大鼠肝癌发生蛋白激酶/磷酸肌醇-3-激酶信号通路的表达及健脾解毒法的调节作用

[目的]探讨中药复方健脾解毒方对二乙基亚硝胺(diethylnitrosamine,DEN)诱导的大鼠肝癌的预防作用及其调控蛋白激酶/磷酸肌醇-3-激酶(AKT/PI3K)信号通路的机制。[方法]雄性Wistar大鼠,随机分为正常组(n=25)、模型组(n=40)及中药组(n=40)。除正常组外,其他组1~12周饮用含DEN 80 mg/L水[8 mg/(kg.d)]诱癌;中药组同时给予含生药1.75 g/ml的健脾解毒方(10 ml/kg)灌胃,正常组给予10 ml/kg 0.85%氯化钠灌胃,1次/d,共12周。于4、8、12、16周时相点,各组随机取5只大鼠处死取肝,20周时剩余大鼠全部处死取肝,观察肝脏外观,计算病死率和腹水生成率,肝组织进行苏木精-伊红染色,应用RT-PCR半定量检测肝组织AKT、PI3K及p70s6k mRNA的表达,Western blot法检测肝组织p-AKT的表达。[结果]在20周实验结束时,正常组无死亡,模型组死亡率为42.5%(17/40),中药组死亡率为17.5%(7/40);16~20周时模型组腹水发生率为87.5%(7/8),中药组为44.4%(8/18),2组比较差异有统计学意义(P0.05)。正常组没有肿瘤形成,模型组和中药组在16周后成瘤率均为100%;同时模型组肝癌Ⅲ级发生率为100%(5/5),而中药组Ⅰ、Ⅱ、Ⅲ级肝癌发生率分别为40%(2/5)、40%(2/5)及20%(1/5),2组比较差异有统计学意义(P0.05)。RT-PCR结果显示,与正常组比较,模型组4~20周的AKT mRNA均显著上调,p70s6k mRNA均显著下调,PI3K mRNA除第8周外均显著上调。与模型组比较,中药组可阶段性下调AKT/PI3K及上调p70s6k的表达。Western blot显示,模型组与中药组的p-AKT显著上调,尤其在8~12周最为明显,其中药组的上调幅度显著低于模型组。[结论]AKT/PI3K信号通路与肝癌的发生密切相关,表现为AKT、PI3K表达上调,p70s6k表达下调,健脾解毒方有预防DEN诱导大鼠肝癌的作用,其机制可能部分与中药下调AKT/PI3K及上调p70s6k的表达有关。     

DEN诱导大鼠肝癌形成中miR-199a的表达机制及健脾解毒法的干预作用

目的:探讨中药复方健脾解毒方对二乙基亚硝胺(DEN)诱导的大鼠肝癌的预防作用及其调控miR-199a的表达机制.方法:♂Wistar大鼠,随机分为正常组(n=25)、模型组(n=40)及中药预防组(n=40).除正常组外,其他组在1-12wk用含DEN 80 mg/L的饮水[mg/(kg·d)]以诱癌,中药组同时给予含生药1.75 g/mL的健脾解毒方灌胃(10 mL/kg),正常组给予10 mL/kg生理盐水灌胃,每日1次,共12wk.于4、8、12、16 wk时相点,各组随机取5只大鼠处死取肝,20 wk将剩余大鼠全部处死取肝,观察肝脏外观,计算死亡率和腹水生成率,比较肝脾指数,肝组织进行HE染色,应用Realtime PCR检测肝组织miR-199a的表达.结果:在20 wk实验结束时,正常大鼠无死亡,而模型大鼠死亡率为42.5%(17/40),预防组死亡率为17.5%(7/40),16.20 wk时模型组腹水发生率为87.5%(7/8),预防组为44.4%(8/18),与模型组有差异性(P<0.05).正常组没有肿瘤形成,模型组和预防组在16 wk后成瘤率均为100%.16 wk模型组肝癌Ⅲ级发生率为100%(5/5),而预防组Ⅰ、Ⅱ、Ⅲ级肝癌发生率分别为40%(2/5)、40%(2/5)及20%(1/5),两组比较有差异(P<0.05).与模型组比较,中药预防组有显著降低肝脾指数的作用(均P<0.01).PCR结果显示.模型组肝组织miR-199a较正常组明显上调,中药预防组除16 wk外均有显著下调miR-199a表达的作用(均P<0.01).结论:健脾解毒方有预防DEN诱导大鼠肝癌发生的作用,其机制可能部分与下调miR-199a的表达有关.     

