肝纤维化肝窦内皮细胞免疫功能改变的研究进展

正常肝脏是免疫耐受大于免疫原性的器官,肝窦内皮细胞(HSEC)在参与正常肝脏免疫耐受方面的研究是热点。在肝脏疾病中,很少有关HSEC免疫方面的研究。肝纤维化过程中,HSEC结构和功能的改变,可能造成其免疫功能的转变(免疫耐受转化为免疫应答),但HSEC免疫功能的转变,也可能影响肝纤维化的进程,研究纤维化肝脏肝窦内皮细胞(FH-SEC)免疫功能,对应用免疫手段治疗肝纤维化有重要意义。     

肝纤维化肝窦内皮细胞对肝星状细胞的影响

肝纤维化是肝损伤后修复性应答。当损肝因素导致肝损伤时,肝内相关细胞分泌多种细胞冈子经旁分泌和自分泌的形式激活肝星状细胞(hepatic stellate cell,HSC),致使其分泌大量细胞外基质(extracellular matrix,ECM),导致ECM的生成大于降解,以致ECM过度沉积,从而引起肝窦毛细血管化,促使肝纤维化形成,故HSC的激活是     

p-moesin在血吸虫病肝纤维化鼠肝窦内皮细胞中的表达及意义

目的:动态观察血吸虫病肝纤维化鼠肝窦内皮细胞moesin表达水平的时相变化,探讨其与肝窦内皮细胞失窗孔化的关系。方法:采用腹部敷贴法感染血吸虫尾蚴建立血吸虫病性小鼠肝纤维化模型,除正常对照组(A组,10只)外,78只成模小鼠分为血吸虫病组(B组,24只)、吡喹酮(Biltricide)杀虫片治疗组(C组,18只)、Rock抑制剂(Hydroxyfasudil)治疗组(D组,18只)、Biltricide+Hydroxyfasudil治疗组(E组,18只),分别于治疗第16周、19周、21周分批剖腹取各组小鼠肝组织进行HE、Masson染色;同时Western blotting检测p-moesin蛋白表达,透射电镜观察超微结构。结果:与A组相比,B组肝组织p-moesin蛋白表达量增加。给予药物干预后,D组、E组p-moesin蛋白降低,16周时最为明显,但D组于19周开始逐渐恢复到原水平,而E组继续下降,明显低于D组。肝窦超微结构观察,药物干预21周时,C组与D组相比,肝组织炎性细胞明显减少,Disse腔内胶原纤维有所减少,但肝窦内皮细胞窗孔、肝窦内皮下基底膜无明显好转。E组与C组比较肝细胞器形态明显恢复,可见窗孔,未见基底膜。结论:血吸虫病肝纤维化小鼠肝窦内皮细胞通过p-moesin蛋白表达上调,参与肝窦内皮细胞失窗孔化,从而引起肝内微循环障碍。     

结缔组织生长因子在血吸虫病肝纤维化鼠肝窦内皮细胞中的表达及意义

目的:动态观察结缔组织生长因子(CTGF)蛋白在血吸虫病肝纤维化鼠肝窦内皮细胞表达水平的时相变化,探讨其与肝窦内皮细胞下基底膜形成的关系。方法:采用腹部敷贴法建立血吸虫病肝纤维化模型,HE、Masson染色和透射电镜观察其病理变化;免疫组化技术检测CTGF、Ⅳ型胶原(ColⅣ)和层粘连蛋白(LN)在小鼠肝脏组织中的表达和分布;并应用彩色图像分析仪进行定量分析。结果:与正常对照组比较,模型组鼠肝窦内皮细胞表达CTGF蛋白阳性或强阳性,肝窦壁LN、ColⅣ表达水平增高,且随着肝纤维化的发展,CTGF和LN、ColⅣ表达逐渐增强,肝窦内皮下基底膜逐渐增厚。图像定量分析两组平均吸光度值、平均灰度值和阳性面积比具有统计学差异;CTGF蛋白与LN、ColⅣ水平呈正相关。结论:血吸虫病肝纤维化时小鼠肝窦内皮细胞通过CTGF蛋白表达上调,调控细胞外基质产生,导致ColⅣ、LN分泌增加,参与肝窦内皮下基底膜形成,从而引起肝内微循环障碍。     

