天晴甘平减轻急性肝衰竭小鼠肝细胞损伤的机制

目的: 探讨天晴甘平减轻急性肝衰竭肝细胞损伤的机制.方法: 54只健康雌性昆明小白鼠随机分为实验组、对照组、正常对照组3组, 每组18只. 腹腔注射D-氨基半乳糖(D-galn)和脂多糖(LPS)制作急性肝衰竭模型. 注射前3 h, 实验组给予天晴甘平灌胃, 分别于注射后不同时间点用彗星实验检测肝细胞核DNA的损伤.结果: 实验组和对照组的Olive尾矩值在0.5 h即开始增大, 且随时间延伸逐渐增加, 与正常对照组比较, 差异均具显著意义(0.5 h: 3.95±1.42, 4.79±1.62 vs 0.95±0.56, 均P<0.05); 实验组与对照组比较, 实验组Olive尾矩值明显变小, 差异亦具显著意义(0.5-8 h: 10.81±2.85vs 19.36±3.95, P<0.05).结论: 急性肝衰竭小鼠肝细胞损伤的发生机制之一为肝细胞核DNA的损伤, 天晴甘平具有减轻急性肝衰竭小鼠肝细胞核DNA损伤的作用.     

Oridonin对急性肝衰竭小鼠肝细胞凋亡的影响及其机制研究*

目的 探讨冬凌草甲素（oridonin）对脂多糖/D-氨基半乳糖氨（LPS/D-Gal）联合诱导的急性肝衰竭（ALF） 小鼠肝细胞凋亡的影响,并探讨其可能的机制。方法 取25只小鼠,随机分成5组,每组5只。采用LPS/D-Gal腹腔注射建立小鼠ALF模型,设生理盐水对照组、LPS/D-Gal诱导模型组、LPS/D-Gal诱导和不同剂量oridonin干预组及oridonin处理组。采用末端转移酶介导的缺口末端标记法（TUNEL）检测肝细胞凋亡,采用real-time PCR法检测肝组织TNF-α mRNA水平,采用Western blot法检测凋亡相关蛋白的变化。结果 模型组小鼠肝细胞凋亡率为（36.4±1.8） %,显著高于两个oridonin干预组的[（19.4±3.3）%和（11.4±0.3）%,P<0.01];模型组小鼠肝组织TNF-α mRNA水平显著高于正常对照组（P<0.01）,而两个oridonin干预组肝组织TNF-α mRNA水平显著低于模型组（P<0.01）;模型组小鼠线粒体凋亡通路相关的促凋亡蛋白c-Jun氨基末端激酶（JNK）、bax、细胞色素C、cleaved caspase9/3活化水平显著高于两个oridonin干预组（P<0.01）,模型组小鼠抗凋亡蛋白bcl-xl水平显著低于两个oridonin干预组（P<0.01）,模型组小鼠死亡受体凋亡通路相关的促凋亡蛋白caspase8活化水平与两个oridonin干预组并无明显差异（P>0.01）。结论 Oridonin可抑制LPS/D-Gal诱导的ALF小鼠肝细胞凋亡,其机制可能与下调促凋亡细胞因子TNF-α水平和抑制JNK介导的线粒体凋亡信号通路有关。     