正肝方对大鼠癌前病变肝组织细胞周期蛋白D1及依赖性激酶4的影响

[目的]观察正肝方对黄曲霉毒素B1（AFB1）诱发的大鼠癌前病变肝组织细胞周期蛋白D1（Cyclin D1）、细胞周期蛋白依赖性激酶4（CDK4）的影响。[方法]Wistar大鼠100只随机分为4组：模型、正肝方小剂量、正肝方大剂量组各30只、正常组10只。除正常组外,先后用AFB1和2-乙酰氨基芴（2-AAF）处理各组大鼠造模。正肝方大、小剂量组在造模期间,将正肝方水剂（0.6、0.3 g/ml）按10 ml/kg分别灌胃;模型组用无菌蒸馏水按10 ml/kg灌胃。8周后处死大鼠,肝组织取材。分别采用免疫组化法和RT-PCR法,检测大鼠肝组织中Cyclin D1、CDK4蛋白和mRNA表达。[结果]模型组Cyclin D1、CDK4染色阳性细胞百分率（LI）和表达强度（PU）最高,Cyclin D1、CDK4mRNA表达水平也最高（P〈0.01）。正肝方能降低大鼠癌前病变肝组织Cyclin D1、CDK4蛋白和mRNA的异常高表达（P〈0.01或〈0.05）,且呈现出一定的量效关系。[结论]正肝方能通过下调Cyclin D1、CDK4异常表达,改善细胞周期G1~S期调控点失控,从而减缓肝细胞异常增生。     

蕲艾提取液对大鼠肝组织细胞周期蛋白Cyclin D1表达的影响

[目的]研究蕲艾提取液对免疫性肝纤维化大鼠肝组织细胞周期蛋白Cyclin D1表达的影响。[方法]72只Wister大鼠随机分为正常对照（正常）组,肝纤维化模型（模型）组,蕲艾提取液低、中、高剂量组和丹参组。猪血清腹腔内注射法构建肝纤维化大鼠模型。采用荧光定量PCR和Western blot法检测大鼠肝脏组织细胞周期蛋白Cyclin D1蛋白和mRNA表达水平。[结果]与模型组比较,蕲艾提取液组可下调Cyclin D1 mRNA及蛋白表达（P〈0.05,〈0.01）。[结论]蕲艾提取液可下调Cyclin D1的表达,发挥对细胞周期的调控,使细胞复制停滞于G0/G1期,抑制细胞增殖,进而达到抗肝纤维化的作用。     

EBNA1BP2基因在肝癌形成过程中的表达及不同中医治法的调控作用

目的:探讨EBNA1BP2基因在肝癌形成过程中不同阶段的表达情况及不同中医治法对其在肝癌中表达的影响。方法:①以DEN诱发大鼠肝癌模型,分别观察造模4、8、16、20周后其肝脏组织病理形态学的改变以及EB-NA1BP2基因表达的差异。②应用基因芯片检测健脾益气、清热解毒、活血化瘀等常用中医治法对大鼠EBNA1BP2基因表达的影响。结果:①肝组织病理形态学观察结果表明,大鼠肝癌造模4、8周时肝组织主要表现为炎性病变,而到16周后呈现典型的增生病变,20周后已全部发展为肝细胞癌。②芯片结果显示,EBNA1BP2基因在DEN诱发大鼠肝癌形成过程中的表达量持续增加,至16周后形成表达高峰。③不同中医治法对EBNA1BP2基因在大鼠肝癌中具有不同程度的调控作用,其中健脾益气法下调作用较明显。结论:成功应用DEN诱发大鼠肝癌模型,EBNA1BP2基因在肝癌形成过程中表达持续增加,健脾益气法对其具有明显的下调作用。     

