100％。结论在1．040～1．060g／mL密度范围内采用多重多次密度梯度离心方法,避免了HSC分离过程中因密度配比误差及HSC自身密度相对不均一性所导致的细胞流失,建立了一种改良的肝星状细胞分离方法,HSC得率和纯度更加稳定,达到国外文献报道水平。     

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。     

甘草次酸靶向肝星状细胞治疗肝纤维化的体内研究

目的观察以6-磷酸甘露糖修饰的白蛋白(M6P26-HSA)作为特异性载体,将甘草次酸(GA) 靶向释放到肝星状细胞治疗肝纤维化的效果。方法用125I记由M6P26-HSA和GA在体外合成新的偶合物GA-HSA-M6P26,观察其在体内的器官分布情况,用双重免疫组织化学的方法观察星状细胞对GA- HSA-M6P26的选择性摄取;选用Sirius红染色观察GA-HSA-M6P26对肝纤维化时胶原沉积的影响,用定量聚合酶链反应检测GA-HSA-M6P26对Ⅰ型前胶原mRNA表达的影响。结果静脉注射后10min,GA-HSA- M6P26选择性地分布于肝脏,摄取高峰可达(5 5.093±5.404)%。双重免疫组织化学染色证实GA-HSA- M6P26主要被星状细胞选择性摄取,GA-HSA-M6P26治疗后肝脏胶原沉积明显减少,Ⅰ型前胶原和α-平滑肌肌动蛋白mRNA表达明显降低。结论GA-HSA-M6P26可以选择性地分布于肝脏星状细胞,有显著的抗肝纤维化作用。     

酒精性肝病患者血浆白细胞介素-21水平及重组IL-21对LX-2肝星状细胞增殖和活化的影响

目的：分析酒精性肝病患者血浆白细胞介素-21（IL-21）水平及重组IL-21体外对LX-2肝星状细胞增殖和活化的影响。方法采用ELISA法检测17例酒精性肝炎、51例酒精性肝硬化患者和20例健康人血浆IL-21水平;体外培养LX-2肝星状细胞,以IL-21（1ng/ml或10ng/ml）处理24 h或48 h,检测LX-2细胞增殖及α-平滑肌肌动蛋白表达。结果与健康对照人群比,酒精性肝炎和酒精性肝硬化患者血浆IL-21水平均显著升高（P＆lt;0.05）,但酒精性肝炎和肝硬化患者之间无显著性差异,不同Child分级的肝硬化患者之间也无显著性差异;在IL-21作用24～48 h后,LX-2细胞增殖水平与对照组比无显著性差异,但α-平滑肌肌动蛋白表达均较对照组显著升高。结论血浆IL-21可能通过促进肝星状细胞活化参与了酒精性肝病患者肝纤维化的发生和发展。     

肝星状细胞激活与信号转导

肝纤维化是多种慢性肝病向肝硬化发展的必经阶段,是所有慢性肝病的共同病理基础.目前认为肝星状细胞的激活是肝纤维化形成的关键,各种致病因素作用下引起肝损伤,释放多种细胞因子,如转化生长因子β、血管紧张素、瘦素,通过各种信号转导使肝星状细胞激活.本文就肝星状细胞激活过程中一些重要的膜受体、核受体信号转导途径及其研究进展作一综述.     

Alpha-SMA expression in hepatic stellate cells and quantitative analysis of hepatic fibrosis in cirrhosis and in recurrent chronic hepatitis after liver transplantation

BACKGROUND: The alpha isotype of actin expressed by hepatic stellate cells reflects their activation to myofibroblast-like cell and has been directly related to experimental liver fibrogenesis, and indirectly to human fibrosis in chronic liver disease. AIMS: To evaluate the changes in distribution and percentage of alpha-smooth muscle actin-positive hepatic stellate cells and the correlation with the degree of the fibrosis in cirrhotic livers, as well as in patients with recurrent HCV chronic hepatitis after liver transplantation. METHODS: Human liver biopsies were divided in four groups: (1) normal livers obtained from cadaveric liver donors (n=35), (2) cirrhosis post-HBV hepatitis (n=11), (3) cirrhosis post-HCV hepatitis (n=10), and (4) post-transplant recurrent HCV chronic hepatitis (n=13). Samples were stained with anti-alpha-smooth muscle actin antibody by immunoperoxidase method and semi-quantitatively evaluated. Liver fibrosis was assessed from specimens stained with Masson's trichrome and quantified by computer image analysis. RESULTS: The percentage of alpha-smooth muscle actin-positive hepatic stellate cells was significantly higher in the HBV cirrhosis, HCV cirrhosis and post-transplant HCV recurrent hepatitis groups (36.1+/-15.2, 23.8+/-19.7 and 27.8+/-16.4%, respectively) compared to the liver donor group (2.9+/-4.0%). The alpha-smooth muscle actin-positive hepatic stellate cells to fibrous tissue ratio were significantly higher in the post-transplant recurrent HCV hepatitis group (2.36+/-1.12) compared to both the donor livers and the HCV cirrhosis groups (0.74+/-1.09 and 1.03+/-0.91, respectively). The alpha-smooth muscle actin-positive hepatic stellate cell percentage and fibrosis correlated positively in the post-transplant recurrent HCV hepatitis group and negatively in the HCV cirrhosis group. No difference in the immunohistochemical and morphometrical variables was found between the HCV cirrhosis and HBV cirrhosis groups. CONCLUSIONS: These results indirectly confirm that, in vivo, alpha-smooth muscle actin expression is a reliable marker of hepatic stellate cells activation which precedes fibrous tissue deposition even in the setting of recurrent HCV chronic hepatitis after liver transplantation, and it could be useful to identify the earliest stages of hepatic fibrosis and monitoring the efficacy of the therapy. In the presence of advanced cirrhosis other factors, rather than alpha-smooth muscle actin-positive hepatic stellate cells, may sustain fibrosis deposition.