蓝藻提取物对原代培养大鼠肝细胞的氧化损伤作用研究

为探讨微囊藻毒素对原代培养大鼠肝细胞的毒作用机理,用蓝藻提取物处理这种细胞,然后测定乳酸脱氢酶渗出率、丙二醛含量、总活性氧自由基及超氧化物阴离子的形成情况。结果,前３种指标都呈现明显的剂量和时间反应关系,超氧化物阴离子的形成也随剂量增加而增加。提示脂质过氧化引起的氧化损伤作用是微囊藻毒素致原代培养大鼠肝细胞毒性的重要原因。这一机理的了解可为使用抗氧化剂治疗及预防这种毒素对机体的危害提供理论依据     

微囊藻提取物对大鼠原代培养的肝细胞酶学和形态学影响研究

目的 深入探讨以微囊藻毒素（MC）为代表的蓝藻提取物对肝脏的毒性作用特征及其机制。方法 采用不同剂量的蓝藻提取物染毒原代培养的大鼠肝细胞,观察由此所致的细胞酶学和形态学变化。结果 当微囊藻毒素含量为0．1、1、10μg/ml时,肝细胞培养液中乳酸脱氢酶（LDH）、谷草转氨酶（AST）含量增加,碱性磷酸酶（AKP）、γ－谷氨酰转移酶（GGT）、谷丙转氨酶（ALT）和谷胱甘肽（GSH）尚未见明显变化。染毒后的肝细胞增生活跃,并伴有凋亡或坏死样表现,扫描电镜显示染毒后的肝细胞变得不规则,呈空洞样变并有特征性的胞膜疱出现,与培养细胞所表现的膜损伤相一致。结论 微囊藻提取物可明显促进肝细胞增生并影响肝细胞的生理生化功能和形态的完整性。     

三叶青提取物对肝癌细胞HepG2及原代大鼠肝细胞的体外毒作用研究

目的探讨三叶青提取物对肝癌细胞Hep G2及原代大鼠肝细胞的体外毒作用.方法采用体外细胞培养技术,以细胞活性、细胞乳酸脱氢酶(LDH)活力的改变为指标,观察三叶青提取物对肝癌细胞Hep G2,体外毒作用的时效、量效关系,共观察其对正常原代培养大鼠肝细胞的影响.结果三叶青提取物以1×10-4g/L的浓度与Hep G2共同培养,在24h较空白对照组的细胞活性有显著降低;不同浓度的提取物和Hep G2共同培养,乙酸乙酯提取部位对细胞活性及酶学改变有显著性影响;对原代培养大鼠肝细胞活性及LDH也有一定的影响.结论三叶青提取物对Hep G2细胞具有明显的体外细胞毒作用,其毒性作用与药物的浓度和作用时间呈明显正相关,但对正常原代大鼠细胞毒性较小.     

铜绿微囊藻毒素的肝细胞毒性及活性氧生成作用

目的探讨微囊藻毒素的肝细胞毒性及其预防措施。方法利用原代培养的大鼠肝细胞，对水华提取物微囊藻毒素进行了细胞毒性和活性氧类（ＲＯＳ）生成的研究。结果在微囊藻毒素处理的肝细胞中，可以引起明显的依赖时间和剂量的乳酸脱氢酶（ＬＤＨ）释放率的升高，同时还可以引起明显的时间依赖的ＲＯＳ的升高。在去铁敏（ＤＦＯ）的作用下，可以分别使微囊藻毒素处理的肝细胞的ＬＤＨ释放率和ＲＯＳ升成率降低。结论微囊藻毒素具有明显的肝细胞毒性，其所导致的ＲＯＳ的生成很有可能是其产生的肝细胞毒性的原因之一     

