骨髓基质细胞在小鼠肝脏内分化及癌变的潜能

目的:探讨骨髓基质细胞(BMSCs)在诱癌小鼠模型中向肝细胞分化及癌变的可能性.方法:♂BALB/c小鼠BMSCs分离培养及经门静脉移植到♀BALB/c小鼠肝脏.二乙基亚硝胺诱导肝癌.6mo后处死小鼠,取肝脏标本.用免疫组织化学检测胎盘型谷胱苷肽转移酶、甲胎蛋白和角蛋白19的表达及用荧光原位杂交(fluorescence in situ hybridisation,FISH)检测Y染色体阳性细胞.结果:BMSCs在添加肝细胞生长因子的培养基中体外培养能分化为肝细胞样细胞.诱癌6mo后26%的小鼠存活并成功诱导肝细胞性肝癌.免疫组织化学显示肝癌细胞表达胎盘型谷胱苷肽转移酶和甲胎蛋白,而不表达角蛋白19.FISH结果显示骨髓基质细胞移植及诱癌6mo后小鼠肝脏内有Y染色体阳性的肝细胞.而无二乙基亚硝胺诱癌的小鼠.BMSCs移植6mo后肝脏内未发现Y染色体阳性的肝细胞.另外,FIsH检测未发现Y染色体阳性的肝癌细胞.结论:在肝癌的小鼠诱癌模型中,移植的BMSCs能分化为肝细胞,但癌变的可能性小.     

肝细胞色素P450 2E1在大鼠非酒精性脂肪肝形成中的作用

目的 研究肝细胞色素P450 2E1在大鼠非酒精性脂肪肝形成中的作用。方法 Wistar大鼠40只，随机分为正常对照组和高脂饲料2、4、8和12周组(其中每组各8只)：HE染色光镜观察肝脏组织病理改变；硫代巴比妥酸法测定肝脏组织丙二醛(MDA)的含量变化：免疫组织化学和Westemblot方法研究高脂饲料诱导的大鼠脂肪肝形成中肝细胞色素P450 2E1表达变化。结果 高脂饲料组大鼠肝脏内MDA含量明显高于对照组，随时间延长，逐渐增加：随着脂肪肝程度的加重，MDA含量逐渐增强，肝细胞色素P450 2E1蛋白表达亦明显增强。结论 非酒精性脂肪肝大鼠肝细胞色素P450 2E1表达变化与脂肪肝引起的脂质过氧化损伤程度密切相关。     

复方半枝莲防治二乙基亚硝胺诱发大鼠肝癌的研究

目的 :研究复方半枝莲 (SBC)对二乙基亚硝胺 (DEN)诱发大鼠肝癌的防治作用。方法 :利用免疫组化、流式细胞仪、血清和组织生化等检测方法 ,分别在第 14周和第 2 4周观察 SBC对 DEN诱发的肝癌形成过程的影响。结果 :中药组 14周时大鼠肝脏异型性增生灶明显较模型组少 ,2 4周时形成的肝癌结节小而少 ,模型组、中药组肝癌发生率分别为 75 .0 %、5 0 .0 % ;免疫组化显示中药组大鼠肝组织谷胱甘肽 - S-转移酶胎盘型阳性灶面积明显低于模型组 ;肝组织匀浆上清液谷胱甘肽 - S-转移酶含量以及血清 λ-谷氨酰转移酶、碱性磷酸酶、谷丙转氨酶含量也明显低于模型组 ;流式细胞仪检测结果显示 ,中药组大鼠肝细胞 G0 - G1 期比例下降 ,G2 - M期比例升高。结论 :SBC能抑制癌前病变 ,延缓肝癌的形成 ,降低肝癌发病率 ,其作用机制之一可能为阻滞 G2 - M期细胞进展 ,从而抑制DEN引起的肝细胞的去分化和恶性增殖     

肝细胞肝癌谷胱甘肽硫转移酶P1活性和甲基化研究

与环境中致癌物黄曲霉毒素B1（AFB1）密切接触可增加肝细胞肝癌（HCC）的危险性。进入体内的AFB1在肝内经细胞色素P4501A2活化为AFB1-8.9环氧化物（前致癌物），在肝细胞胞浆中与谷胱甘肽S-转移酶（GST）携带的活性极团谷胱苷肽结合后，水溶性增加，     