卵巢切除对四氯化碳诱导大鼠肝纤维化形成的影响

为探讨卵巢切除对CCl4 诱导大鼠肝纤维化形成的影响 ,采用CCl4 诱导雌性大鼠肝纤维化动物模型 ,观察卵巢切除及雌激素替代治疗 (苯甲酸雌二醇 1mg kg)对肝脏胶原沉积和I、Ⅲ型胶原蛋白表达的影响 ,并分别检测血清学标志及肝脏组织学等变化。结果显示CCl4 模型组大鼠肝脏发生典型的肝纤维化改变 ,卵巢切除组的肝脏胶原沉积更为明显 ,肝脏表达I、Ⅲ型胶原及血清肝纤维化指标也明显高于CCl4 摸型组 (P <0 0 5 ) ,而雌激素干预及替代治疗则可抑制肝纤维化的形成。表明卵巢切除加速CCl4 诱导大鼠肝纤维化的形成 ,其发生可能与卵巢分泌的雌激素对肝纤维化的抑制作用有关。     

槲皮素对四氯化碳诱导的大鼠肝纤维化抑制作用和肝保护作用

目的:探讨槲皮素对四氯化碳(CCl4)诱导大鼠肝纤维化的保护作用,并阐明槲皮素的抗肝纤维化机制.方法:Wistar大鼠随机分为5组:对照组、模型组、低剂量组(EXP-L)、中剂量组(EXP-M)、高剂量组(EXP-H).HE染色检测肝组织病理损伤情况;试剂盒检测谷丙转氨酶(ALT)和谷草转氨酶(AST)活力;羟脯氨酸(...     

肝窦内皮细胞的分离纯化及鉴定方法

肝窦内皮细胞(liver sinusoidal endothelial cells,LSEC)是肝脏非实质细胞(nonparenchymal cell,NPC)的主要细胞群,占这些细胞数的40％左右,由LSEC组成的肝窦壁是全身毛细血管壁中唯一缺乏基底膜的毛细血管,同时LSEC具有特殊的标志性结构-窗孔.肝窦内皮细胞不仅在形态上与其他血管内皮细胞不同,在功能上也有不同.肝窦内皮细胞组成肝窦壁,构成血流与肝细胞间的屏障,在调节肝窦血流与周围组织的物质交换中起有效的中枢性作用,对于维持正常的肝功能起十分重要的作用.在物质代谢方面,L.SEC在脂质、胆固醇和维生素A的平衡和分配方面具有重要的作用,同时能通过特异的内吞作用清除变性的大分子脂质、某些细胞外基质成分,在维持肝窦的正常生理功能方面发挥重要作用;在免疫方面,ISEC的位置使其可以第一时间与抗原接触,启动免疫应答,同时近年来发现LSEC能诱导T细胞免疫耐受,这在肝移植中有重要意义;此外,研究发现LSEC损伤在肝脏冷保藏和缺血再灌注损伤中占中心地位,而LSEC在缺血再灌注损伤以及冷保藏中的损伤机制也是近几年的研究热点.     

肝纤维化大鼠肝细胞内质网形态及GRP78表达的研究

目的: 观察四氯化碳(CCl₄)致大鼠肝纤维化过程中内质网形态及内质网应激标志性蛋白——葡萄糖调节蛋白78(GRP78)的表达变化, 探讨内质网应激在肝纤维化发病机制中可能的作用。方法: 雄性Wistar大鼠皮下注射CCl₄制备肝纤维化模型,分别在4周及8周处死大鼠测定肝脏指数、血清丙氨酸氨基转移酶(ALT)和天冬氨酸氨基转移酶(AST)活性, 观察肝组织病理改变和肝细胞内质网形态, 免疫组化和real-time PCR分别检测肝组织GRP78蛋白及mRNA表达变化。结果: 肝纤维化组大鼠肝脏指数、血清ALT和AST活性显著高于正常对照组(P<0.01),肝纤维化明显, 电镜下见肝细胞内质网扩张,数量明显减少; 肝细胞胞浆中GRP78蛋白表达量及mRNA表达量较正常组显著增加(P<0.01)。结论: 在CCl₄诱导的肝纤维化发生过程中肝细胞内质网形态有明显损伤性变化, 内质网应激蛋白GRP78蛋白及基因表达水平明显增加, 提示内质网应激参与肝纤维化发生发展。     