大黄对急性肝衰竭大鼠肝细胞凋亡的影响

目的 探讨大黄对急性肝衰竭大鼠肝细胞凋亡的影响及可能机制.方法 Wistar大鼠随机分为4组:正常对照组、模型组、大黄组和促肝细胞生长素(PHGF)组.大黄组和PHGF组于造模前3d分别每天灌服大黄水煎液和皮下注射PHGF 1 ml/100 mg,正常组和模型组每天灌服0.9％氯化钠注射液1 ml/100 mg.采用D-氨基半乳糖(D-GalN)/内毒素脂多糖(LPS)腹腔注射制备大鼠急性肝衰竭模型,造模8h后开腹取大鼠肝组织作病理检查,HE染色光镜下观察大鼠肝组织病理学变化,应用TUNEL法检测肝细胞凋亡情况,并应用免疫组化法分别检测肝组织中Fas、FasL和caspase-3的表达.结果 D-GαlN和LPS可引起严重的急性肝坏死并有广泛的肝细胞凋亡,伴Fas、FasL和caspaSe-3在肝细胞中强烈表达,大黄能减轻肝组织损伤,并可降低肝细胞凋亡及Fas、FasL和caspase-3表达(P均＜0.05).结论 大黄可抑制急性肝衰竭大鼠肝细胞凋亡,其机制可能与减轻肝细胞Fas、FasL和caspase-3表达有关.     

胎肝细胞培养上清液对小鼠急性肝衰竭的保护作用

为探讨胎肝细胞治疗重型肝炎的机制,将84只小鼠随机分为3组(每组28只),经D-氨基半乳糖/内毒素(D-galn/LPS)诱导急性肝衰竭后,分别给予生理盐水(NS)、RPMI1640培养液(RPMI)和人胎肝细胞培养上清液(FHCS)治疗,各组分别取14、8、6只小鼠进行存活率、血清谷丙酶(ALT)及肝脏病理观察。结果显示,NS组和RPMI组24小时存活率分别为14.3％和21.4％;ALT升高达207.2±41.3IU/L和188.4±52.6IU/L。FHCS组小鼠存活率为57.1％,ALT为92.5±28.7IU/L,与对照组相比差异显著(P<0.05,P<0.01)。此外,对照组分别有5(83.3％)只和4(66.7％)只动物肝组织病理改变在Ⅲ级以上,而FHCS组仅有2(33.3％)只。上述结果表明,FHCS对急性肝衰竭有保护作用,同时提示胎肝细胞分泌产物可能与保护肝细胞、治疗重型肝炎及肝衰竭的机制有关。     

肝细胞移植治疗急性肝衰竭

“20世纪是药物治疗的年代，21世纪却是细胞治疗的年代”。细胞治疗是指将干细胞或由其分化产生的功能细胞植入病变部位代偿病变细胞丧失的功能，或将细胞经体外遗传操作后用于疾病治疗的方法。急性肝衰竭（acute liver failure，ALF）常规药物治疗疗效差，是一种病死率极高的临床综合征，在我国主要由病毒性肝炎引起。近几年开展的较为有效的治疗方法主要有原位肝移植、人工肝支持系统和肝细胞移植（hepatocyte transplantation，HTx）。肝移植被认为是目前治疗ALF的最有效方法，但供肝短缺、免疫排斥和高额费用限制了受益者范围。     

凉血化瘀方对急性肝衰竭大鼠肝细胞凋亡的影响

目的：研究凉血化瘀方防治急性肝功能衰竭的作用机制。方法：将SD大鼠36只随机分为6组，除正常组外每组大鼠分别连续灌胃给药4天。末次给药后1小时，每组腹腔注射GaIN＋LPS，造成大鼠急性肝功能衰竭。6小时后采用流式细胞术检测大鼠肝细胞凋亡，同时采用原位末瑞标记（TUNEL）法半定量检测肝细胞凋亡情况。结果：流式细胞仪检测结果发现凉血化瘀方与阳性对照组细胞凋亡率比较模型组显著下降（P〈0．01，P〈0．05），并且模型组大量细胞阻滞在s期，G2期细胞减少，凉血化瘀方中剂量组与阳性药物对照组较模型组其细胞周期阻滞改善，凉血化瘀方组与模型组比较，差异有显著性意义（P〈0．01）。TUNEL半定量检测细胞凋亡与流式细胞仪结果基本一致，凉血化瘀方与模型组比较细胞凋亡率显著下降。结论：凉血化瘀方能够抑制急性肝损伤肝细胞凋亡，调节细胞周期阻滞，其机制可能为修复DNA复制损伤。     

