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采用胶原酶二步灌流法获取怀孕大鼠的原代肝细胞,以Aroclor1254诱导肝细胞损伤,用不同剂量的槲皮素分别处理损伤的肝细胞2472h,RT-PCR及Western-blot法检测肝细胞中细胞色素酶P450(CYP450)的表达。结果显示,槲皮素处理损伤的肝细胞后,肝细胞CYP1A1、CYP2B1及CYP2E1的表达随槲皮素浓度的增加和处理时间的延长而呈先升高后降低的趋势。10mg/L Aroclor1254是诱导体外培养的原代肝细胞损伤的最适质量浓度,10μmol/L槲皮素是对损伤的怀孕大鼠肝细胞的最佳保护浓度。结果表明,槲皮素对损伤的怀孕大鼠肝细胞具有保护作用。 相似文献
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为了研究槲皮素对Aroclor 1254损伤的孕鼠子宫内膜细胞是否具有保护作用,试验通过分离、培养怀孕大鼠的子宫内膜细胞,以Aroclor 1254诱导子宫内膜细胞损伤模型,用不同剂量的槲皮素分别处理损伤的子宫内膜细胞24~72h,MTT法测细胞的活力,RT-PCR及Western blot法检测细胞中CYP450的表达,并通过ELISA法检测细胞培养基中TNF-α、IL-6、雌二醇(estradiol,E2)和孕酮(progesterone,P4)的含量,来确定槲皮素是否对损伤的细胞具有保护作用。结果显示,处理24h后,随着槲皮素浓度的升高,子宫内膜细胞的成活率也随之增高,其中50μmol/L槲皮素时成活率最高。CYP1A1、CYP2B1和CYP2E1在子宫内膜细胞mRNA中的表达随槲皮素浓度的升高呈上升的趋势。CYP1A1和CYP2E1在细胞的蛋白中不表达,CYP2B1的表达随槲皮素浓度的增加而呈升高的趋势。50μmol/L槲皮素作用24h对损伤的怀孕大鼠子宫内膜细胞CYP1A1、CYP2B1和CYP2E1的表达效果最明显。Aroclor 1254损伤的子宫内膜细胞中TNF-α和IL-6的含量比对照组显著升高(P0.05),E2和P4的含量则比对照组显著降低(P0.05)。50μmol/L槲皮素处理后TNF-α和IL-6的含量比Aroclor 1254损伤组显著降低(P0.05),E2和P4则显著升高(P0.05)。这表明槲皮素对损伤的怀孕大鼠子宫内膜细胞具有保护作用。 相似文献
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为建立Aroclor 1254致孕鼠子宫内膜细胞损伤模型,通过分离、培养怀孕大鼠的原代子宫内膜细胞,用不同浓度的Aroclor 1254处理子宫内膜细胞48 h,倒置显微镜下观察细胞形态,MTT法检测细胞的活力,Western blot法检测细胞中CYP450的表达,并通过ELISA法检测细胞培养基中TNF-α、IL-6、雌二醇(estradiol,E2)和孕酮(progesterone,P4)的含量,来确定Aroclor 1254是否对细胞造成损伤。结果表明,随着Aroclor 1254浓度的升高,子宫内膜细胞的损伤及抑制率也随之增高,CYP450的表达随Aroclor 1254浓度的升高而升高。TNF-α和IL-6的水平比对照组显著升高(P0.05),E2和P4的水平则比对照组显著降低(P0.05)。这表明Aroclor 1254对体外培养子宫内膜细胞损伤程度呈浓度依赖性,10 mg/L的质量浓度作用48 h可建立可靠的子宫内膜细胞损伤模型。 相似文献
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为了探讨脱氢表雄酮(DHEA)对大鼠原代卵巢颗粒细胞激素生成及相关甾体生成酶的影响,采用机械法分离培养大鼠原代卵巢颗粒细胞,放射免疫分析法检测培养液中雌二醇、孕酮和睾酮的含量;Real-time PCR法检测类固醇代谢途径中3β-羟基固醇脱氢酶(3β-HSD)、17β-羟基固醇脱氢酶(17β-HSD)、CYP450 mRNA的表达变化。结果显示:10μmol/L DHEA处理48 h后可显著促进大鼠原代卵巢颗粒细胞雌二醇(P<0.05)、孕酮(P<0.01)和睾酮的分泌量(P<0.01);显著促进3β-HSD(P<0.01)和17β-HSD(P<0.05)mRNA的表达,CYP450 mRNA表达略有降低,但无统计学意义。说明外源性DHEA处理可能通过调节3β-HSD、17β-HSD和CYP450等相关酶的基因表达,进而影响大鼠原代卵巢颗粒细胞雌激素和雄激素的分泌。 相似文献
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为探讨丹参对脂肪变性奶牛肝细胞的作用机制,采用胶原酶两步灌流法分离培养奶牛肝细胞,不同浓度DL-乙硫氨酸处理,油红染色观察其脂滴数量,MTT法测定肝细胞的活性,生化法检测TG含量。用不同浓度的丹参处理变性肝细胞后,生化法检测ALT、AST、TG、SOD、MDA的含量或活性,ELISA法测定TNF-α的含量,Western blot法检测NF-κB和CYP450的蛋白表达水平。结果显示,筛选出2 mmol/L的DL-乙硫氨酸作用24 h作为体外造模的最佳条件。用丹参处理后,能降低ALT、AST、TG和MDA的活性或含量,抑制TNF-α的释放,增加SOD活性。随丹参浓度增加和处理时间的延长,CYP450的表达呈逐渐降低的趋势。24 h时,丹参对NF-κB的表达无明显影响;48 h时,随丹参浓度的增加,NF-κB的表达呈逐渐降低的趋势。这表明丹参对奶牛脂肪肝有一定的治疗作用,其作用机制与促进脂质代谢、抑制肿瘤细胞坏死因子、抗脂质过氧化及降低NF-κB和CYP450的表达密切相关。 相似文献
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蛹虫草是一种药用真菌。从人工培养的蛹虫草中提取蛹虫草多糖(CMPS),采用Ⅳ型胶原酶灌流法分离大鼠肝细胞进行原代培养,研究蛹虫草多糖对CCl4诱导大鼠原代肝细胞损伤的保护作用。结果表明:蛹虫草的子实体和基质都含有丰富的多糖,其中基质中的多糖含量最高,约为子实体的2～4倍;CMPS可明显降低由CCl4诱导的损伤肝细胞培养上清液中谷草转氨酶(AST)、谷丙转氨酶(ALT)的活性水平和丙二醛(MDA)的含量水平,显著提高由CCl4诱导的损伤肝细胞培养上清液中谷胱甘肽过氧化物酶(GSH-Px)活性和诱导损伤肝细胞的存活率。试验结果提示CMPS对大鼠原代培养肝细胞损伤有直接保护作用,该作用可能与其抗氧化作用有关。 相似文献
