硒对T—2毒素所致体外培养大鼠肝细胞损伤的影响

通过测定大鼠肝细胞培养液中乳酸脱氢酶、白蛋白及细胞内ＤＮＡ总量，我们对Ｔ—２毒素所致的肝细胞损伤作用及硒对其影响进行了探索。结果表明：Ｔ—２毒素可引起ＬＤＨ升高、白蛋白及ＤＮＡ合成下降；加硒０．１～０．５μｇ／ｍｌ１２小时后上述指标部分恢复。揭示Ｔ—２毒素可造成大鼠肝细胞膜系统的破坏，并由此导致细胞合成能力下降；适量补硒可部分拮抗Ｔ—２毒素的细胞毒作用，其机理可能与硒保护细胞膜的作用有关。     

内毒素／肿瘤坏死因子所致肝细胞损害机制的体外研究

为了解内毒素（ＬＰＳ）／肿瘤坏死因子（ＴＮＦ）所致肝细胞损害的机制，以乳酸脱氢酶（ＬＤＨ）及噻唑蓝染色（ＭＴＴ）为细胞毒性指标，利用Ｗｉｓｔａｒ大鼠肝细胞进行了ＬＰＳ及其介质ＴＮＦ等的肝细胞损害机制研究。结果发现：ＬＰＳ、ＴＮＦ－α、ＩＬ－１、ＩＬ－６等对肝细胞ＬＤＨ漏出及ＭＴＴ无显著影响（Ｐ＞０．０５），仅在ＬＰＳ、ＬＰＳ／ＴＮＦ－α加入肝细胞－Ｋｕｐｆｆｅｒ细胞混合培养组后有所变化；经ＧａｌＮ／ＬＰＳ体内作用后分离的Ｋｕｐｆｆｅｒ细胞与正常肝细胞混合培养，加入ＬＰＳ、ＴＮＦ及ＬＰＳ／ＴＮＦ－α均可致ＬＤＨ漏出显著增高（Ｐ＜０．０ｌ），多粘菌素Ｂ及抗－ＴＮＦ能分别完全和部分阻断此效应；经ＬＰＳ或ＴＮＦ－α体内作用后抽取的大鼠血清，加入培养肝细胞可致ＬＤＨ漏出显著增高（Ｐ＜０．０１）与ＭＴＴ显著降低（Ｐ＜０．０５），经５６℃灭活后此细胞毒性作用完全消失。上述结果提示，ＬＰＳ及其介质ＴＮＦ无肝细胞直接毒性作用，而经其活化的Ｋｕｐｆｆｅｒ细胞可能引起一系列瀑布效应，导致肝细胞损害。     

TG,VLCL和OX—VLDL对L—02和HLF细胞增殖及合成细胞外基质的影响

目的 探讨脂质对肝细胞和成纤维细胞生物学活性的影响。方法 以ＭＴＴ法、透明质酸（ＨＡ）放免测定法及＾３Ｈ－脯氨酸掺入法，观察４种不同浓度的甘油三酯（ＴＧ）、极低密度脂蛋白（ＶＬＤＬ）和化极低密度脂蛋白（ＯＸ－ＶＬＤＬ）对无血清培养正常人成人肝细胞（Ｌ－０２细胞）和人胚肺成纤维细胞（ＨＬＦ细胞）增殖及合成ＨＡ和胶原蛋白的影响，结果 一定浓度的ＴＧ、ＶＬＤＬ和ＯＸ－ＶＬＤＬ可影响ＨＬＦ和Ｌ－０２细胞的     

黑加仑油对大鼠血脂的影响

目的 研究在高脂血症形成过程中黑加仑油的调节血脂作用。方法 在高脂饲料中加入６％的黑加仑油喂饲Ｗｉｓｔａｒ雄性大鼠３ｗ，观察其对大鼠血脂的早衰亲高脂血症大鼠血清甘油三酯（ＴＧ）、高胆固醇（ＴＣ）、低密度脂蛋白胆固醇（ＬＤＬ－Ｃ）含量、ＬＤＬ－Ｃ与高密度脂蛋白胆固醇（ＨＤＬ－Ｃ）比值（ＬＤＬ－Ｃ／ＨＤＬ－Ｃ）和ＴＣ／ＨＤＬ－Ｃ比值降低，而ＨＤＬ－Ｃ、高密度脂蛋白亚组分Ⅱ胆固醇（ＨＤＬ２－Ｃ）水平、Ｈ     