赤芍承气汤对急性肝衰竭大鼠肝细胞凋亡的影响

目的：观察赤芍承气汤对内毒素脂多糖（LPS）致急性肝衰竭大鼠肝细胞凋亡的影响。方法：以内毒素（LPS）＋D－氨基半乳糖（D－GalN)）联合制备大鼠急性肝衰竭模型,模型组用生理盐水、治疗组用复方中药赤芍 承气汤分别灌胃,4天后开腹取鼠肝组织作病理检查,以HE染色观察大鼠肝组织病理学变化;以免疫组化法（SP法）检测Fas,FasL蛋白在大鼠肝 的表达情况,并观察赤芍承气汤对肝细胞凋亡的影响。结果：LPS＋D－GalN可引起严重的急性肝坏死并有广阔物肝细胞凋亡,伴Fas,FasL蛋白在肝细胞中强烈表达;肝细胞Fas／FasL阳性率,模型组为83％：92％,中药组为53％：50％,经统计学分析,P＜0．01;其阳性表达率随肝细胞坏死程度加重而增加,结论：赤芍承气汤可减轻肝细胞Fas／FasL的表达,抵抗内毒素诱发的肝细胞凋亡。     

腹腔移植微载体粘附人肝细胞对急性肝衰竭小鼠的保护作用

为了探讨腹腔移植微载体粘附培养人肝细胞对D氨基半乳糖(GalN)诱导的急性肝衰竭小鼠的保护作用．本实验采用胶原酶灌流法分离人肝细胞，胶原被覆的微载体Cytodex3加以培养，经腹腔注射肝细胞及载体，观察其对急性肝衰竭小鼠的影响。结果显示．与只接受微载体而无粘附肝细胞的对照组比较，腹腔移植培养肝细胞对肝衰竭小鼠的存活率有明显改善，其血清ALT及总胆红素较低(P<0．05)。肝组织病理改变较轻。该初步观察结果提示．腹腔移植微载体牯附培养肝细胞可提供代谢支持作用，从而使GalN损伤的小鼠肝功能得以恢复。     

抗TNFR1中和抗体对急性肝衰竭小鼠肝细胞的保护作用

目的探讨抗TNFR1中和抗体对急性肝衰竭小鼠肝细胞的作用。方法联合应用D-氨基半乳糖和肿瘤坏死因子-α造模，酶联免疫吸附试验测定血清ALT、AST浓度。HE染色检查肝脏组织病理变化、脱氧核糖核苷酸转移酶介导的缺口末端标记（TUNEL）技术检测肝细胞的凋亡程度。结果模型组各时间点（4、8、12h）均可见明显的肝细胞凋亡，治疗组各时间点亦可见少量凋亡细胞，但与模型组比较各时间点均明显减少（P〈0．01），其血清ALT、AST浓度较模型组也有下降。结论抗TNFR1中和抗体对TNF—α能诱导肝细胞损伤具有保护作用肝。     

牛肝细胞刺激物质的成分及对小鼠急性肝衰竭的保护作用

应用液相色谱及等离子体发射光谱法分析新生牛肝匀浆提取液中的游离氨基酸及微量元素成分;观察该提取液腹腔注射后对CCl_4中毒小鼠急性肝衰竭的保护作用。结果表明提取液中含有13种以上的游离氨基酸,其中6种为必需氨基酸,牛磺酸含量为正常成人血浆的8倍,支/芳氨基酸比值为3.2。微量元素以Mn、Zn、Mg、Se及Fe的含量较高。该提取液能明显提高实验动物的成活率(60％,对照组为10％,P相似文献   

