肝星状细胞SSeCKS的表达及其在肝纤维化中的作用

目的 了解src抑制的蛋白激酶C底物(SSeCKS)在肝星状细胞(HSC)活化中的变化和作用.方法 (1)分离正常大鼠的HSC,以实时定量PCR检测其在体外培养过程中SSeCKS mRNA的表达变化,以蛋白质印迹法和细胞免疫荧光法检测HSC中SSeCKS蛋白的表达变化.(2)制作肝纤维化的大鼠模型,以免疫荧光法检测肝组织内SSeCKS和α-平滑肌肌动蛋白(α-SMA)的表达变化及定位.结果 初分离的HSC表达低水平的SSeCKS mRNA,经培养活化后SSeCKS的表达增强.在纤维化肝组织中,SSeCKS阳性细胞增多,阳性细胞多分布于肝窦周围,与α-SMA阳性细胞的分布一致.结论 在体外活化过程中HSC中SSeCKS表达增强.SSeCKS可能参与HSC在肝脏炎症和纤维化中的作用.     

整合素αvβ3在肝星状细胞和肝纤维化组织中的表达

目的 研究整合素avβ3在肝星状细胞和纤维化肝组织中的表达,探讨能反映肝星状细胞活化状态的表型受体.方法 分离大鼠肝星状细胞,体外培养激活.硫代乙酰胺175 mg/kg每周2次,连续12周腹腔注射诱导大鼠肝纤维化.免疫染色观察细胞和组织中整合avβ3和a-平滑肌肌动蛋白(a-SMA)的表达.实时PCR和Western印迹法分析avβ3在星状细胞中、正常和肝纤维化组织中的表达.结果 活化星状细胞中avβ3表达明显上调,与第1天相比,第14天的mRNA和蛋白质水平分别增加18倍和5.2倍.免疫染色整合素avβ3成簇极性表达于活化星状细胞的胞膜.肝纤维化模型中avβ3在mRNA和蛋白水平明显高于正常对照(t=2.39,P＜0.05;t=2.74,P＜0.05),正常肝组织中a-SMA和avβ3局限表达于门静脉周围,肝纤维化组织中a-SMA和avβ3表达于扩大的门管区和纤维间隔.结论 体内外活化星状细胞中整合素avβ3表达水平上调,它是星状细胞活化的表型受体,参与肝纤维化的发生.     

姜黄素对肝纤维化大鼠肝组织中NF-κB及ERK-1 mRNA表达的影响

目的 探讨核转录因子-κB(nuclear factor kappa B,NF-κB)及细胞外信号调节激酶(extracegular signal-reglulated kinase,ERK)mRNA在CCl4致实验性肝纤维化大鼠肝组织中的表达及姜黄素(curcumin,CUR)对它们的影响.方法 建立CCl4致大鼠实验性肝纤维化的模型,用免疫组化法比较正常组、模型组及CUR组的α平滑肌肌动蛋白(α-SMA)的表达,用RT-PCR法比较各组肝组织NF-κB及ERK-1 mRNA的表达.结果 模型组中,ERK-1及NF-κB mRNA的表达均增高,而CUR组α平滑肌肌动蛋白则较模型组低,NF-κB及ERK-1 mRNA的表达也降低.结论 CUR可能通过抑制α-SMA的表达,减弱NF-κB诱导的肝星状细胞的活化及抑制ERK-1促肝星状细胞的增殖,从而产生抗四氯化碳诱导的大鼠肝纤维化的作用.     

高血糖对大鼠肝星状细胞活化及转化生长因子-β1和结缔组织生长因子表达的影响

目的探讨高血糖加重四氯化碳中毒大鼠肝纤维化进展的机制.方法24只SD大鼠分为对照组、高血糖组、正常血糖 四氯化碳组、高血糖 四氯化碳组,通过链脲佐菌素诱导高血糖,4周后用四氯化碳诱导肝纤维化,第9周处死.免疫组化法检测肝组织α-平滑肌肌动蛋白表达(反映肝星状细胞活化);实时RT-PCR及免疫印迹法测量肝组织转化生长因子-β1(TGF-β1)、结缔组织生长因子(CTGF)mRNA及蛋白表达.结果肝星状细胞活化、TGF-β1和CTGFmRNA和蛋白表达在对照组和高血糖组无明显改变,正常血糖 四氯化碳组、高血糖 四氯化碳组较对照组升高,后者较前者变化更为明显.这些改变与肝纤维化积分相平行.结论高血糖可通过诱导肝脏TGF-β1、CTGF表达以及星状细胞活化,加重四氯化碳中毒大鼠肝纤维化的程度.     