合成多肽体外诱导乙肝病毒抗原特异性T杀伤细胞实验研究

目的及方法 为探讨ＨＢＶ抗原特异性细胞毒Ｔ细胞（ＨＢＶ－ＣＴＬ），根据ＨＢｃＡｇ的ＨＬＡ分子结合关键序列合成抗原多肽２段，分别与转铁蛋白（Ｔｆ），抗ＣＤ单克隆抗体（ＣＤ３ｍＡｂ）和ＩＬ－２联合体外诱导ＨＢＶ－ＣＴＬ，应用＾３Ｈ－ＴｄＲ释放法分别测定ＨＢＶ－ＣＴＬ对ＨＢＶＤＮＡ转染的ＨｅｐＧ２．２．１５细胞特异性活性及无ＨＢＶＤＮＡ转染的ＨｅＰＧ２细胞的非特异性杀伤率。结果 ＨＢＶ－ＣＴＬ对ＨＢＶＤ     

肿瘤坏死因子—α诱导体外培养大鼠肝细胞凋亡的作用

目的 了解肿瘤坏死因子－α（ＴＮＦ－α）对体外培养大鼠肝细胞凋亡的作用。方法 在半乳糖胺（ＧａＩＮ）或放线菌素Ｄ（ＡｃｔＤ）致敏情况下，加入ＴＮＦ－α，通过光镜、电镜及ＤＮＡ电泳等方法，观察ＴＮＦ－α对体外培养肝细胞凋亡的作用。结果 在大鼠注射ＧａｌＮ后分离肝细胞加入ＴＮＦ－α，ＡＬＴ上升，电镜下可见细胞坏死及凋亡；给正常鼠肝细胞ＡｃｔＤ加入ＴＮＦ－α，光镜下出现典型凋亡细胞，ＤＮＡ电泳出现凋亡的     

TG、VLDL和OX-VLDL对L-02和HLF细胞增殖及合成细胞外基质的影响

目的探讨脂质对肝细胞和成纤维细胞生物学活性的影响。方法以ＭＴＴ法、透明质酸（ＨＡ）放免测定法及３Ｈ－脯氨酸掺入法，观察４种不同浓度的甘油三酯（ＴＧ）、极低密度脂蛋白（ＶＬＤＬ）和氧化极低密度脂蛋白（ＯＸ－ＶＬＤＬ）对无血清培养正常成人肝细胞（Ｌ－０２细胞）和人胚肺成纤维细胞（ＨＬＦ细胞）增殖及合成ＨＡ和胶原蛋白的影响。结果一定浓度的ＴＧ、ＶＬＤＬ和ＯＸ－ＶＬＤＬ可影响ＨＬＦ和（或）Ｌ－０２细胞的增殖，促进其胶原蛋白和ＨＡ的合成，其中以ＯＸ－ＶＬＤＬ的作用最为显著。结论肝内脂质，特别是ＯＸ－ＶＬＤＬ过多，可通过促进肝细胞和成纤维细胞合成细胞外基质诱导肝纤维化形成。     

氧化低密度和极低密度脂蛋白对单核细胞血小板源性生长因子B链表达的影响

在单核细胞的培养基中分别加入２５ｍｇ·Ｌ￣（－１）低密度脂蛋白（ｌｏｗｄｅｎｓｉｔｙｌｉｐｏｐｒｏｔｅｉｎ，ＬＤＬ）、氧化ＬＤＬ（ｏｘｉｄｉｚｅｄＬＤＬ，ＯＬＤＬ）、极低密度脂蛋白（ｖｅｒｙｌｏｗｄｅｎｓｉｙｌｉｐｏｐｒｏｔｅｉｎ，ＶＬＤＬ）和氧化极低密度脂蛋白（ｏｘｉｄｉｚｅｄＶＬＤＬ，ＯＶＬＤＬ），培养２４ｈ后再用无血浆脂蛋白培养基收集条件培养基，并观察此条件培养基对￣３Ｈ－ＴｄＲ投入血管壁平滑肌细胞ＤＮＡ的影响。用抗血小板源性生长因子Ｂ链抗体（抗ＰＤＧＦ－Ｂ抗体）作疫组织化学染色。结果表明，单核细胞能表达ＰＤＧＦＢ，ＯＬＤＬ和ＯＶＬＤＬ能明显地促进单核细胞ＰＤＧＦ－Ｂ的表达，其条件培养基亦能促进￣３Ｈ－ＴｄＲ掺入平滑肌细胞ＤＮＡ内。上述结果提示，ＯＬＤＬ和ＯＶＬＤＬ通过加强单核细胞分泌ＰＤＧＦ－Ｂ并促进平滑肌细胞增殖而在动脉粥样硬化的发病过程中起作用。     