直链烷基苯磺酸钠致人张氏肝细胞损伤的毒性作用

目的通过检测不同剂量的直链烷基苯磺酸钠(LAS)对人张氏肝细胞(Chang liver cells)增殖、损伤、凋亡及坏死的影响,探讨LAS对人张氏肝细胞的毒性作用。方法体外培养人张氏肝细胞,将细胞分为对照组及LAS不同剂量组,分别为正常对照组(不含LAS的正常培养基培养细胞)、LAS低、中、高剂量组(37.5、75.0及150.0 mg/L)。各组细胞培养24 h后,倒置显微镜下观察细胞形态及结构;四甲基偶氮唑盐(MTT)法检测细胞生存率;检测丙二醛(MDA)、乳酸脱氢酶(LDH)、天门冬氨酸氨基转移酶(AST)水平以了解肝细胞损伤程度;Hoechst 33342及PI染色观察细胞凋亡及坏死情况。结果 LAS处理后细胞表面出现黑色颗粒,部分细胞变圆脱落并产生碎片,高剂量组出现细胞脱落、死亡。LAS各处理组细胞存活率明显低于对照组(P0.01),MDA、LDH及AST水平也均明显高于对照组;细胞凋亡率与坏死率明显增加(P0.01),且这些变化随LAS剂量增加更加明显(P0.01)。结论 LAS可抑制人张氏肝细胞增殖,改变细胞形态,并导致细胞损伤、凋亡和坏死。     

槲皮素对氧化应激大鼠肝细胞的保护作用

目的研究大鼠肝细胞对槲皮素的吸收以及吸收后对氧化应激损伤的作用。方法用高效液相色谱法（HPLC）测定原代培养的大鼠肝细胞对槲皮素的吸收。于培养液中加入过氧化氢诱导肝细胞氧化应激，观察槲皮素预处理后，氧化应激肝细胞乳酸脱氢酶（LDH）释放量、培养液总抗氧化能力（T-AOC）、细胞匀浆脂质过氧化终产物丙二醛（MDA）含量以及细胞凋亡率的变化。结果大鼠肝细胞对槲皮素有一定量的吸收，24小时达到高峰。过氧化氢可导致大鼠肝细胞损伤，培养液中LDH活性增加、T-AOC增强，细胞匀浆的MDA含量增加，细胞凋亡率增高。槲皮素预处理能减少氧化应激肝细胞LDH的释放，进一步增强T-AOC，降低MDA含量，并降低细胞凋亡率。结论大鼠肝细胞对槲皮素有一定量的吸收，槲皮素预处理对氧化应激肝细胞有一定的保护作用。     

牛磺酸对海马神经细胞的营养和保护作用

目的 :　利用无血清培养的新生大鼠原代海马神经细胞 ,观察牛磺酸 ,谷氨酸和过氧化氢对神经细胞损伤的保护作用。方法 :　用含不同剂量牛磺酸的培养基培养神经元 ,动态观察神经细胞的生长情况以了解牛磺酸对神经细胞的营养作用 ;不同剂量牛磺酸与神经细胞预孵育 2 4 h后 ,分别加入损伤剂 (谷氨酸和过氧化氢 ) ,观察神经细胞存活数及乳酸脱氢酶的漏出率的变化 ;并且利用双波长荧光分光光度计观察谷氨酸对神经细胞内钙稳态的改变及牛磺酸的拮抗作用。结果 :　牛磺酸能促进体外培养的海马神经元的生长 ,并延长其存活时间 ;而且能剂量依赖性地拮抗过氧化氢和谷氨酸引起的神经细胞存活数下降及乳酸脱氢酶漏出率增高 ;牛磺酸还可抑制谷氨酸引起的升钙作用。结论 :　牛磺酸能促进神经细胞的生长 ,对谷氨酸和过氧化氢引起的细胞损伤均有明显的保护作用。     

水体有机物和蓝灌对BALB／c 3T3细胞的毒性作用

目的：研究水体有机物和蓝藻提取物的毒作用机理。方法：通过形态学观察,MTT法和中性红摄入法,从多个终点检测二者对BALB／c3T3细胞的毒性作用。结果：二者达一定浓度时,可引起细胞形态改变;有机物在各个浓度,各个培养时相均可引起细胞的存活率下降,有明显的剂量－时间－效应关系;而藻毒素在低学短时间染毒下可引起细胞数目的增加,在高浓度或长时间作用下,细胞存活率却明显下降。结论：两种比色法可敏感检测有机物及藻毒素对BALB／c 3T3细胞的毒性作用,两者对细胞膜均有损伤作用;有机物对线粒体有损伤作用,而藻毒素对线粒体具有刺激和损伤的双重作用。     

微囊藻毒素-LR诱导肝细胞氧化应激

目的 研究微囊藻毒素-LR在不引细胞毒性的剂量（3～30μg／L）时对肝细胞的氧化应激。方法以HL-7702细胞株为模型，检测细胞内的酶系统损伤情况。结果细胞内过氧化氢酶（CAT）、谷胱甘肽（GSH）和超氧化物歧化酶（SOD）随着毒素剂量的升高和作用时间的延长而降低，而乳酸脱氢酶（LDH）渗出率及丙二醛（MDA）含量同对照组相比差异无统计学意义。结论微囊藻毒素-LR在非细胞毒性的剂量下可引起肝细胞的氧化应激，但尚不足以导致脂质过氧化。     