STAT3、Cyclin D1在大鼠肝癌组织中的表达变化及意义

目的观察信号转导和转录活化因子3（STAT3）和细胞周期素D1（Cyclin D1）在大鼠肝癌组织中的表达变化,并探讨其意义。方法将72只大鼠随机分为模型组和对照组各36只,模型组自由饮用0.1 mg/mL的二乙基亚硝胺（DEN）溶液,对照组饮用等量灭菌蒸馏水,连续饮用20周。处死大鼠取癌组织,用RT-PCR法检测STAT3、Cyclin D1 mRNA,Western blot法和免疫组化法检测STAT3、Cyclin D1蛋白。结果对照组STAT3、Cyclin D1mRNA表达水平分别为0.32±0.12、0.18±0.05,模型组分别为0.72±0.25、0.57±0.15,P均〈0.05。Western blot法检测模型组STAT3、Cyclin D1蛋白表达量分别为0.58±0.25、0.65±0.14,对照组分别为0.32±0.19、0.41±0.15,P均〈0.05。免疫组化法检测模型组STAT3、Cyclin D1蛋白IOD分别为2 373.87±258.79、668.44±63.10,对照组分别为487.81±55.31和138.86±31.50,P均〈0.05。结论 STAT3、Cyclin D1在肝癌大鼠癌组织中表达上调,二者可促进肝癌的发生和发展。     

肝素结合表皮生长因子对部分肝移植大鼠肝细胞CyclinD1表达的影响

目的观察肝素结合表皮生长因子(heparin-binding epidermal growth falrtor like growth factor,HB-EGF)对部分肝移植大鼠肝细胞Cyclin D1表达的影响,并探讨其促进肝再生的机制。方法首先采用改良的"两袖套"法构建大鼠部分(60%)肝移植模型,共75只,受体随机平均分为A、B、C三组。其中,A组经皮下注射生理盐水(100 mg/kg),B组皮下注射HGF(100 mg/kg),C组为皮下注射HB-EGF(100 mg/kg),1次/d。手术后第1天、第2天、第3天、第5天和第7天各处死5只受体大鼠,采用实时荧光定量PCR、Western blotting检测肝组织中Cyclin D1 mRNA及蛋白的表达。结果 RT-PCR检测结果表明,C组术后第1天Cyclin D1 mRNA表达就达到高峰,且显著高于A、B组(P0.05),之后1周逐渐降低;Western blotting检测结果表明,三组Cyclin D1蛋白表达水平均有上升,但C组术后第1天即达到高峰,且显著高于A、B组(P0.05)。结论 HE-EGF可能通过上调肝移植后肝细胞Cyclin D1的表达来促进肝再生。     

叶酸、维生素B12对高同型半胱氨酸血症大鼠ICAM-1及VCAM-1表达的影响

目的观察叶酸、维生素B12对高同型半胱氨酸血症(hyperhomecysteinemia,Hhcy)大鼠主动脉壁细胞间黏附分子-1(ICAM-1)及血管细胞黏附分子-1(VCAM-1)表达的影响。方法 24只雄性SD大鼠随机分为三组:空白对照组、Hhcy模型组、叶酸、维生素B12干预组。8 w后检测血浆同型半胱氨酸(Hcy)水平,观察主动脉壁形态学变化并检测胸主动脉血管壁ICAM-1及VCAM-1的表达。结果 Hhcy模型组大鼠的血浆Hcy水平明显高于空白对照组(P<0.01),主动脉壁ICAM-1及VCAM-1表达较空白对照组明显上调(P<0.01)。Hhcy模型组血浆Hcy水平明显高于叶酸、维生素B12干预组(P<0.01),主动脉壁ICAM-1及VCAM-1的表达较空白对照组明显上调(P<0.01)。结论高蛋氨酸饮食可诱发Hhcy,主动脉壁ICAM-1及VCAM-1表达上调。叶酸、维生素B12可以下调血浆Hcy水平及主动脉壁ICAM-1及VCAM-1的表达,对Hhcy致AS具有明显预防作用。     