粒细胞集落刺激因子对恶性血液病化疗患者血清LDH和ALP及 …

目的：观察恶性血液病患者使用粒细胞集落刺激因子（Ｇ－ＣＳＦ）后对血清乳酸脱氢酶（ＬＤＨ）、碱性磷酸酶（ＡＬＰ）及β２微球蛋白（β２－ＭＧ）的影响。方法：将化疗后的恶性血液病患者分为治疗组及对照组,观察随白细胞升高ＬＤＨ、ＡＬＰ、β２－ＭＧ的变化。结果：随着白细胞的升高,ＬＤＨ、ＡＬＰ也不升,而β２－ＭＧ无变化。结论：当将ＬＤＨ、ＡＬＰ作为肿瘤指标时,要注意与由Ｇ－ＣＳＦ引起的ＬＤＨ、ＡＬＰ升高相区     

HBV与AFB1诱发树Qu肝癌前病变过程中癌基因的表达

目的 动态观察黄曲霉毒素Ｂ１（ＡＦＢ１）与乙型肝炎病毒（ＨＢＶ）诱发树Ｑｕ肝细胞癌（ＨＣＣ）病变过程，肝癌前病变γ－谷氨酰转肽酶阳性肝细胞灶（ＧＧＴ阳性灶）和 岛素样生长因子Ⅱ（ＧＦ－Ⅱ）、ｐ２１和ｐ５３表达，探讨ＨＣＣ发生的可能机制。方法实验动物分４组：Ａ组ＨＢＶ感染阳性和摄入ＡＦＢ１；Ｂ组ＨＢＶ阳性；Ｃ组摄入ＡＦＢ１；Ｄ组空白对照。采用ＬＳＡＢ、组织化学和原位杂交检测。结果 （１）各实验组肝癌     

Relationship between cellular ATP content and cellular functions of primary cultured rat hepatocytes in hypoxia

The importance of oxygen in maintaining the functional integrity of hepatocytes has been well established in a variety of experimental models, such as in vivo , perfused liver and isolated hepatocytes. However, one of the shortcomings of these systems is their short life span. Therefore, we have examined the effects of long-term hypoxia on cellular adenine nucleotide content and cellular functions, such as albumin production, urea production and DNA synthesis, in adult rat hepatocytes in primary culture. Hepatocytes were cultured at a density of 11 × 10⁴ and 5 × 10⁴ cells/0.18 mL per cm² for the study of albumin and urea production and DNA synthesis, respectively, at various oxygen tensions (20, 12, 8 and 5%) for 24 h. Cellular ATP content in cultured hepatocytes in hypoxia gradually declined, corresponding to the decrease in oxygen tension, and the cellular ATP level at 5% oxygen was approximately 20% of that at 20% oxygen. Albumin production also decreased in parallel with the decrease in cellular ATP content in cultured hepatocytes in hypoxia. However, even when cellular ATP content gradually declined corresponding with the decrease in oxygen tension in cultured hepatocytes in hypoxia, such as at 8 or 5% oxygen, urea production remained at a high level; in contrast, DNA synthesis was completely suppressed. These results suggest that the cellular ATP content decreases in cultured hepatocytes during long-term hypoxia in relation to oxygen tension and that the relationship between decreased ATP levels and liver function in cultured hepatocytes during hypoxia differs for albumin production, urea production and DNA synthesis.     

Inhibitory activity of the serum from patients with fulminant hepatitis against liver regeneration

The effect of sera from 8 patients with fulminant hepatitis, including 2 survival cases, on DNA and protein synthesis in primary cultured rat hepatocytes was studied. The serum from patients at an early stage or within 10 days after onset tended to intensify DNA synthesis in isolated hepatocytes, whereas the serum from patients with a history of over 50 days distinctly inhibited synthesis. When the serum was fractionated by gel filtration or free-flow electrophoresis, only the albumin fraction inhibited DNA synthesis in cultured hepatocytes. The suppressive effect of the albumin fraction was demonstrated even in patients suffering for only a short period of time. The inhibitory activity against DNA and protein synthesis in cultured hepatocytes was demonstrated in a substance extracted with a chloroform and methanol mixture from the albumin fraction of patients with fulminant hepatitis. The extract from the patients' sera also inhibited acceleration of DNA synthesis by epidermal growth factor (EGF) in the same cells.     