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《中国兽医杂志》2020,(5)
应用肽聚糖(PGN)诱导大鼠肠黏膜微血管内皮细胞(RIMMVECs)炎性损伤模型,探究槲皮素对RIMMVECs炎性损伤的保护作用并探讨其作用机制。通过CCK8法筛选槲皮素作用浓度,荧光定量PCR(FQ-PCR)法测量Toll样受体2(TLR2)、Toll样受体4(TLR4)的mRNA表达水平,Western Blot法测量TLR2、TLR4和其下游MyD88蛋白表达水平,和ELISA法评估炎性因子TNF-α分泌水平,综合评价槲皮素对PGN诱导的RIMMVECs细胞炎性损伤的保护作用。结果表明,不同浓度的槲皮素能显著降低TLR2和TLR4 mRNA和蛋白水平,降低MyD88蛋白表达水平,进一步降低炎性因子TNF-α释放量(P0.05)。槲皮素能保护RIMMVECs免受PGN导致的细胞炎性损伤。 相似文献
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为探讨内毒素（ET）对大鼠肝脏c—myc基因的表达影响及阳离子（cA）对该过程的保护效应,本试验将72只清洁级SD大鼠随机分为对照组、内毒素组和阳离子A保护组,3组经相应处理后分别在3、4、8、12h采集肝脏作为样本。采用实时荧光定量RT—PCR检测法和免疫组化法分析,结果显示：在mRNA水平和蛋白水平上,ET可上调C—myc基因的表达,从而促进肝细胞凋i-,最终导致肝细胞损伤,CA则能明显下调c—myc基因的表达,从而抑制肝细胞凋亡,对ET诱导的肝损伤具有明显的保护效应。 相似文献
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S B Hooser V R Beasley L L Waite M S Kuhlenschmidt W W Carmichael W M Haschek 《Veterinary pathology》1991,28(4):259-266
The morphologic effects of microcystin-LR (MCLR) were examined in vitro and in vivo to identify the specific cell type(s) affected and to characterize the actin filament changes occurring in hepatocytes. Male Sprague Dawley rats were used for all studies. For in vitro studies, hepatic cells were isolated by collagenase perfusion of liver, while parenchymal cells (hepatocytes) and nonparenchymal cells were prepared by pronase digestion and metrimazide gradient centrifugation. Cell suspensions and and primary hepatocyte monolayer cultures were treated with MCLR at doses up to 10 micrograms/ml; cultured hepatocytes were also treated with phalloidin or cytochalasin B at a dose of 10 micrograms/ml; and rats were treated intraperitoneally with MCLR at 180 mg/kg. Cultured hepatocyte preparations and frozen liver sections were stained with rhodamine-labeled phalloidin for filamentous actin. In cell suspensions, MCLR did not affect nonparenchymal cells but caused rapid, progressive, blebbing of the plasma membrane in hepatocytes. In cultured hepatocytes, MCLR caused plasma membrane blebbing as well as marked reorganization of actin microfilaments. These alterations were dose and time dependent. Cultured hepatocytes treated with phalloidin or cytochalasin B also showed extensive plasma membrane blebbing and actin filament alterations; however, actin filament changes were morphologically distinct from those induced by MCLR. In vivo, MCLR-induced hepatocyte actin alterations occurred at the same time as, or slightly preceded, histologic changes that began 30 minutes after dosing. These studies suggest that early MCLR-induced morphologic changes occurring both in vivo and in vitro are due to alterations in hepatocyte actin filaments. 相似文献