在肝脏癌变过程中试验小鼠转录因子Nrf2／MafK可调节胎盘谷胱甘肽S-转移酶基因

试验小鼠的胎盘谷胱甘肽S-转移酶(GST-P)是一种Ⅱ期的解毒酶，正常肝细胞中不表达，但是在早期肝脏癌变过程及肝细胞癌细胞中被大量和特异性地诱导出来。先前研究显示GST-P基因活化作用主要受增强子GPE1(GST-P增强子1)控制，但是GST-P基因的特异性活化作用机制现在不是十分清楚。     

甲氰咪呱对非酒精性脂肪性肝炎(NASH)大鼠肝微粒体细胞色素P450的影响

目的研究甲氰咪呱对非酒精性脂肪性肝炎（NASH）大鼠肝微粒体细胞色素P450的影响。方法通过高脂饮食制作非酒精性指肪性肝炎（NASH）动物模型，给予甲氰咪呱灌胃治疗，观察肝组织病理形态变化，进行炎症活动计分，同时测定细胞色素P450（CYP450）和细胞色素2E1（CYP2EI）含量及ALT、AST，并与对照组比较。结果与正常组比较，模型组大鼠ALT、AST及CYP450和CYP2EI含量明显升高；与模型组比较，甲氰咪呱治疗组CYP450和CYP2EI含量均明显下降，（P〈0．05），ALT、AST及炎症活动计分明显低于模型组（P〈0．05）。结论甲氰咪呱能显著改善NASH大鼠肝脏功能及形态损伤，可能通过抑制肝细胞微粒体CYP450和同功酶CYP2EI的表达发挥作用。     

CYP2E1在肝细胞癌中的作用研究进展

细胞色素P450 2E1(CYP2E1)是肝脏的重要代谢酶,参与肝病和肝细胞癌的发生发展,在底物的诱导下其过表达产生大量活性氧,通过脂质过氧化、损伤生物膜功能,协同细胞因子和肝细胞凋亡等诱发和加重肝细胞的炎症、坏死、纤维化甚至癌变;通过增加花生四烯酸等环节参与HCC侵袭转移.对CYP2E1转录水平的调节研究有助于在肝癌治疗中寻找新的抗肿瘤靶点.     

二甲基奶油黄诱发大鼠肝癌的病理形态学观察

应用二甲基奶油黄(DAB)诱发大鼠肝癌,整个过程分为非特异性炎症、肝硬化、肝癌3个阶段。诱癌后第12周开始出现肝癌,第20周时诱癌率达92.86%(13/14)。非特异性炎症阶段光镜下改变为肝细胞气球样变性、炎性细胞侵润。肝硬化阶段表现为肝细胞和胆管腺瘤样增生,假小叶形成,肝组织炎性细胞侵润。肝癌有肝细胞癌、胆管细胞癌和混合细胞癌3核膜增厚,线粒体减少。胆管细胞癌中发现致密核心颗粒,其意义有待进一步研究。     

实验性肝细胞癌变过程中嗜碱性小细胞病灶的癌变趋势

目的动态观察肝癌的发生、发展过程，探讨肝细胞癌前期病变的形态特点及其癌变过程。方法145只雄性SD大鼠随机分成模型组和正常对照组，模型组大鼠以质量体积分数为1％的二乙基亚硝胺60mg／kg灌胃，每周1次，第12周后改为40mg／kg至14周后停药；正常对照以等渗盐水灌胃。于实验开始后第2，3，5，8，10，12，14、18周分批处死大鼠，剩余大鼠第26周全部处死。肝组织苏木精-伊红，Masson三色，过碘酸雪夫，谷胱甘肽-S-转移酶免疫组织化学、增殖细胞核抗原免疫组织化学染色，动态观察肝组织病理形态变化，以寻找肝细胞癌前期病变的特点。结果肝癌病理变化可分为3个阶段：肝细胞损伤阶段（2～5周）：主要表现为中央静脉周围肝细胞坏死，坏死塌陷区少量细胞外基质沉积；细胞增生及肝硬化形成阶段（8～12周）：肝细胞呈结节状再生，伴肝细胞异型增生，坏死塌陷区细胞外基质沉积，并逐渐形成肝硬化。肝癌形成阶段（第14周以后）：此期有肝癌形成，18周成癌率为62．5％。从实验第10周开始，嗜碱性细胞灶明显增多，部分胞质内见苍白小体，嗜碱性小细胞改变从第12周出现，增殖细胞核抗原免疫染色呈阳性，部分小细胞改变，细胞质内含脂泡，向周围肝细胞间浸润生长。结论细胞质内含苍白小体和脂肪泡，证明嗜碱性细胞灶和小细胞改变与肝细胞癌有关，其向肝细胞间的浸润生长，进一步证明其可癌变。     