微囊化肝细胞移植对急性肝功能衰竭大鼠细胞因子的影响

目的 观察微囊化肝细胞移植对急性肝功能衰竭(ALF)大鼠脂多糖(LPS)、TNF-α、IL-1β和IL-6的影响.方法 用D-氨基半乳糖诱导大鼠ALF模型.造模后18 h,60只大鼠均分为模型组、裸肝细胞移植组和微囊化肝细胞移植组,72 h取血标本,检测血常规、Cr、肝功能、PT和血氨;鲎试剂检测LPS;ELISA法检测TNF-α、IL-1β和IL-6.多组比较采用单因素方差分析.结果 裸肝细胞移植组和微囊化肝细胞移植组大鼠Cr、肝功能、PT及血氨较模型组有明显改善(P<0.01),且微囊化肝细胞移植组较裸肝细胞移植组改善更明显,差异有统计学意义(P<0.01).裸肝细胞移植组和微囊化肝细胞移植组LPS为(1.2±0.3)和(0.5±0.1)ng/L,TNF-α为(27.7α4.2)和(21.7±3.2)μg/L,IL-1β为(298.7±13.9)和(247.7±14.1)ng/L,IL-6为(275.7±51.8)和(208.7±48.1)ng/L;模型组分别为(2.1±0.6)ng/L、(37.7±5.1)μg/L、(355.5±26.4)ng/L和(424.8±67.8)ng/L,裸肝细胞移植组LPS、TNF-α、IL-1β、IL-6与模型组比较,差异有统计学意义(F=6.24,F=67.3,F=8.38,F=7.59,均P<0.01);微囊化肝细胞移植组与模型组比较,差异亦有统计学意义(F=11.73,F=10.75,F=15.91,F=10.83,均P<0.01);微囊化肝细胞移植组与裸肝细胞移植组比较,差异亦有统计学意义(F=5.49,F=4.01,F=7.53,F=3.35,均P<0.01).结论 微囊化肝细胞移植可通过降低LPS、TNF-α、IL-1β及IL-6而发挥抗ALF的作用.     

腹腔肝细胞移植治疗大鼠急性肝功能衰竭

目的观察腹腔肝细胞移植（HCT）治疗大鼠急性肝功能衰竭（ALF）疗效。方法取10只大鼠用胶原酶消化法分离肝细胞;另取50只大鼠随机分为A组20只、B组20只、C组10只。A、B组均采用D-氨基半乳糖（D-GalN）制作ALF模型,造模后分别腹腔注射肝细胞悬液2．5ml／只、不含肝细胞的培养液2．5ml／只;C组不予任何处理。观察移植后48h肝功能情况、肝脏病理改变及7d存活率。结果A、B组ALT、AST、AMM均高于C组（P均〈0．01）,但A、B组间比较,P〉0．05;A组7d存活率为50．00％,B组为14．28％,两组比较,P〈0．01。A组7d肝小叶结构基本恢复正常,仅见少量肝细胞脂肪样变性,汇管区见少量炎性细胞;B组肝小叶结构仍紊乱,较多细胞呈脂肪样变性,可见较多炎性细胞浸润。结论经腹腔HCT治疗D—GalN诱导的大鼠ALF有效。     

Treatment of acetaminophen-induced acute liver failure in the mouse with conditionally immortalized human hepatocytes

BACKGROUND/AIMS: Liver failure is a life threatening condition currently treated by palliative measures and, when applicable, organ transplantation. The use of a bioartificial organ capable of fulfilling the main functions of the liver would represent an attractive alternative. However, the shortage of suitable donor cells, and their limited growth ability have impeded the development of this strategy. We investigated whether lentiviral vectors allow for conditional immortalization of human hepatocytes and whether these immortalized hepatocytes could reverse lethal acute liver failure. METHODS: We exposed primary human hepatocytes to Cre-excisable lentiviral vectors coding for SV40T Antigen, telomerase, and/or Bmi-1 and tested the functionality of the resulting cell lines. Therapeutic potential of immortalized hepatocytes were tested in a murine model of acetaminophen-induced hepatic injury. RESULTS: The immortalized hepatocytes grew continuously yet were non-tumorigenic, stopped proliferating when exposed to Cre recombinase, and conserved defining properties of primary hepatocytes, including the ability to secrete liver-specific proteins and to detoxify drugs. The implantation of encapsulated immortalized human hepatocytes rescued mice from lethal doses of acetaminophen. CONCLUSIONS: Lentiviral vectors represent tools of choice for immortalization of non-dividing primary cells, and lentivirally immortalized human hepatocytes are promising reagents for cell-based therapy of acute liver failure.     