化浊解毒益气方对肝纤维化大鼠细胞因子的影响

目的 观察化浊解毒益气方对肝纤维化大鼠转化生长因子β1(TGF-β1)和组织金属蛋白酶抑制因子-1(TIMP-1) mRNA表达的影响.方法 采用腹腔注射猪血清的方法建立免疫性肝纤维化大鼠动物模型,以化浊解毒益气方分别于造模的开始和第8周进行干预,以秋水仙碱作为阳性对照.分别于造模的8和12 w处死5只大鼠,提取肝组织行RT-PCR法检测TGF-β1和TIMP-1 mRNA的表达.结果 腹腔注射猪血清成功复制了免疫性肝纤维化大鼠模型,与正常对照组相比,模型对照组TGF-β1和TIMP-1 mRNA的表达明显增高(P＜0.05);中药干预可降低TGF-β1和TIMP-1 mRNA的表达,其作用优于秋水仙碱(P＜0.05).但中药预防组与中药治疗组之间,秋水仙碱预防组与秋水仙碱治疗组之间,差异无显著性(P＞0.05).结论 化浊解毒益气方可以减轻猪血清诱导的大鼠免疫性肝纤维化,其作用可能与降低肝组织TGF-β1和TIMP-1 mRNA的表达有关.     

解聚复肾宁对糖尿病大鼠肾组织基质金属蛋白酶-9、金属蛋白酶组织抑制剂-1表达的影响

目的观察解聚复肾宁(JJFSN)对糖尿病(DM)大鼠肾组织基质金属蛋白酶-9(MMP-9)、金属蛋白酶组织抑制剂-1(TIMP-1)表达及肾脏保护作用机制。方法建立STZ诱导的DM SD大鼠模型,将成模DM大鼠随机分成4组:模型组、JJFSN组、厄贝沙坦组、JJFSN+厄贝沙坦组,同时设正常对照组。各组大鼠采用相应的干预措施处理12 w。检测各组大鼠第12周时肾重/体重、血糖、尿素氮、血肌酐、尿白蛋白排泄率(UAER),免疫组化法检测肾组织MMP-9、TIMP-1表达,透射电镜观察肾脏超微结构。结果模型组肾脏超微结构改变明显,血糖、UAER、尿素氮、血肌酐、肾重/体重显著增高。模型组大鼠肾组织TIMP-1的表达较正常对照组明显上调(P<0.01),JJFSN组、厄贝沙坦组、JJFSN+厄贝沙坦组肾组织TIMP-1表达明显下调(P<0.01),但仍高于正常对照组(P<0.01),JJFSN+厄贝沙坦组下调最明显(P<0.01)。DM模型组大鼠肾组织MMP-9的表达较正常对照组明显下调(P<0.01),解聚复肾宁组、厄贝沙坦组、解聚复肾宁+厄贝沙坦组肾组织MMP-9表达明显上调(P<0.01),但仍低于正常对照组(P<0.01),解聚复肾宁+厄贝沙坦组上调最明显(P<0.01)。结论 MMP-9、TIMP-l的表达变化与肾小球细胞外基质(ECM)降解减少相关,可能促进了糖尿病肾病(DN)的发生,JJFSN可能通过干预这种表达变化,减缓DN的发生和发展。     

1-Naphthyl isocyanate and 1-naphthylamine as metabolites of 1-naphthylisothiocyanate