鱼藤酮对体外培养星型胶质细胞毒性作用

目的观察鱼藤酮对体外培养星型胶质细胞毒性作用的特点。方法体外培养大鼠中脑星型胶质细胞,采用胶质纤维酸性蛋白（GFAP）免疫荧光法鉴定。观察染毒星型胶质细胞形态,检测细胞活力,通过生长曲线分析鱼藤酮对星型胶质细胞增殖的影响以及RT-PCR检测缝隙连接蛋白（connexin43,CX43）mRNA表达。结果0.5μmol/L鱼藤酮染毒24 h即可引起明显的细胞形态改变;0.75,1.0,2.0μmol/L鱼藤酮染毒时星型胶质细胞活力明显降低,乳酸脱氢酶释放量显著增加;0.5μmol/L以上鱼藤酮可明显抑制胶质细胞增殖,CX43 mRNA表达降低。结论鱼藤酮对体外培养星型胶质细胞可以产生明显的毒性损伤作用,但与神经元比较,星型胶质细胞对相同剂量的鱼藤酮染毒具有更强的耐受能力。     

Microcystic cyanobacteria extract induces cytoskeletal disruption and intracellular glutathione alteration in hepatocytes

Microcystins are a group of highly liver-specific toxins, although their exact mechanisms of action remain unclear. We examined the effects of microcystic cyanobacteria extract (MCE) collected from a contaminated water source on the organization of cellular microtubules (MTs) and microfilaments (MFs) in hepatocytes. We also investigated the effects on lactate dehydrogenase (LDH) leakage and intracellular glutathione (GSH). Primary cultured rat hepatocytes exposed to MCE (equivalent to 125 microg/mL lyophilized algae cells) showed a characteristic disruption of MTs and MFs in a time-dependent manner. Under these conditions, MCE caused aggregation of MTs and MFs and a severe loss of MTs in some cells. Moreover, MCE-induced cytoskeletal alterations preceded the LDH leakage. On the other hand, the treatment of cells with MCE led to a dose-dependent increase of intracellular GSH. However, time-course study showed a biphasic change of intracellular GSH levels with a significant increase in the initial stage followed by a decrease after prolonged treatment. Furthermore, pretreatment with N-acetylcystein (NAC), a GSH precursor, significantly enhanced the intracellular GSH level and decreased the MCE-induced cytotoxicity as well as cytoskeleton changes. In contrast, buthionine-(S, R)-sulfoximine, a specific GSH synthesis inhibitor, increased the cell susceptibility to MCE-induced cytotoxicity by depleting the intracellular GSH level. These findings suggest that intracellular GSH plays an important role in MCE-induced cytotoxicity and cytoskeleton changes in primary cultured rat hepatocytes. Increasing intracellular GSH levels protect cells from MCE-induced cytotoxicity and cytoskeleton changes.     

Microcystic cyanobacteria causes mitochondrial membrane potential alteration and reactive oxygen species formation in primary cultured rat hepatocytes.

Cyanobacteria contamination of water has become a growing public health problem worldwide. Microcystis aeruginosa is one of the most common toxic cyanobacteria. It is capable of producing microcystins, a group of cyclic heptapeptide compounds with potent hepatotoxicity and tumor promotion activity. The present study investigated the effect of microcystic cyanobacteria on primary cultured rat hepatocytes by examining mitochondrial membrane potential (MMP) changes and intracellular reactive oxygen species (ROS) formation in cells treated with lyophilized freshwater microcystic cyanobacteria extract (MCE). Rhodamine 123 (Rh-123) was used as a fluorescent probe for changes in mitochondrial fluorescence intensity. The mitochondrial Rh-123 fluorescence intensity in MCE-treated hepatocytes, examined using a laser confocal microscope, responded in a dose- and time-dependent manner. The results thus indicate that the alteration of MMP might be an important event in the hepatotoxicity caused by cyanobacteria. Moreover, the parallel increase of ROS formation detected using another fluorescent probe, 2',7'-dichlorofluorescin diacetate also suggests the involvement of oxidative stress in the hepatotoxicity caused by cyanobacteria. The fact that MMP changes precede other cytotoxic parameters such as nuclear staining by propidium iodide and cell morphological changes suggests that mitochondrial damage is closely associated with MCE-induced cell injury in cultured rat hepatocytes.     