移植培养人肝细胞对急性肝衰竭小鼠的保护作用

目的探讨腹腔移植微载体粘附培养人肝细胞对Ｄ氨基半乳糖（ＤＧａｌ）诱导的急性肝衰竭小鼠的保护作用．方法采用胶原酶灌流法分离人肝细胞，胶原被覆的微载体Ｃｙｔｏｄｅｘ３加以培养，经腹腔注射２×１０６个肝细胞及载体，观察急性肝衰竭小鼠（ｎ＝３２，２５ｇ／ｋｇＤＧａｌ诱导４ｈ）７２ｈ存活率、血清ＡＬＴ、总胆红素以及肝脏病理变化．结果与只接受微载体而无粘附肝细胞的对照组比较，腹腔移植培养肝细胞对肝衰竭小鼠的存活率有明显改善（６５％ｖｓ０％），其血清ＡＬＴ（ＩＵ／Ｌ，２１７４４±２６３０ｖｓ４２６３１±４９２８，Ｐ＜００５）及总胆红素（ｍｍｏｌ／Ｌ，２６９１±３７６ｖｓ４１６８±３８３，Ｐ＜００５）较低．肝组织病理改变较轻．结论腹腔移植微载体粘附培养肝细胞可提供代谢支持，使ＤＧａｌ损伤的小鼠肝功能恢复．     

磷酸化p38丝裂原活化蛋白激酶在急性肝衰竭小鼠模型及HBV相关慢加急性肝衰竭患者肝组织中的表达及意义磷酸化p38丝裂原活化蛋白激酶在急性肝衰竭小鼠模型及HBV相关慢加急性肝衰竭患者肝组织中的表达及意义

目的：研究磷酸化后的p38丝裂原活化蛋白激酶（p－p38MAPK）在氨基半乳糖（D－GalN）或脂多糖（LPS）诱导的急性肝衰竭小鼠模型以及HBV相关慢加急性肝衰竭（ACLF）患者肝脏中的表达及其意义。方法采用D－GalN／LPS诱导C57BL／6小鼠构建急性肝衰竭模型,分别在给药0、0．5、1、2、4、6、8 h设立实验组,每组4只,处死小鼠取肝组织标本进行HE染色观察肝组织结构的病理变化,分别运用蛋白免疫印迹技术半定量检测及免疫组化染色定位检测肝组织中p－p38MAPK的表达;同时对照研究p－p38MAPK在HBV－ACLF、乙型肝炎肝硬化、慢性乙型肝炎（CHB）患者肝组织中的表达情况。组间比较采用独立样本t检验。结果蛋白免疫印迹法检测p－p38MAPK在肝衰竭小鼠肝组织匀浆中的表达随时间变化持续增高,给药6 h组半定量分析表达量显著高于正常对照组,差异具有统计学意义（t＝－2.727,P＝0．034）。免疫组化染色结果显示随存活时间的延长,小鼠肝组织炎症程度逐渐加重,炎症早期主要为窦细胞表达p－p38MAPK,随着肝组织损害加重,肝细胞表达p－p38MAPK渐多,坏死肝组织附近分布大量p－p38MAPK阳性肝细胞;正常人肝组织中p－p38MAPK的表达量很低,CHB患者肝组织内可见浸润淋巴细胞及肝细胞表达p－p38MAPK,并且随病程进展呈增多趋势,与临床观察病变程度逐渐加重相一致。结论 D－GalN／LPS诱导的小鼠急性肝衰竭模型中,p－p38MAPK表达随肝组织损害加重,表明其在肝衰竭病变过程中占重要地位;在HBV－ACLF患者致病过程中,p－p38MAPK信号通路可能发挥重要作用。     