大鼠纤维化肝组织中PTEN的动态表达及其与肝星状细胞增殖和活化的关系

目的 探讨第10号染色体缺失的磷酸酶张力蛋白同源物基因(PTEN)在大鼠纤维化肝组织中的动态表达及其对在体肝星状细胞(HSC)活化、增殖的影响. 方法 采用胆总管结扎法建立大鼠肝纤维化模型;应用免疫组织化学染色、Western blot和实时荧光定量PCR技术测定大鼠肝组织中PTEN的表达;应用免疫荧光双标记共聚焦激光扫描显微术测定大鼠肝组织中活化HSC的PTEN表达;采用免疫组织化学染色检测大鼠肝组织中α-平滑肌肌动蛋白的表达.结果 免疫组织化学染色显示正常大鼠肝组织中PTEN有广泛表达,主要表达于细胞质,随着肝纤维化的发展,PTEN表达逐渐减少(P<0.01),而α-平滑肌肌动蛋白阳性细胞明显增多(P<0.01);造模1、2、3周及4周不同时间大鼠纤维化肝组织中PTEN的mRNA(分别为假手术组的0.66、0.53、0.44和0.37)及蛋白质表达(吸光度比值分别为1.20±0.13,1.07±0.16,0.88±0.08,0.73±0.07)均低于假手术组(P<0.01),并随着肝纤维化的进展逐渐降低(P<0.01);免疫荧光双标记共聚焦激光扫描显微术显示PTEN在活化HSC广泛表达,主要表达于细胞质,随着肝纤维化的进展,表达PTEN的活化HSC占总的活化HSC的比例逐渐减少(P<0.01). 结论 大鼠纤维化肝组织中PTEN的mRNA及蛋白质表达均下调;在体HSC的PTEN表达亦降低;肝组织中PTEN的动态表达与HSC的活化、增殖呈显著负相关.     

姜黄素预防肝纤维化作用与肝星状细胞的关系

目的观察姜黄素预防大鼠肝纤维化作用及活化肝星状细胞(HSC)的数目、分布、凋亡等变化,并探讨两者间的关系。方法以四氯化碳制作大鼠肝纤维化模型,同时按每100 g体重分别给予20、10、5 mg姜黄素灌胃处理,设立正常对照组、肝纤维化组和阳性对照组;8周后处死大鼠,留取肝左叶行HE、Masson染色,参照肝纤维化半定量计分系统进行肝纤维化程度评分,免疫组织化学方法检测α-平滑肌肌动蛋白以了解活化HSC的数量变化, TUNEL与肌源性特异性标志物结合蛋白(Desmin)免疫组织化学双染法检测HSC凋亡。结果姜黄素可明显改善四氯化碳所致大鼠肝纤维化的病理学改变;α-平滑肌肌动蛋白在肝纤维化时表达明显增多,姜黄素使活化HSC数量减少, }[SC凋亡增加,与对照组比较差异具有统计学意义(P<0．05),且具有量效关系。结论姜黄素可抑制HSC活化、增殖,诱导HSC凋亡,可能为预防肝纤维化的作用机制之一。     

脂联素对肝星状细胞增殖和活化的影响

目的 观察外源性脂联素对体外培养大鼠肝星状细胞(HSC)增殖和活化的影响,探讨脂联素对肝纤维化的影响.方法 外源性脂联素(0.1 μtg/mL、1μtg/mL、10 μg/mL)处理体外培养的HSC-T6,MTT法分析其对HSC-T6增殖的影响,Western blot法检测HSC-T6细胞中α-平滑肌肌动蛋白(α-SMA)的表达.结果 脂联素对HSC的抑制作用随药物浓度的增加而增强,α-SMA的蛋白表达随脂联素浓度增加而下降.结论 脂联素能抑制HSC-T6的增殖,从而发挥抗肝纤维化作用.     