槲皮素对大鼠原代肝细胞酒精性氧化损伤的防护作用

目的:观察槲皮素(quercetin)对大鼠原代肝细胞酒精性氧化损伤的防护作用。方法:经二步胶原酶技术分离培养大鼠原代肝细胞,用100mmol/L无水乙醇染毒细胞8h,作为酒精损伤模型。乙醇染毒前经不同剂量槲皮素(25～200μmol/L)预作用不同时间(0～8h),观察槲皮素干预对细胞培养上清液中乳酸脱氢酶(LDH)、谷草转氨酶(AST)活性,丙二醛(MDA)、谷胱甘肽(GSH)水平和超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性的影响。结果:大鼠原代肝细胞经100mmol/L酒精暴露8h后,LDH、AST活性,MDA、GSH含量和SOD、CAT活性与对照组相比,均有非常显著性差异;酒精暴露前1～4h经50～75μmol/L槲皮素干预效果最明显,LDH、AST、MDA和GSH、SOD、CAT水平分别较酒精组非常显著性降低和升高。结论:槲皮素可能通过减少GSH耗竭,提高抗氧化酶活性,抑制脂质过氧化来保护大鼠原代肝细胞抗酒精性氧化损伤,保护作用呈剂量与时间效应关系。     

Dual effect of ethanol on cell death in primary culture of human and rat hepatocytes

AIMS: In-vivo and in-vitro studies have shown that ethanol induces hepatocyte damage. The aim of the present study was to evaluate the effect of a broad range of ethanol concentrations on apoptosis and necrosis in primary culture of human and rat hepatocytes. METHODS: Human and rat hepatocytes were isolated from human hepatectomies and male Wistar rats (200-250 g) using the classical collagenase perfusion method. After stabilization of cell culture, ethanol (0-10 mmol/l) was administered and the parameters were measured 24 h after ethanol addition. Apoptosis was studied by DNA fragmentation, iodide propidium-DNA staining, caspase-3 activity and annexin V binding in hepatocytes. Necrosis was evaluated by lactate dehydrogenase (LDH) release. Malondialdehyde (MDA) and GSH/GSSG were used as parameters of oxidative stress. RESULTS: Ethanol enhanced dose-dependently all the parameters associated with apoptosis in human and rat hepatocytes. Low or high ethanol concentrations induced an opposite action against cell necrosis in cultured hepatocytes. Low concentrations of ethanol (1-2 mmol/l) reduced LDH release from human and rat hepatocytes. However, the highest ethanol concentration (10 mmol/l) induced a sharp increase in cell necrosis. The effect of ethanol on cell necrosis was related to lipid peroxidation in hepatocytes. CONCLUSIONS: Ethanol differentially regulates apoptosis or necrosis in cultured hepatocytes. Although ethanol exerted a dose-dependent induction of apoptosis, low ethanol concentrations were able to reduce basal lipid peroxidation and necrosis in hepatocytes. The highest ethanol concentration (10 mmol/l) induced apoptosis and necrosis in human and rat cultured hepatocytes.     

赤豆荚果总黄酮提取物对原代培养大鼠肝细胞氧化损伤的保护作用

目的:探讨赤豆荚果总黄酮提取物抗氧化作用以及对培养大鼠原代肝细胞氧化损伤的保护作用。方法:采用Photochem抗氧化测定仪对赤豆荚果总黄酮提取物体外抗氧化作用进行评价,同时评价其对培养大鼠原代肝细胞Fe2+氧化损伤保护作用的量效、时效关系。结果:赤豆荚果总黄酮提取物具有较强的抗氧化作用,在40,80,120ng剂量下,抗氧化作用大于芹菜素(P<0.05)。培养的原代肝细胞中加入100 μmol/L Fe2+后肝细胞活性减弱,培养液中ALT活力升高(P<0.05),脱落明显。加入提取物后,可有效抑制上述损伤作用。论:赤豆荚果总黄酮提取物具有较强的体外抗氧化作用,对Fe2+导致的大鼠原代肝细胞氧化损伤具有保护作用。     

Diallyl trisulfide modulates cell viability and the antioxidation and detoxification systems of rat primary hepatocytes