Abstract: The importance of the bioactivation of 1-naphthylisothiocyanate was studied. Forty minutes after 1-naphthylisothiocyanate administration to rats, bile was collected over a 2.5-h period; the liver was then excised and homogenized. 1-naphthylisothiocyanate and its metabolites in bile and liver of rats were identified and quantified using coupled gas chromatography-mass spectrometry. Three main compounds were found in all 1-naphthylisothiocyanate-treated animals. They were identified as 1-naphthyl isocyanate, 1-naphthylamine and the parent compound, 1-naphthylisothiocyanate. When rats were given cycloheximide, which attenuates 1-naphthylisothiocyanate toxicity, 30 min before 1-naphthylisothiocyanate (300 mg/kg), 1-naphthyl isocyanate concentration was significantly lower than in rats receiving only 1-naphthylisothiocyanate. The appearance of 1-naphthylamine was also inhibited by cycloheximide, although not to the same extent as 1-naphthyl isocyanate. On the other hand, phenobarbital, which potentiates 1-naphthylisothiocyanate hepatotoxicity, enhanced 1-naphthyl isocyanate and 1-naphthylamine formation. It is suggested that 1-naphthyl isocyanate, 1-naphthylamine and the highly reactive sulfur released from 1-naphthylisothiocyanate might be involved in the hepatotoxic effect of 1-naphthylisothiocyanate.     

The target of ezetimibe is Niemann-Pick C1-Like 1 (NPC1L1)

Ezetimibe is a potent inhibitor of cholesterol absorption that has been approved for the treatment of hypercholesterolemia, but its molecular target has been elusive. Using a genetic approach, we recently identified Niemann-Pick C1-Like 1 (NPC1L1) as a critical mediator of cholesterol absorption and an essential component of the ezetimibe-sensitive pathway. To determine whether NPC1L1 is the direct molecular target of ezetimibe, we have developed a binding assay and shown that labeled ezetimibe glucuronide binds specifically to a single site in brush border membranes and to human embryonic kidney 293 cells expressing NPC1L1. Moreover, the binding affinities of ezetimibe and several key analogs to recombinant NPC1L1 are virtually identical to those observed for native enterocyte membranes. KD values of ezetimibe glucuronide for mouse, rat, rhesus monkey, and human NPC1L1 are 12,000, 540, 40, and 220 nM, respectively. Last, ezetimibe no longer binds to membranes from NPC1L1 knockout mice. These results unequivocally establish NPC1L1 as the direct target of ezetimibe and should facilitate efforts to identify the molecular mechanism of cholesterol transport.     

甲型H1N1与季节性H1N1流感病毒血凝素基因比较分析

目的分析泰安市2008～2009年度季节性流感与2009年度甲型H1N1流感病原学检测结果 ,比较季节性H1N1与甲型H1N1血凝素基因变异情况。方法选择国家级流感监测哨点医院以及暴发疫情的疫点,采集流感样病例的鼻咽拭子标本,通过RealtimePCR进行病毒检测,用MDCK细胞进行病毒分离,通过RT-PCR扩增血凝素HA1片段的基因并测序,利用生物信息学进行序列分析。结果 2008～2009年共检测鼻咽拭子标本283份,分离出流感病毒33株,分离阳性率为11.67%,其中季节性H1N1亚型31株。2009年5月1日～12月31日,检测鼻咽拭子标本996份,流感核酸检测阳性417份,阳性率为41.86%,其中甲型H1N1337份,季节性H1N1亚型1份。6株季节性H1N1病毒均在多个氨基酸位点上发生变异,与疫苗株A/Brisbane/59/2007(H1N1)比较,有11个位点发生了突变,其中5个位点位于抗原决定簇上;测序成功的6株甲型H1N1病毒在多个氨基酸位点发生变异,与疫苗株A/California/07/2009(H1N1)比较,有6个位点发生突变,其中1个位点位于抗原决定簇的B区。结论 2008～2009年度季节性H1N1为优势株,甲流暴发后,甲型H1N1成为绝对优势毒株。季节性H1N1分离株有多处氨基酸替换,抗原决定簇B区变异频繁;甲型H1N1病毒分离株的基因有变异,但关键位点第222位仍为D(天冬氨酸),与疫苗株相比抗原决定簇的关键位点变化不大。     