四氯化碳诱导性肝纤维化大鼠肝组织骨形态发生蛋白-7的表达

目的 探讨四氯化碳(CCl4)诱导性肝纤维化大鼠的肝组织纤维化与骨形态发生蛋白-7(BMP-7)表达的相互关系.方法 将健康雄性 Wistar 大鼠随机分为对照组和肝纤维化组.对照组大鼠每周一、四背部皮下注射橄榄油(0.12ml/100g),肝纤维化组每周一、四背部皮下注射60% CCl4橄榄油(0.3ml/100g),连续注射6、10、16 和21周.实验结束时处死大鼠,取肝中叶,石蜡包埋、切片.行天狼猩红染色,观察肝组织胶原纤维的增生情况;行BMP-7免疫组织化学染色和 Western blotting 检测,观察肝组织.BMP-7的表达.结果 天狼猩红染色显示对照组大鼠仅在肝门静脉及门管区看到少量的胶原纤维,各肝纤维化组大鼠均可见肝组织呈明显的胶原纤维增生,且随着时间的延长,肝纤维化程度逐渐加重;免疫组织化学染色显示,对照组大鼠肝组织中可见极少量的BMP-7表达阳性的细胞,6周肝纤维化组大鼠可见较多的BMP-7阳性表达细胞,但随着实验时间的延长,BMP-7阳性细胞呈递减的趋势,至21周肝纤维化组大鼠肝组织中基本呈阴性表达;Western blotting 法检测大鼠肝组织BMP-7的表达结果与免疫组织化学的结果基本一致.结论 CC14诱导性肝纤维化大鼠肝组织BMP-7的表达与肝纤维化的程度呈负相关的趋势,推测BMP-7可能对肝纤维化具有一定的保护作用.     

Effect of cholinergic denervation on hepatic fibrosis induced by carbon tetrachloride in rats

Various factors involved in the development of liver fibrosis, including hepatic stellate cells (HSCs), cholinergic nervous activity and fibrogenetic cytokines. The present study aims to investigate the role of cholinergic regulation in the promoting of liver fibrogenesis relating to bone morphogenetic protein-6 (BMP-6) and/or transforming growth factor-beta1 (TGFbeta1). We treated carbon tetrachloride (CCl(4)) into rats for eight weeks to induce liver fibrosis and arranged these rats for cholinergic denervation, hepatic branch vagotomy or atropine administration. Acetylcholinesterase (AChE) staining showed the distribution of cholinergic nerve around fibrosis scaring septa. The immunohistochemical staining for alpha smooth muscle actin (alphaSMA) indicated the less HSCs in CCl(4) treated rat liver with cholinergic denervation as compared to the sham-operated CCl(4) treated rats. It seems that cholinergic nerve not only innervates around the fibrosis area but also promotes HSCs. We also detected TGFbeta1 and BMP-6 expressions using RT-PCR and immunohistochemistry. The obtained results show that cholinergic denerveration decreases BMP-6 and TGF-beta1 expressions in CCl(4) induced liver fibrosis of rats. In conclusion, cholinergic nerve may influence HSCs in addition to the lowering of BMP-6 and TGF-beta1 gene expressions to modify liver fibrosis.     