Resistance of three immortalized human hepatocyte cell lines to acetaminophen and N-acetyl-p-benzoquinoneimine toxicity

BACKGROUND/AIMS: Acetaminophen toxicity in hepatocytes is attributed to generation of the toxic metabolite N-acetyl-p-benzoquinoneimine, leading to depletion of intracellular glutathione, alteration of redox potential and ultimately, cellular necrosis. We aimed to determine the effect of acetaminophen and N-acetyl-p-benzoquinoneimine on three human hepatocyte cell lines HH25, HH29 and HHY41, and for comparison, on primary rat hepatocytes, a cell type that is relatively resistant to acetaminophen-induced toxicity. METHODS: We investigated the effect of incubation of rat hepatocytes and 3 hepatocyte cell lines with acetaminophen or N-acetyl-p-benzoquinoneimine on LDH release, glutathione status, mitochondrial function, CYP1A activity, albumin synthesis and DNA content. RESULTS: We demonstrated that HH25, HH29 and HHY41 are resistant to the toxic effects of acetaminophen under conditions that induce cytotoxicity in rat primary hepatocytes, as indicated by maintenance of glutathione levels and basal LDH release. Incubation with N-acetyl-p-benzoquinoneimine caused a dose-dependent cytotoxicity in rat hepatocytes. Under comparable conditions N-acetyl-p-benzoquinoneimine had no effect on any of the hepatocyte cell lines. Nevertheless, when culturing the cells for a further 48 h, a decrease in glutathione levels, albumin synthesis, CYP1A activity, DNA content and mitochondrial function was apparent. CONCLUSION: HH25, HH29 and HHY41 cells are highly resistant to acetaminophen and N-acetyl-p-benzoquinoneimine-induced toxicity. They tolerate a much higher concentration of both toxins for a longer period of time compared to rat primary hepatocytes. These results are of relevance in the use of these cell lines to investigate acetaminophen hepatotoxicity, and may be of importance in the choice of cells for use in bioartificial liver support systems.     

Response of porcine hepatocytes in primary culture to plasma from severe viral hepatitis patients

AIM: To observe the effects of plasma from patients with severe viral hepatitis (SVHP) on the growth and metabolism of porcine hepatocytes and the clinical efficiency of bioartificial liver device. METHODS: Hepatocytes were isolated from male porcines by collagenase perfusion. The synthesis of DNA and total protein, leakages of AST and LDH, changes in glutathione (GSH), catalase and morphology of porcine hepatocytes exposed to SVHP were investigated to indicate the effect of plasma from patients with severe hepatitis on the growth, injury, detoxification, and morphology of porcine hepatocytes. RESULTS: The synthesis of DNA and protein was inhibited in the medium containing 100% SVHP compared to the controls. The leakages of LDH and AST increased in porcine hepatocytes following exposure to 100% SVHP for 5 h. The difference between 100% SVHP and 10% newborn calf serum (NCS) was significant in t-test (LDH: t = 24.552, P = 0.001; AST: t = 4.169, P = 0.014). After exposure to SVHP for 24 h, alterations in GSH status were significant (F = 2.746, P<0.05) between porcine hepatocytes in 100% SVHP and 10% NCS, but no alteration occurred in the culture medium after 48 h (F = 4.378, P<0.05). A similar profile was observed in catalase activity. Many round vacuoles were observed in porcine hepatocytes cultured in SVHP. The membranes of these cells became indistinct and almost all the cells died on d 5. CONCLUSION: Plasma from patients with severe hepatitis inhibits the growth, injures membrane, disturbs GSH homeostasis and induces morphological changes of porcine hepatocytes. It is suggested that SVHP should be pretreated to reduce the toxin load and improve the performance of porcine hepatocytes in extracorporeal liver-support devices.     

原代猪肝细胞无血清培养

目的研究原代猪肝细胞无血清培养及肝细胞的功能。方法采用EGTA和胶原酶P两步法经肝静脉逆行灌注分离乳猪肝细胞,在无血清培养基中培养,并对不同培养时间的肝细胞生化合成及生物转化功能进行检测。结果肝细胞产量为（1．5±0．1）×10^10／肝,活率为（90．3±1．5）％,接种培养后肝细胞增生旺盛,LDH漏出第2天达到高峰,然后逐渐下降并稳定于一定水平。随着时间的延长及肝细胞数量的增加,自蛋白合成及利多卡因转化率逐渐增加,呈时间及肝细胞数量的依赖关系。结论采用本法分离获取的猪肝细胞产率高、活性强、具有良好的生物合成及生物转化功能,可作为生物人工肝较理想的肝细胞来源。     