Inhibition of procarcinogen-bioactivating human CYP1A1, CYP1A2 and CYP1B1 enzymes by melatonin

Abstract:  Administration of melatonin to rodents decreases the incidence of tumorigenesis initiated by benzo[ a ]pyrene or 7,12-dimethylbenz[ a ]anthracene, which requires bioactivation by cytochrome P450 enzymes, such as CYP1A1, CYP1A2 and CYP1B1, to produce carcinogenic metabolites. The present study tested the hypothesis that melatonin is a modulator of human CYP1 catalytic activity and gene expression. As a comparison, we also investigated the effect of melatonin on the catalytic activity of CYP2A6, which is also a procarcinogen-bioactivating enzyme. Melatonin (3–300 μ m ) decreased 7-ethoxyresorufin O -dealkylation catalyzed by human hepatic microsomes and recombinant CYP1A1, CYP1A2 and CYP1B1, whereas it did not affect coumarin 7-hydroxylation catalyzed by hepatic microsomes or recombinant CYP2A6. Melatonin inhibited CYP1 enzymes by mixed inhibition, with apparent K _i values (mean ± S.E.M.) of 59 ± 1 (CYP1A1), 12 ± 1 (CYP1A2), 14 ± 2 (CYP1B1) and 46 ± 8 μ m (hepatic microsomes). Additional experiments indicated that melatonin decreased benzo[ a ]pyrene hydroxylation catalyzed by hepatic microsomes and CYP1A2 but not by CYP1A1 or CYP1B1. Treatment of MCF-10A human mammary epithelial cells with melatonin (up to 300 μ m ) did not affect basal or benzo[ a ]pyrene-inducible CYP1A1 or CYP1B1 gene expression. Consistent with this finding, melatonin did not influence reporter activity in aryl hydrocarbon receptor-dependent pGudluc6.1-transfected MCF-10A cells treated with or without benzo[ a ]pyrene, as assessed in an in vitro cell-based luciferase reporter gene assay. Overall, melatonin is an in vitro inhibitor of human CYP1 catalytic activity, and it may be useful to develop potent analogues of melatonin as potential cancer chemopreventive agents that block CYP1-mediated chemical carcinogenesis.     

Characterization of MTHFR,GSTM1, GSTT1, GSTP1, and CYP1A1 genotypes in childhood acute leukemia

The role of methylenetetrahydrofolate reductase (MTHFR C677T), glutathione S-transferases (GSTM1 and GSTT1 null, GSTP1 Ile105Val), and cytochromes p450 (CYP1A1*2A) genotypes in the etiology of childhood leukemia was simultaneously investigated. 144 Turkish children with acute lymphoblastic leukemia (ALL) and 33 with acute nonlymphoblastic leukemia (ANLL) were studied and compared with 185 healthy pediatric controls. The frequency of MTHFR genotype was insignificantly higher in ALL (7.7%) and ANLL (6.3%) than in controls (4.4%). Equal distribution of the GSTM1 null genotype was detected between ALL patients and controls (55%), while its incidence was slightly higher in ANLL patients (61.3%). Although GSTT1 null genotype was insignificantly lower in ALL patients (20.9%) than controls (22.7%), it was significantly underrepresented in ANLL patients (6.5%) (P = 0.05, OR 0.24, 95% CI 0.05-1.03). The homozygous frequency of GSTP1 genotype did not differ significantly between groups of ALL (3.7%), ANLL patients (9.1%) and controls (4.9%). Homozygous CYP1A1*2A genotype was underrepresented in ALL patients (1%) as compared to control (4.8%) but the differences did not reach to statistical significance (OR 0.21; 95% CI 0.03-1.72). Homozygosity for this genotype was not detected in ANLL patients. No particular association was noted between different combinations of combined genotypes and risk of development of childhood ALL and ANLL. These results suggested that there are no significant associations between the studied genotypes and the risk of developing either form of acute leukemia except GSTT1 null and homozygosity for CYP1A1 genotypes that may play protective roles in the development of ANLL in Turkish children.