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本文对鼠伤寒沙门氏菌致突变试验在国内实验条件下方法标准化进行了研究。国产试剂配制的营养肉汤可满足试验菌株的营养需求,烧瓶振荡培养10小时即可达1～2×10~9存活菌/ml,肉汤培养物稀释后测定OD650可监测细菌密度。国产五氯联苯和Aroclor 1254诱导的Sprague-Dawley及Wistar大鼠肝S9活化苯并(a)芘及2-乙酰氨基芴的能力相似(P>0.05)。本文还提出以点试验估计受试物毒性及以参比物减小或消除多次实验间的误差。 相似文献
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R C Beier J O Norman T R Irvin D A Witzel 《American journal of veterinary research》1987,48(4):583-585
Components of white snakeroot, a plant toxic to livestock and human beings, were activated by Aroclor 1254-induced rat liver microsomes. The toxic products of microsomal activation were evaluated in murine melanoma (B16F1) cell cultures. Toxic products in white snakeroot were inactive in cell culture systems without microsomal activation. This activation system revealed that at least 2 fractions of white snakeroot were metabolically activated to cytotoxic agents. The autocatalytic inactivator of cytochrome P-450, 1-aminobenzotriazole, inhibited activation of white snakeroot constituents by rat liver microsomes. 相似文献
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实验建立了鸡胚下丘脑星形胶质细胞体外培养模型,研究了大豆黄酮对多氯联苯Aroclor 1254(A1254)引起的下丘脑星形胶质细胞损伤的缓解作用。结果表明:0.1～1μg/mL的A1254引起的细胞损伤不显著,高剂量时(10μg/mL)能引起星形胶质细胞大量死亡。抗氧化剂大豆黄酮10μg/mL能明显缓解A1254对星形胶质细胞的损伤作用。 相似文献
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Hirai T Nunoya T Ihara T Saitoh T Shibuya K Nakamura K 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2006,68(2):179-182
Infectivity of porcine circovirus (PCV) 1 and PCV2 was examined in primary porcine hepatocyte culture by comparing that of PCV in primary kidney cell culture. The virus titer of PCV2-infected hepatocyte cultures was higher than that of the PCV1-infected hepatocyte cultures and the PCV-infected kidney cell cultures. The number of virus-positive cells was most abundant in PCV2-infected hepatocyte cultures as determined by immunohistochemistry and/or in situ hybridization. The results of our data suggest that PCV2 preferably infects cultured hepatocytes as observed in the liver of pigs with postweaning multisystemic wasting syndrome. 相似文献
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G. A. E. VAN T KLOOSTER B.J. BLAAUBOER J- NOORDHOEK‡ A. S.J. P. A. M. VAN MIERT 《Journal of veterinary pharmacology and therapeutics》1993,16(4):454-461
van't Klooster, G.A.E., Blaauboer, B.J., Noordhoek, J. & van Miert, A.S.J.P.A.M. Sulfadimidine metabolism in vitro: II. comparative studies in cultured rat, goat, sheep and cattle hepatocytes. J. vet. Pharmacol. Therap. 16, 454–461.