This study investigated the effects of various concentrations of diallyl trisulfide (DATS) and incubation times on cell viability, glutathione (GSH) content, and GSH-related enzyme activity in rat primary hepatocytes. Isolated and cultured primary rat hepatocytes were used as an experimental model. Cells were treated with 0 (control), 0.025, 0.05, or 0.25 mmol/L DATS for 0, 4, 8, or 24 h. After 24 h of treatment, some cells were incubated in fresh medium without DATS for an additional 24 h (48-h incubations). Based on lactate dehydrogenase (LDH) leakage and morphological examination, hepatocytes treated with 0.025 mmol/L DATS did not differ from the control cells at 4, 8, 24, and 48 h of incubation. However, LDH leakage was higher than in the control cells (P < 0.05) when the hepatocytes were treated with 0.05 or 0.25 mmol/L DATS for 4 h or more. The intracellular GSH levels of hepatocytes treated with 0.025 or 0.05 mmol/L DATS were higher than those of the control cells (P < 0.05), whereas those treated with 0.25 mmol/L DATS did not differ. The activity of glutathione reductase (GRd) was higher than in the control cells at 24 h (P < 0.05) when the hepatocytes were treated with 0.025 mmol/L DATS. When the hepatocytes were treated with 0.025 mmol/L DATS, the activity of glutathione S-transferase (GST) was higher than in the control cells at 48 h (P < 0.05). In hepatocytes treated with 0.05 mmol/L DATS, the activity of GST and glutathione peroxidase (GPx) was higher than in the control cells (P < 0.05) at 24 and 48 h of incubation. The results indicate that 0.025 or 0.05 mmol/L DATS could enhance antioxidation and detoxification capabilities by increasing the intracellular GSH level and the activity of GPx, GRd, or GST in rat primary hepatocytes. However, 0.05 or 0.25 mmol/L DATS might adversely affect the viability of hepatocytes.     

X射线对体外培养乳鼠心肌细胞损伤的实验研究

目的 研究X射线对体外培养的心肌细胞活性及凋亡的影响。方法 体外培养新生大鼠心肌细胞,分为对照组和放射组,放射组分别用X射线10Gy、20Gy、30Gy、40Gy单剂量照射心肌细胞,采用MTT法观察X射线对乳鼠心肌细胞活性的影响;血生化自动分析仪定量测定X射线作用后心肌细胞乳酸脱氢酶(LDH)的改变;吖啶橙/碘化丙啶(AO/PI)双染色法在激光扫描共聚焦显微镜下观察心肌细胞凋亡的形态学改变;流式细胞仪定量检测心肌细胞的凋亡率。结果 ①X射线剂量在10~40Gy均能抑制心肌细胞活性并成剂量依赖性;②细胞心肌酶谱检测到LDH释放量与X射线剂量相关;③流式细胞仪发现心肌细胞凋亡率与X射线照射剂量有相关性。结论 本研究结果显示X射线对心肌细胞有毒性作用,可致心肌细胞损伤,表现为心肌细胞坏死和凋亡发生率增加。     

活性氧在微囊藻毒素-LR诱导细胞凋亡效应中的作用探讨

[目的]研究微囊藻毒素．LR（MC—LR）对原代大鼠肝脏细胞的促凋亡效应以及探讨活性氧（ROS）在这一效应中的作用。[方法]采用胶原酶灌注法获取原代大鼠肝脏细胞,用William’s E培养液重悬后接种于铺被鼠尾胶原的孔板内,分为1个对照组和4个染毒组,370C、5％CO2条件下培养3h后换液并染毒各梯度浓度的MC—LR,使各染毒组毒物终浓度分别为1×10^-5、1×10^-6、1×10^-7、1×10^-8mol／L。染毒相应时间后,采用中性红比色实验检测细胞存活率,采用流式细胞术检测细胞凋亡和细胞内ROS含量。[结果]中性红比色实验发现,至染毒期末观察到1×10^-5、1×10^-6mol／L两个染毒组的肝脏细胞几乎全部死亡（〉95％）,1×10^-7mol／L染毒组部分肝脏细胞死亡（约30％）;流式细胞术观察到MC—LR染毒后早期（5min）诱导细胞调亡,同时观察到肝脏细胞内短时间（15min）快速产生大量的ROS。[结论]MC—LR诱导原代大鼠肝脏细胞快速产生大量的ROS,这可能是MC-LR诱导细胞凋亡并最终导致细胞死亡的毒性作用机制之一。