复方半枝莲防治二乙基亚硝胺诱发大鼠肝癌的研究

目的研究复方半枝莲(SBC)对二乙基亚硝胺(DEN)诱发大鼠肝癌的防治作用.方法利用免疫组化、流式细胞仪、血清和组织生化等检测方法,分别在第14周和第24周观察SBC对DEN诱发的肝癌形成过程的影响.结果中药组14周时大鼠肝脏异型性增生灶明显较模型组少,24周时形成的肝癌结节小而少,模型组、中药组肝癌发生率分别为75.0%、50.0%;免疫组化显示中药组大鼠肝组织谷胱甘肽-S-转移酶胎盘型阳性灶面积明显低于模型组;肝组织匀浆上清液谷胱甘肽-S-转移酶含量以及血清λ-谷氨酰转移酶、碱性磷酸酶、谷丙转氨酶含量也明显低于模型组;流式细胞仪检测结果显示,中药组大鼠肝细胞G0-G1期比例下降,G2-M期比例升高.结论SBC能抑制癌前病变,延缓肝癌的形成,降低肝癌发病率,其作用机制之一可能为阻滞G2-M期细胞进展,从而抑制DEN引起的肝细胞的去分化和恶性增殖.     

Overexpression of CYP3A aggravates endotoxin-induced liver injury in hypophysectomized female rats.

Aim: CYP3A2 is a male-specific isoform of cytochrome P450 enzyme which is expressed abundantly in male rats but not in intact female rats. Having previously reported that hepatic CYP3A2 promotes lipopolysaccharide (LPS)-induced liver injury in male rats, we further examined the impact of CYP3A on LPS-induced liver injury by comparing intact and hypophysectomized female rats. In hypophysectomized female rats, phenobarbital (PB), a cytochrome P450 inducer, markedly increased the hepatic content and activity of CYP3A1/2, but did not do so in intact rats. CYP2B1 increased to similar levels in PB-treated hypophysectomized and intact rats. Methods: Rats were administered 10 mg/kg LPS intravenously and some were given PB for three days before LPS injection. Liver injury was analyzed 8 h after LPS injection. Results: PB-LPS increased plasma alanine aminotransferase significantly more in hypophysectomized female rats than in intact female rats. Ketoconazole, a CYP3A inhibitor, inhibited the increase of liver injury. Hepatic 8-hydroxydeoxyguanosine in nuclei and 4-hydroxy-2-nonenal-modified proteins, measured to evaluate oxidative stress by LPS treatment, increased markedly more in PB-treated, hypophysectomized female rats, than in intact female rats. Conclusion: Overexpression of CYP3A aggravated LPS-induced liver injury in rats, apparently through the formation of reactive oxygen species.     