苦瓜对实验性大鼠肝纤维化的干预作用及可能机制

目的: 探讨苦瓜(BM)对四氯化碳(CCl₄)诱导大鼠肝纤维化的干预作用及相关机制。方法: 随机将32只雄性健康Wistar大鼠分为4组:对照组(C组);模型组(CCl₄,M组);BM低剂量组(BM 100g/kg饲料+CCl₄,BM-L组)、BM高剂量组(BM 200g/kg饲料+CCl₄,BM-H组)。饲养中除C组外的各组大鼠均皮下注射50%CCl₄-橄榄油溶液2 mL/kg,2次/周,共8周,诱导肝纤维化动物模型。8周后处死大鼠,留取大鼠肝脏和血清。计算肝体指数;测定血清丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性;测定肝匀浆总蛋白(TP)和白蛋白(Alb)含量、谷胱甘肽过氧化物酶(GSH-Px)活性、羟脯氨酸(HYP)含量和单胺氧化酶(MAO)活性;胶原纤维染色观察大鼠肝组织变性与胶原沉积病理改变。结果: 与M组比较,摄入BM后的各剂量组大鼠肝体指数显著降低(P<0.01);血清MDA含量及肝匀浆HYP含量和MAO活性均明显降低(P<0.01),而血清SOD活性、肝组织TP和Alb含量、GSH-Px活性明显增强(P<0.01)。与正常大鼠相比,模型大鼠肝脏有明显胶原沉积与肝纤维化,伴有不同程度的肝细胞炎性损伤坏死;BM组明显减轻模型大鼠肝组织损伤坏死与胶原沉积等病理变化,以高剂量组更明显。结论: BM具有抗CCl₄诱导大鼠肝纤维化作用,其机制可能与其抗脂质过氧化、降低肝HYP含量及MAO活性的作用有关。     

Danshensu-mediated protective effect against hepatic fibrosis induced by carbon tetrachloride in rats

The culprit of hepatic fibrosis (HF) is linked to suprathreshold deposition of collagen. Thus, collagen reduction by improved metabolism contributes to HF management. In this study, we aimed to investigate the hepatoprotective effects of Danshensu (DSS) against carbon tetrachloride (CCl₄)-induced HF rats. The results showed that DSS-administrated rats resulted in decreasing in hepatosomatic indexes, and lowering serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Meanwhile, the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) were increased, while the content of malonaldehyde (MDA) was lessened in liver tissue of DSS administration group. In addition, the pro-fibrotic markers of hydroxyproline (Hyp), type III procollagen (PCIII) and hyaluronic acid (HA) contents were decreased. Histopathological examination confirmed that the hepatotoxicity in CCl₄-injured rats was alleviated following the DSS administration. Furthermore, intrahepatic protein expressions of alpha-smooth muscle actin (α-SMA), phosphorylated JAK2 (p-JAK2) and phosphorylated STAT3 (p-STAT3) were effectively down-regulated, respectively. Overall, this work demonstrates that DSS played the protective effect against CCl₄-induced cytotoxicity in liver tissue, which the probable mechanism is associated with attenuation of lipid peroxidation, collagen accumulation and enhancement of anti-oxidative defense capability, as well as regulation of intrahepatic JAK/STAT pathway for maintaining collagenic homoeostasis.     

肝纤维化大鼠肝脏中组蛋白修饰水平的变化

目的:观察肝纤维化大鼠肝脏中组蛋白修饰的变化,并探讨其在肝纤维化发生发展过程中可能的作用。方法:雄性Wistar大鼠20只,随机分为正常对照组和肝纤维化组,其中肝纤维化组采用CCl_4皮下注射以制备大鼠肝纤维化模型,正常组注射等量植物油溶液。实验第8周末,股动脉放血处死大鼠,取2组血清,采用生化和放射免疫法测定血清肝功能指标丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST),以及肝纤维化标志物血清透明质酸(HA)、层粘连蛋白(LN)、Ⅳ型胶原(Col Ⅳ)和Ⅲ型前胶原(PCⅢ)的水平;取2组大鼠肝脏,测定肝脏指数;取肝组织常规固定,HE染色和Masson染色观察组织病理改变及胶原纤维沉积情况;Western blot检测2组大鼠肝脏组织中α-平滑肌肌动蛋白(α-SMA)和I型胶原(ColⅠ)表达情况,以及acH4K12、acH3K9、H3K4me2和H3K9me2修饰水平的变化。结果:与对照组相比,模型组大鼠肝脏指数及ALT、AST、HA、LN、ColⅣ和PCⅢ水平明显增高(P0.05);Western blot检测发现,与对照组比较,肝纤维化组大鼠肝组织的acH4K12修饰水平减少(P0.05),acH3K9和H3K9me2修饰水平及α-SMA和ColⅠ表达明显增加(P0.05),H3K4me2修饰水平的差异无统计学显著性。结论:肝纤维化大鼠肝脏中acH4K12、acH3K9和H3K9me2修饰水平改变可能与某些细胞外基质代谢相关基因转录调控有关,从而参与了大鼠肝纤维化发生。     