胶原凝胶固定培养大鼠肝细胞的功能与形态观察

目的：探索混合胶原凝胶培养肝细胞的方法，观察培养鼠肝细胞的功能与形态特征。方法：两步法分离大鼠肝细胞，与I型鼠尾胶原溶液混合接种于培养瓶，待胶原液形成凝胶后，加培养液常规培养，观察培养鼠肝细胞的形态学特征和尿素合成及酶漏出量。结果：成功将大鼠肝细胞混合固定于胶原中，形成凝胶状进行培养。培养期间，始终能检测出鼠肝细胞合成分泌的尿素，而肝细胞乳酸脱氢酶漏出量较少，倒置相差显微镜下观察到典型的形态特征。结论：混合胶原凝胶培养方法能为肝细胞提供更接近体内的培养环境，可能适用于生物人工肝研究。     

Glucose loading during primary culture has opposite effects on the viability of hepatocytes exposed to potassium cyanide and to iodoacetic acid

Whether or not to apply nutritional pretreatment and how to do so are controversial issues with respect to the liver about to undergo aggressive intervention. We studied the effects of glucose loading on the viability of hepatocytes that were subsequently exposed to the inhibitors of carbohydrate metabolism, potassium cyanide (KCN) and iodoacetic acid (IAA). After rat hepatocytes were cultured for 24 hours in Leibovitz's L-15 medium containing 0, 10, 20, and 30 mmol/L glucose, the medium was replaced with modified Hanks-HEPES buffer with or without 2.5 mmol/L KCN or 0.5 mmol/L IAA. Lactate dehydrogenase (LDH) activity, lactate concentration, and pH of the supernatant were measured after 0, 2, 4, and 6 hours of exposure to KCN and after 0, 20, 40, and 60 minutes of exposure to IAA. Glycogen and adenosine triphosphate (ATP) contents in the hepatocytes were measured simultaneously. Hepatocytes cultured with various concentrations of glucose for 24 hours stored levels of glycogen in proportion to the glucose concentration in the culture medium without any significant difference in viability. The hepatocytes cultured with higher glucose concentrations maintained a higher ATP content and released less LDH and more lactate, and the pH decreased in the supernatant during exposure to KCN. Conversely, hepatocytes cultured with lower glucose concentrations maintained a higher ATP content and released less LDH during exposure to IAA. In conclusion, prior glucose loading appears to be beneficial for hepatocytes if oxidative phosphorylation is to be inhibited, whereas withholding glucose appears to be beneficial if glycolysis is to be inhibited.     

库普弗细胞条件培养基对原代培养肝细胞的细胞毒性

目的 探讨内毒素血症时库普弗细胞激活对肝细胞损伤的作用机制.方法 用链霉蛋白酶和胶原酶原位灌流,Percoll密度梯度离心分离、纯化SD大鼠肝细胞和库普弗细胞,制备内毒素(LPS)刺激的库普弗细胞的条件培养基(KCCM)并作用于肝细胞,检测肝细胞培养基丙氨酸转氨酶(ALT)、天门冬氨酸转氨酶(AST)、乳酸脱氢酶(LDH)的含量,四甲基偶氮唑蓝(MTT)法检测KCCM对大鼠原代肝细胞活性的影响.结果 KCCM作用于肝细胞2 h后,肝细胞培养基中ALT、AST及LDH含量均明显升高(P＜0.05),在2～4 h内随时间延长ALT、AST及LDH含量均逐渐增加,呈明显的时间-效应关系;MTT结果显示,KCCM作用后12 h、24 h时间点对肝细胞具有明显损伤作用(P＜0.05).结论 内毒素诱导库普弗细胞释放的可溶性因子作用一定时间后可直接对肝细胞造成损伤.     

Protective effects of a hibernation-inducer on hepatocyte injury induced by hypothermic preservation

Background/Purpose

For hepatocyte-based cell therapy to be realistic, the method chosen for cryopreservation or hypothermic preservation is critical. The aim of the present study was to clarify whether D-Ala2-Leu5-enkephalin (DADLE), a hibernation inducer, has protective effects on hepatocytes with regard to hypothermic preservation injury.

Methods

A suspension of rat hepatocytes was stored at 4?°C for 24?h with or without DADLE. Their viability was measured by the trypan blue dye exclusion method, and alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) levels in the preservation solution were measured. After 24?h of cold storage, viable hepatocytes were cultured at 37?°C for another 24?h. Then albumin production and lidocaine clearance were measured.

Results

DADLE significantly improved the survival rate of hepatocytes. The levels of ALT and LDH in the preservation solution with DADLE were significantly lower than those in the preservation solution without DADLE. The treated viable hepatocytes maintained both albumin synthesis and lidocaine clearance.

Conclusions

DADLE appears to have protective effects on hepatocytes with regard to hypothermic preservation injury in vitro. This hibernation-inducer is useful in prolonged hypothermic preservation for hepatocyte-based therapy.