细胞色素P450 2E1在大鼠急性肝损伤中的表达及其意义

目的研究细胞色素P4502E1在大鼠急性肝损伤中的表达变化及其意义。方法随机将Wista大鼠分成正常对照组和急性肝损伤组，采用四氯化碳制备急性肝损伤模型，并按染毒时间分为3、6、12、24、36和48h6个亚组，每组5只大鼠。采用western blot方法测定染毒后不同时间点肝组织细胞色素P4502E1蛋白的表达变化；测定大鼠血清ALT、AST水平和肝组织MDA浓度、SOD活性的变化以及采用电子自旋共振（ESR）技术测定大鼠肝组织自由基（ROS）浓度变化，HE染色观察肝组织病理形态学改变；结果四氯化碳可明显导致大鼠肝脏损伤，表现为：血清ALT、AST水平显著升高，肝组织MDA浓度和ROS含量显著增加。SOD活性明显下降，和正常对照组相比，差异均十分显著（P〈0．01）；western blot结果显示：细胞色素P4502E1在正常大鼠肝组织中有表达，染毒3h后表达增强，12h达到高峰，明显高于正常对照组（P〈0．05），其表达趋势与ROS浓度变化相一致。结论细胞色素P4502E1蛋白在大鼠急性肝损伤时表达显著增强，提示其在中毒性肝损伤的发病中可能具重要的病理生理意义，并与四氯化碳诱导的氧化应激反应密切相关。     

Selective mitochondrial glutathione depletion by ethanol enhances acetaminophen toxicity in rat liver

Chronic alcohol consumption may potentiate acetaminophen (APAP) hepatotoxicity through enhanced formation of N-acetyl-p-benzoquinone imine (NAPQI) via induction of cytochrome P450 2E1 (CYP2E1). However, CYP2E1 induction appears to be insufficient to explain the claimed magnitude of the interaction. We assessed the role of selective depletion of liver mitochondrial glutathione (GSH) by chronic ethanol. Rats were fed the Lieber-DeCarli diet for 10 days or 6 weeks. APAP toxicity in liver slices (% glutathione-S-transferase alpha released to the medium, GST release) and NAPQI toxicity in isolated liver mitochondria (succinate dehydrogenase inactivation, SDH) from these rats were compared with pair-fed controls. Ethanol induced CYP2E1 in both the 10-day and 6-week groups by approximately 2-fold. APAP toxicity in liver slices was higher in the 6-week ethanol group than the 10-day ethanol group. Partial inhibition of NAPQI formation by CYP2E1 inhibitor diethyldithiocarbamate to that of pair-fed controls abolished APAP toxicity in the 10-day ethanol group only. Ethanol selectively depleted liver mitochondrial GSH only in the 6-week group (by 52%) without altering cytosolic GSH. Significantly greater GSH loss and APAP covalent binding were observed in liver slice mitochondria of the 6-week ethanol group. Isolated mitochondria of the 6-week ethanol group were approximately 50% more susceptible to NAPQI (25-165 micromol/L) induced SDH inactivation. This increased susceptibility was reproduced in pair-fed control mitochondria pretreated with diethylmaleate. In conclusion, 10-day ethanol feeding enhances APAP toxicity through CYP2E1 induction, whereas 6-week ethanol feeding potentiates APAP hepatotoxicity by inducing CYP2E1 and selectively depleting mitochondrial GSH.     

Chemoprevention of 2-amino-1-methyl-6-phenyli-midazo 4,5-b pyridine-induced carcinogen-DNA adducts by Chinese cabbage in rats

ＭＥＴＨＯＤＳＳｐｒａｇｕｅＤａｗｌｅｙｒａｔｓｗｅｒｅｍａｉｎｔａｉｎｅｄｆｏｒ１０ｄａｙｓｏｎｂａｓａｌｄｉｅｔｏｒｄｉｅｔｃｏｎｔａｉｎｉｎｇ２０％（ｗ／ｗ）ｆｒｅｅｚｅｄｒｉｅｄｃａｂｂａｇｅｐｏｗｄｅｒｐｒｉｏｒｔｏａｄｍｉｎｉｓｔ...     