Cell biology and pathology of liver sinusoidal endothelial cells

Growing evidence revealed that liver sinusoidal endothelial cells (SEC) play several important roles in physiology and pathology of the liver. It has been well understood that their structural characteristics, such as the membrane sieve and lack of basement membrane, facilitate direct contact of soluble and insoluble serum substances with hepatic parenchymal cells, resulting in enhancement of hepatic metabolic activity. In addition, SEC is now regarded as a member of the scavenger endothelial cells, which have potential to eliminate a variety of macromolecules from the blood circulation by receptor-mediated endocytosis. It is reported that molecules preferentially eliminated by SEC are denatured or modified proteins such as advanced glycation end products, extracellular matrix components including hyaluronic acid, and some lipoproteins. The nature of the scavenger receptors corresponding to these molecules remains to be clarified. Recently, it was noted that SEC has an antigen-presenting function similar to dendritic cells. Taken together, it is suggested that SEC, cooperating with Kupffer cells and hepatic dendritic cells, may partake of immunoregulatory functions in the liver. SEC also plays a pivotal role in the pathological process of ischemia-reperfusion injury following liver surgery and liver transplantation. Thus, it is of importance to elucidate the mechanisms of apoptosis and proliferation of SEC. Recent results on the regulation of growth and apoptotic signaling of SEC are discussed.     

DDR2与MMP2在酒精性肝病肝窦毛细血管化中的协同表达

目的:探讨盘状区域受体2(DDR2)与基质金属蛋白酶(MMP2)在酒精性肝纤维化大鼠肝窦毛细血管化病理进程中的表达及可能发挥的作用。方法:橄榄油拌平衡饲料喂养大鼠的基础上给予60%(V/V)白酒胃内灌注制备酒精性肝纤维化模型,分别于4周、8周、12周和16周末观察肝组织网状纤维染色、免疫组化染色(Ⅰ、Ⅳ型胶原及层黏连蛋白),荧光定量-PCR和Western blotting检测肝组织DDR2及MMP2基因和蛋白表达并与肝窦毛细血管化各项评价指标进行相关性分析。结果:大鼠白酒灌胃4周出现肝脏脂肪变性,随灌胃时间延长逐渐加重为肝脏坏死、炎症及纤维化。酒精性肝病模型组大鼠DDR2 mRNA和蛋白表达量显著高于对照组,且随造模时间延长表达增加(P0.01)。MMP2 mRNA和蛋白表达自造模4周开始升高,于12周达峰值,造模16周下降,各模型组MMP2 mRNA和蛋白表达较正常对照组明显升高(P0.05)。相关性分析显示DDR2与MMP2、网状纤维、Ⅰ、Ⅳ型胶原及层黏连蛋白表达均呈显著正相关。结论:在酒精性肝病肝窦毛细血管化病程中DDR2表达呈时间依赖性,其可能通过效应蛋白MMP2在肝窦毛细血管化的发生和进展中发挥重要作用。     