Toll样受体4在CCI4诱导的大鼠慢性肝损伤过程中的表达

背景：Toll样受体4（TLR4）在内毒素的信号转导过程中具有重要作用。目的：动态观察在CCl4诱导的大鼠慢性肝损伤过程中。肝组织和Kupffer细胞TLR4基因表达的变化，探讨TLR4在肝损伤中的作用。方法：以CCl4诱导慢性肝损伤纤维化大鼠模型，分离肝Kupffer细胞。以逆转录聚合酶链反应（RT-PCR）检测肝组织和Kupffer细胞TLR4 mRNA的表达；将Kupffer细胞分别与不同浓度的脂多糖（LPS）孵育，以酶联免疫吸附测定（ELISA）检测细胞培养上清的肿瘤坏死因子（TNF）-α水平。以基质显色法测定大鼠血浆内毒素水平。结果：正常大鼠肝组织TLR4 mRNA表达水平较低，Kupffer细胞未检测到TLR4 mRNA表达；CCl4处理2～6周大鼠的肝组织和Kupffer细胞TLR4 mRNA表达水平显著增高（P〈0．05）。CCl4处理4周和6周大鼠Kupffer细胞的TNF-α基础分泌水平显著高于正常大鼠（P〈0．05）；在LPS的刺激下，TNF-α的分泌水平较基础值进一步增高（P〈0．05），呈浓度依赖性。大鼠血浆内毒素水平在肝损伤过程中逐渐增高，相关分析显示在慢性肝损伤的早、中期。肝组织和Kupffer细胞TLR4 mRNA的表达与血浆内毒素水平呈正相关。结论：在CCl4诱导的慢性肝损伤过程中，大鼠肝脏TLR4基因表达上调，与Kupffer细胞活化和肝脏的炎症损伤有关。     

锌α2糖蛋白抑制肝星状细胞活化和增殖的体外研究

目的探讨锌α2糖蛋白(AZGP1)在肝星状细胞活化和肝纤维化发生和发展中的作用机制。方法采用转化生长因子-β1刺激诱导人肝星状细胞株LX2活化和构建四氯化碳小鼠肝纤维化模型,观察细胞和肝组织中AZGP1的表达情况。应用质粒转染法过表达和抑制表达AZGP1后,分别检测LX2细胞的活化、增殖和凋亡功能及相关因子的改变。多组间比较采用单因素方差分析。结果免疫荧光染色结果显示在活化的LX2细胞中,AZGP1蛋白减少而α-平滑肌肌动蛋白增多,二者呈负相关。AZGP1基因和蛋白在活化的LX2细胞和四氯化碳肝纤维化小鼠的肝组织内显著低表达。过表达AZGP1后,LX2细胞中Ⅰ型胶原、基质金属蛋白酶-2和α-平滑肌肌动蛋白基因和蛋白明显下调;细胞荧光显示过表达AZGP1的细胞活化和α-平滑肌肌动蛋白减少。此外,过表达AZGP1后,LX2细胞的增殖活性和G1/S-特异性周期蛋白D1蛋白显著降低;细胞周期实验显示过表达AZGP1的细胞停滞在G0/G1期比例显著增多、而S期比例显著减少。AZGP1对LX2细胞凋亡无明显影响。结论AZGP1可通过抑制肝星状细胞活化和增殖而逆转肝纤维化;过表达AZGP1有望成为肝纤维化治疗的新靶点。     

一次性大剂量二甲基亚硝胺致大鼠肝纤维化模型中肝星状细胞的病理变化

目的观察一次性大剂量二甲基亚硝胺（DMN）致大鼠肝纤维化模型中肝星状细胞的病理学变化。方法将大鼠分为对照组和模型组。造模组以DMN50mg·kg^-1大鼠体重的剂量一次性腹腔注射。分别于注射后6、12、24和36小时,2、3、5、7、10和14天,1、3、6和12个月取肝组织进行病理形态学观察及α-平滑肌肌动蛋白（α—SMA）、单核／巨噬细胞表面特异性标志抗原（Ectodermal dysplasia-1,ED-1）和转化生长因子151（TGF-β1）免疫组织化学染色,并采用逆转录一聚合酶链式反应（RT—PCR）法检测肝组织内Ⅰ型胶原和TGF-β1 mRNA的表达。结果模型组大鼠在损伤后出现6小时～5天、5—14天和14天～12月三个不同的反应阶段。6小时后中央静脉周围肝实质细胞开始出现点状坏死,于36小时达到急性亚大片出血性坏死的高峰,坏死区内无残存的肝星状细胞;3天时残存肝实质内出现α-SMA阳性的活化肝星状细胞。坏死区内大量ED-1阳性的巨噬细胞聚集,并表达TGF—β1蛋白;第5天时,坏死区内组织碎片和红细胞被巨噬细胞清除。肝星状细胞不断活化、增生、并向坏死区周围及坏死区内移动。部分肝星状细胞开始表达TGF—β1蛋白;第7天时,大量活化的肝星状细胞沿肝窦和中央静脉间纤细的纤维间隔内分布,并与巨噬细胞一起表达TGF-β1蛋白;第14天时,坏死灶完全被再生的肝细胞及纤维组织取代,中央静脉之间桥接纤维化完全形成。纤维间隔内存在大量活化的肝星状细胞和巨噬细胞;此后至12个月,肝纤维化形态始终保持,无其他进展性或可逆性变化发生。结论采用一次性大剂量DMN注射,可获得大鼠肝脏亚大片坏死后性纤维化的稳定模型。研究形成过程中的肝星状细胞、巨噬细胞和肝细胞等实质细胞的形态和功能变化以及它们之间的相互作用对进一步阐明肝纤维化及其相关肝脏疾病的发生机制有重要的意义。     

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.     