Hydroxylation and acetylation of sulphadimidine (SDD) and the deacetylation of N4-acetyl SDD was investigated in cultured hepatocytes from male and female rats, from male and female goats and from female sheep and cattle. Significant sex differences were observed for hydroxylation of SDD in hepatocytes from rat and goat. In goat, sheep and cow hepatocytes, the hydroxylation pathway is relatively important, whereas in rat hepatocytes, acetylation is predominant. Hepatocytes of all four species deacetylated N4-acetyl SDD. In ruminant hepatocytes, deacetylating activity was of considerable importance, whereas in rat hepatocytes, it appeared a minor pathway of metabolism. Similar to the in vivo situation, formation of N4-acetyl SDD in cultured hepatocytes results from an equilibrium of acetylation and deacetylation. A good correlation was found between results in isolated hepatocytes and previous findings in vivo , both in levels of species-related activities and in acetylation-hydroxylation ratios. In conclusion, cultured hepatocytes appear a useful in vitro model to study comparative sulfonamide metabolism. 相似文献
Hydroxylation and acetylation of sulphadimidine (SDD) and the deacetylation of N4-acetyl SDD was investigated in cultured hepatocytes from male and female rats, from male and female goats and from female sheep and cattle. Significant sex differences were observed for hydroxylation of SDD in hepatocytes from rat and goat. In goat, sheep and cow hepatocytes, the hydroxylation pathway is relatively important, whereas in rat hepatocytes, acetylation is predominant. Hepatocytes of all four species deacetylated N4-acetyl SDD. In ruminant hepatocytes, deacetylating activity was of considerable importance, whereas in rat hepatocytes, it appeared a minor pathway of metabolism. Similar to the in vivo situation, formation of N4-acetyl SDD in cultured hepatocytes results from an equilibrium of acetylation and deacetylation. A good correlation was found between results in isolated hepatocytes and previous findings in vivo , both in levels of species-related activities and in acetylation-hydroxylation ratios. In conclusion, cultured hepatocytes appear a useful in vitro model to study comparative sulfonamide metabolism. 相似文献
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Said MM Ogawa K Pitchakarn P Takahashi S Asamoto M Shirai T 《Journal of toxicologic pathology》2009,22(4):263-271
The present study was undertaken to investigate the effect of dietary supplementation with nimesulide or eugenol on N-nitrosodiethylamine (DEN)-initiated early hepatocarcinogenesis in F344 male rats. Both compounds did not alter the expression of cytochrome P450 (CYP) 2E1, the enzyme that plays a major role in the activation of DEN to genotoxic products; however, nimesulide induced the expression of CYP1A1. Western blot analysis revealed that COX-1 and COX-2 protein expressions were not modulated by DEN compared with normal controls. Furthermore, post-initiation feeding with nimesulide or eugenol did not modulate COX-2 protein expression in normal or DEN-treated rats, whereas eugenol significantly increased the liver prostaglandin E(2) (PGE(2)) levels of DEN-injected animals compared with the DEN controls. Ultimately, nimesulide or eugenol did not modify DEN-induced hepatocarcinogenesis as evidenced by insignificant changes in the number and size of preneoplastic placental glutathione S-transferase (GST-P) positive liver foci compared with the DEN controls. These results suggest that COX-2, as well as prostaglandin E(2), may play no role in the post-initiation development of DEN-induced rat hepatocarcinogenesis at an early stage. 相似文献