Differences in Hepatic Microsomal Cytochrome P-450 Isoenzyme Induction by Pyrazole, Chronic Ethanol, 3-Methylcholanthrene, and Phenobarbital in High Alcohol Sensitivity (HAS) and Low Alcohol Sensitivity (LAS) Rats

High and low alcohol sensitivity (HAS and LAS) rats have been selected for their differences in ethanol-induced sleep time. Liver monooxygenase activities were studied in HAS and LAS rats before and after treatments with known inducers such as chronic ethanol, pyrazole, 3-methylcholanthrene (3-MC) and phenobarbital (PB) to determine whether the selection procedure also selected for differences in the cytochrome P-450 (P-450) inducibility. This previously has been shown with long sleep (LS) and short sleep (SS) mice, which were selected using a similar criterion. 3-MC and PB, in conjunction with chronic ethanol treatment, were used in order to evaluate the interactions of ethanol with these inducers. Prior to treatment, total P-450 content was slightly lower in LAS than in HAS rats. However, both lines displayed the same microsomal monooxygenase activities related to different P-450 isozymes. This was demonstrated by ethoxyresorufin deethylation (EROD) for cytochrome P-450 1A1 (CYP1A1), acetanilide hydroxylation (ACET) for CYP1A2, pentoxyresorufin dealkylation (PROD) for CYP2B, 1-butanol oxidation (BUTAN) and N-nitrosodimethylamine demethylation (NDMA) for CYP2E1. After the different treatments, HAS rats did not differ from LAS rats in their CYP2E1 inducibility. However, pyrazole, PB and 3-MC treatment led to differences in CYP1A and CYP2B monooxygenase activities between the two lines. The enhancement of PROD by pyrazole treatment was less prominent in LAS (1.7-fold of the control value) than in HAS rats (3.8-fold).(ABSTRACT TRUNCATED AT 250 WORDS)     

肝细胞色素P450 2E1在实验性肝纤维化组织中的表达

目的 研究肝细胞色素Ｐ４５０ ２Ｅ１在实验性肝纤维化组织中的表达。方法 ８只Ｗｉｓｔａｒ大鼠,建立四氯化碳（ＣＣｌ４）肝纤维化模型,用免疫组织化学方法观察肝纤维化模型肝组织中肝细胞色素Ｐ４５０ ２Ｅ１。结果 正常肝组织内,ＣＹＰ ２Ｅ１表达仅见于中央静脉周围区,主要表达于肝腺泡Ⅲ区,２～３层细胞厚,肝细胞浆和细胞膜可见ＣＹＰ ２Ｅ１表达。在ＣＣｌ４模型肝组织中,ＣＹＰ ２Ｅ１表达的强度增加,分布的     

Biochemical effects of dietary intakes of different broccoli samples. I. Differential modulation of cytochrome P-450 activities in rat liver, kidney, and colon

Modulation of xenobiotic metabolism, including cytochrome P-450 (CYP) enzyme activities, due to dietary intakes of cruciferous vegetables, has been described in animals and humans, and the induction of CYP1A enzymes is suggested mainly to be related to the content of indolyl glucosinolates in these vegetables. The aim of the present study was to evaluate the effects on specific CYP activities of various broccoli samples containing different levels of glucosinolates. Groups of rats were fed 1 of 8 broccoli samples from 2 cultivars grown at different conditions. Thirteen different glucosinolates were quantified. The content of the 4 major glucosinolates, glucoraphanin (GRAP), glucoiberin, glucobrassicin (GB), and neoglucobrassicin (NeoGB) varied 5.6-, 2.7-, 3.2-, and 6.6-fold, respectively, among the broccoli samples. Dietary broccoli induced the CYP1A enzyme activities, 7-ethoxyresorufin-O-deethylase (EROD) and 7-methoxyresorufin-O-demethylase (MROD), in rat liver, weakly in colon, but not in kidney. In concordance, the hepatic metabolism of 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) to the proximate carcinogen N-OH-PhIP, a CYP1A-related activity, was enhanced by broccoli. The 7-pentoxyresorufin-O-depenthylase (PROD) activity, an assay for CYP2B1/2, was weakly induced in colon and kidney but not in liver by broccoli. The 2 beta-OH- and 6 beta-OH-testosterone hydroxylase activities were induced in liver microsomes, showing that broccoli increased CYP3A activity. The observed modulations of CYP activities depended clearly on the broccoli sample used, and significantly different responses were observed for different cultivars and growth conditions. These results indicate that modulation of CYP metabolism by broccoli may vary significantly in humans as well, as the content of glucosinolates and other active substances also varies between commercially available broccoli samples. The different effects depending on the vegetable sample eaten have to be considered in future experiments and dietary recommendations.