CD14-positive hepatic monocytes/macrophages increase in hereditary hemochromatosis.

BACKGROUND/AIMS: Iron overload in hereditary hemochromatosis (HH) may result in hepatic fibrosis and cirrhosis, primarily due to collagen production by hepatic stellate cells that become activated to myofibroblasts. Endotoxin-responsive monocytes/macrophages (CD14-positive) are potential sources of profibrogenic factors. The aims of this study were to determine (1) whether CD14-positive monocytes/macrophages are present in the livers of patients with HH and (2) the potential relationship between CD14-positive cells and hepatic fibrosis in HH. METHODS: HH was diagnosed using standard clinical, biochemical and genotypic parameters. Liver specimens from HH patients and control subjects were immunostained for CD14, CD68 and alpha-smooth muscle actin (alpha-SMA) and the number of cells expressing these antigens was determined. Fibrosis was assessed by routine histological methods. RESULTS: The total number of hepatic CD68-positive monocytes/macrophages was similar in HH patients and control subjects; however, there was a nine-fold increase in the number of CD14-positive monocytes/macrophages in HH patients. Control subjects had very low levels of hepatic CD14 expression. In HH livers with advanced fibrosis, CD14-positive monocytes/macrophages were often associated with fibrous septa containing myofibroblasts expressing alpha-SMA. CONCLUSIONS: There was a substantial increase in hepatic CD14-positive monocytes/macrophages in HH and, in livers with advanced fibrosis, these cells were often associated with fibrous septa and septal myofibroblasts. The total number of monocytes/macrophages was similar in HH and control livers. In control human liver, Kupffer cells had a very low expression of CD14. These findings suggest that CD14-positive monocytes/macrophages may contribute to the process of hepatic fibrogenesis in HH.     

Toll-like receptor 4 mediates inflammatory signaling by bacterial lipopolysaccharide in human hepatic stellate cells

Bacterial lipopolysaccharide (LPS) stimulates Kupffer cells and participates in the pathogenesis of alcohol-induced liver injury. However, it is unknown whether LPS directly affects hepatic stellate cells (HSCs), the main fibrogenic cell type in the injured liver. This study characterizes LPS-induced signal transduction and proinflammatory gene expression in activated human HSCs. Culture-activated HSCs and HSCs isolated from patients with hepatitis C virus-induced cirrhosis express LPS-associated signaling molecules, including CD14, toll-like receptor (TLR) 4, and MD2. Stimulation of culture-activated HSCs with LPS results in a rapid and marked activation of NF-kappaB, as assessed by in vitro kinase assays for IkappaB kinase (IKK), IkappaBalpha steady-state levels, p65 nuclear translocation, NF-kappaB-dependent luciferase reporter gene assays, and electrophoretic mobility shift assays. Lipid A induces NF-kappaB activation in a similar manner. Both LPS- and lipid A-induced NF-kappaB activation is blocked by preincubation with either anti-TLR4 blocking antibody (HTA125) or Polymyxin B. Lipid A induces NF-kappaB activation in HSCs from TLR4-sufficient (C3H/OuJ) mice but not from TLR4-deficient (C3H/HeJ) mice. LPS also activates c-Jun N-terminal kinase (JNK), as assessed by in vitro kinase assays. LPS up-regulates IL-8 and MCP-1 gene expression and secretion. LPS-induced IL-8 secretion is completely inhibited by the IkappaB super repressor (Ad5IkappaB) and partially inhibited by a specific JNK inhibitor, SP600125. LPS also up-regulates cell surface expression of ICAM-1 and VCAM-1. In conclusion, human activated HSCs utilize components of TLR4 signal transduction cascade to stimulate NF-kappaB and JNK and up-regulate chemokines and adhesion molecules. Thus, HSCs are a potential mediator of LPS-induced liver injury.     

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.     

Dynamic expression of extracellular signal-regulated kinase in rat liver tissue during hepatic fibrogenesis

INTRODUCTION Mitogen-activated protein kinase (MAPK) pathway is an important intracellular signal transduction system[1], and extracellular signal-regulated kinase 1 (ERK1) is the critical and classical pathway of MAPK and plays an important role in sever…