Effects of urotensin-Ⅱ on cytokines in early acute liver failure in mice

AIM:To investigate urotensin-Ⅱ(UⅡ) and its effects on tumor necrosis factor(TNF)-α and interleukin(IL)-1β in early acute liver failure(ALF).METHODS:We investigated the time-dependent alteration in UⅡ levels and its effects on TNF-αand IL-1β in liver and blood in the early stage of lipopolysaccharide/D-galactosamine-induced ALF.RESULTS:After lipopolysaccharide/D-galactosamine challenge,UⅡ rose very rapidly and reached a maximal level 0.5 h,and the level remained significantly elevated after 2 h(P 0.05).Six hours after challenge,UⅡ began to degrade,but remained higher than at 0 h(P 0.05).Pretreatment with urantide,an inhibitor of the UⅡ receptor,suppressed the degree of UⅡ increase in liver and blood at 6 h after challenge(P 0.05 vs paired controls).In addition,liver and blood TNF-α increased from 1 to 6 h,and reached a peak at 1 and 2 h,respectively; however,IL-1β did not rise until 6 h after challenge.Urantide pretreatment inhibited the degree of TNF-α and IL-1β increase following downregulation of UⅡ post-challenge(all P 0.05).CONCLUSION:UⅡ plays a role in the pathogenesis and priming of ALF by triggering an inflammatory cascade and driving the early release of cytokines in mice.     

Changes in production of platelet activating factor by adherent hepatocytes of mice with experimental liver failure

It is thought that the platelet activating factor (PAF) enhances the immune response and inflammatory reaction. We studied the production of PAF from liver adherent cells. As a result, liver adherent cells produced PAF, when they were stimulated with calcium ionophore. In addition, when mice were treated with heat-killed Propionibacterium acnes and the mononuclear cells were infiltrated into the liver, a significantly larger amount of PAF was produced compared to normal mice.     

Effects of intraperitoneal transplantation of microcarrier-attached hepatocytes on D-galactosamine-induced acute liver failure in rats.

A study was conducted to investigate morphologic as well as metabolic characteristics of microcarrier-attached hepatocytes in culture, and also to evaluate the effect of intraperitoneal transplantation of the microcarrier-attached hepatocytes on acute hepatic failure in rats induced by D-galactosamine (GalN). Rat hepatocytes were isolated by collagenase perfusion, and cultured on collagen-coated microcarriers. Protein synthesis estimated by [14C] leucine incorporation was four-fold higher in microcarrier culture than in cell suspension. The rates of albumin, transthyretin and bile acid syntheses in hepatocytes cultured on microcarriers were similar to those in monolayer culture. When microcarrier-attached hepatocytes were intraperitoneally transplanted into rats with Galn-induced acute liver failure, a marked improvement in survival rate was observed as compared with control rats which received injections of microcarriers alone (80% vs 0% beyond 6 days of transplantation). Mean serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), methionine and glucose levels were similar in both groups, while serum bilirubin and ammonia levels were lower (P less than 0.1, P less than 0.05) in rats transplanted with the microcarrier-attached hepatocytes. Immunohistochemical examinations revealed that the transplanted hepatocytes around microcarriers had albumin synthesis activity, whereas almost no albumin synthesis was demonstrated in recipient liver. In conclusion, intraperitoneal transplantation of the microcarrier-attached hepatocytes will provide sufficient metabolic support, representing detoxication of ammonia (and presumably bilirubin) and synthesis of albumin, to allow GalN-damaged liver function to restore. Microcarrier culture of isolated hepatocytes seems to be one of the most appropriate tools for an artificial liver support.     

Alginate microencapsulated human hepatocytes for the treatment of acute liver failure in children

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