人脂肪干细胞来源外泌体对四氯化碳诱导肝纤维化模型大鼠的治疗作用

文题释义： 脂肪干细胞：是指从脂肪组织中分离得到的一种间充质干细胞,不但具有跨胚层多向分化潜能,在不同培养条件下可以分化成肌肉、软骨、脂肪组织、神经组织或肝脏组织,而且具备取材方便、来源广阔、增殖能力强、免疫原性低等优点,近年来成为干细胞治疗的热点。 外泌体：是一种细胞主动分泌的大小均一、直径为50-150 nm的脂质双分子层结构囊泡,可由树突细胞、淋巴细胞、成纤维细胞、间充质干细胞和肿瘤细胞等多种不同细胞类型释放。 背景：肝纤维化具有较高的发病率和死亡率,肝星状细胞的活化和增殖是肝纤维化进程中的关键环节。目前还没有针对单一环节或靶点的有效抗纤维化药物。 目的：分析人脂肪干细胞来源外泌体对四氯化碳诱导的大鼠肝纤维化的影响。 方法：①通过酶溶解法获取健康人群来源脂肪中干细胞,体外培养获取一定数量细胞后通过多重超滤法获取外泌体。体外培养的肝星状细胞经转化生长因子β1活化后利用不同浓度外泌体进行处理,通过定量PCR检测细胞内α-平滑肌动蛋白的表达明确其活化程度,以及分别使用CCK-8及流式细胞术检测各组外泌体处理后活化肝星状细胞的生长率及凋亡率。②通过腹腔注射四氯化碳构建肝纤维化大鼠动物模型,尾静脉注射外泌体进行治疗。检测各组动物的肝功能及血清Ⅲ型前胶原、Ⅳ型胶原,肝组织Ishak评分及肝纤维化半定量,以及通过免疫荧光法检测肝组织内基质金属蛋白酶组织抑制剂1、基质金属蛋白酶9及α-平滑肌动蛋白的表达。实验方案于2017年1月经同济大学动物实验伦理委员会以及医学伦理学委员会批准。 结果与结论：人脂肪干细胞来源外泌体可抑制活化的肝星状细胞增殖,其可能的机制为抑制活化巨噬细胞的增殖,减少胶原纤维、α-平滑肌动蛋白及基质金属蛋白酶组织抑制剂1的表达,并促进基质金属蛋白酶9的表达。提示外泌体可治疗四氯化碳诱导肝纤维化。 orcid: 0000-0002-7141-8135 (Li Hongchao) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Cross-presentation of antigens from apoptotic tumor cells by liver sinusoidal endothelial cells leads to tumor-specific CD8+ T cell tolerance

Development of tumor-specific T cell tolerance contributes to the failure of the immune system to eliminate tumor cells. Here we report that hematogenous dissemination of tumor cells followed by their elimination and local removal of apoptotic tumor cells in the liver leads to subsequent development of T cell tolerance towards antigens associated with apoptotic tumor cells. We provide evidence that liver sinusoidal endothelial cells (LSEC) remove apoptotic cell fragments generated by induction of tumor cell apoptosis through hepatic NK1.1+ cells. Antigen associated with apoptotic cell material is processed and cross-presented by LSEC to CD8+ T cells, leading to induction of CD8+ T cell tolerance. Adoptive transfer of LSEC isolated from mice challenged previously with tumor cells promotes development of CD8+ T cell tolerance towards tumor-associated antigen in vivo. Our results indicate that hematogenous dissemination of tumor cells, followed by hepatic tumor cell elimination and local cross-presentation of apoptotic tumor cells by LSEC and subsequent CD8+ T cell tolerance induction, represents a novel mechanism operative in tumor immune escape.     

Glomerular basement membrane alterations induced by gentamicin administration in rats

The widespread therapeutic use of the aminoglycoside antibiotic gentamicin (GM) is limited by its nephrotoxic side effect, which can lead to acute renal failure. This study aimed at examining effects of high, supratherapeutic doses of gentamicin on morphological, structural and functional alterations of the glomerular basement membrane in adult rats. Experiments were done on 30 male Wistar rats, divided into two experimental groups. GM-group (20 rats) received gentamicin at a dose of 100mg/kg intraperitoneally during eight consecutive days. Control or C-group (10 rats) received 1 ml/day saline intraperitoneally. For histological analysis, 5 microm thick sections were stained with hematoxylin and eosin (HE), periodic acid Schiff (PAS), and Jones methenamine silver. Glomerular basement membrane thickness, glomerular area, major and minor axes, perimeter, diameter, roundness and mean optical density were analyzed. Biochemical analyses were used to determine concentrations of blood urea, serum creatinine, sodium and potassium. In GM-group rats, glomeruli were larger and glomerular basement membrane was diffusely and irregularly thickened with neutrophil cell infiltration. Glomerular area, major axis, minor axis, diameter and perimeter were significantly higher in GM-group compared to C-group rats. Opposite to this, glomerular optical density and average roundness were larger in C-group than in gentamicin-treated animals. Our results clearly showed morphological and structural alterations of glomeruli and glomerular basement membrane as well as alterations of proximal tubules in adult rats exposed to high doses of gentamicin.