间断小剂量DEN诱发大鼠肝癌模型研究

目的:建立实验动物死亡率低、诱癌时间短及与人肝癌的发生过程相似的肝癌模型。方法:取3～4个月龄SD大鼠65只,随机分为两组,实验组大鼠给予0.2%二乙基亚硝胺(diethylnitrosa mine,DEN)灌胃,按体质量10mg/kg给药,每周5次,至14周停药。对照组给予等量生理盐水灌胃。结果:实验组大鼠体质量、一般精神状况均不如对照组,P<0.05。诱癌第14周所有实验组大鼠均诱发出肝细胞性肝癌。结论:间断小剂量给予DEN,可以方便、简单、稳定地建立大鼠肝癌模型。     

大鼠诱发肝癌过程中肝脏脂类的变化

脂类是构成细胞内外膜质结构的主要成份。在维持膜结构完整性、信息的跨膜传递和调节代谢等方面起着十分重要的作用。恶性肿瘤时生物膜(包括表面膜、亚细胞膜等)的结构和功能均发生明显改变,导致细胞的各种恶性行为,而这些改变必然伴有脂类组成和代谢的改变。但过去对恶性肿瘤中的蛋白质、酶和核酸研究较多,对脂类则少报道。本实验室曾发现用Solt和Farber法诱发肝癌的鼠肝中,蛋白激酶C在癌前期即见明显升高。因磷脂在蛋白激酶C的调节中起关键作用,故研究肝癌中磷脂的代谢将有助于癌变机理的了解。本文系统地研究了二乙基亚硝胺(DEN)诱发大鼠肝癌过程中,胆固醇和磷脂及其各组分含量的动态变化。     

苦参碱对二乙基亚硝胺诱发大鼠肝癌的预防作用

目的：研究不同剂量苦参碱（MT）对二乙基亚硝胺（DEN）诱发大鼠肝癌的预防阻断作用。方法：采用0．01％的DEN诱发大鼠肝癌90天,同时分别用MT注射液30,15,3mg/kg腹腔注射和3mg/kg灌胃,停止诱癌及给药处理30天后,处死大鼠,观察大鼠肝脏的病理改变,肝表面癌结节数,肝／体重比和血清中丙氨酸氨基转移酶（ALT）,γ-谷氨酰转肽酶（γ－GT）,碱性磷酸酶（ALP）的变化。结果：3mg/kg MT腹腔注射组和灌胃组的体重明显高于模型组,肝表面癌结节数,肝／体重比和血清ALT,γ－GT明显低于模型组和它MT腹腔注射组（P＜0．05）,但腹腔注射的三组中ALP较模型组有不同程度升高,灌胃组则有所下降,结论：MT用于预防阻断DEN诱发大鼠肝癌时,不论剂量大小或给药途径如何,均不能完全阻断DEN诱发大鼠肝癌的发生,但小剂量（3mg/kg)的MT长期口服应用,能保护肝细胞免受损伤,延缓DEN诱发大鼠肝癌的发展。     

二乙基亚硝胺诱发大鼠肝癌组织下调的基因

  目的 运用基因芯片技术，观察二乙基亚硝胺（DEN）诱导大鼠肝癌（含肝）组织与正常对照大鼠肝组织比较表达下调的基因。方法 DEN诱发大鼠肝癌，应用Affymetrix Rat 230A GeneChip进行检测。结果 在芯片的15 710个基因中，剔除正常肝组织表达值读数在50以下的基因，正常肝脏基因表达大于肝癌基因的有248个，其中120个为EST片断，已知基因为128个。结论 DEN诱发大鼠肝癌的后基因组变化是十分复杂的，如何逐渐找到那些起着关键作用的基因，进一步阐释其在肝癌的发生、发展和转归中的作用，是今后工作的重点。     

苦参碱和氧化苦参碱对二乙基亚硝胺诱发大鼠肝癌的预防阻断作用

目的 :研究苦参碱 (MT)和氧化苦参碱 (OMT)分别对二乙基亚硝胺 (DEN)诱发大鼠肝癌的预防阻断作用。方法 :采用 0 .0 1%的DEN诱发大鼠肝癌 90d ,同时分别腹腔注射MT和OMT注射液 15mg/kg ,停止诱癌及给药处理 30d后 ,处死大鼠 ,观察大鼠肝脏的病理改变、肝表面癌结节数、肝 /体重比和血清中丙氨酸氨基转移酶 (ALT)、γ 谷氨酰转肽酶 (γ GT)、碱性磷酸酶 (ALP)的变化。结果 :MT组和OMT组大鼠的体重明显高于模型组 ,肝表面癌结节数、肝 /体重比和血清ALT、γ GT明显低于模型组 (P<0 0 5) ,而ALP较模型组有所升高。另外 ,OMT组的肝重、肝 /体重比和血清ALP均明显低于MT组(P <0 0 5)。结论 :MT和OMT ,尤其是OMT ,尽管不能完全阻断DEN诱发大鼠肝癌的发生 ,但能保护肝细胞免受损伤 ,延缓DEN诱发大鼠肝癌的形成     

柑皮油对二乙基亚硝胺诱发大鼠肝癌前病变的抑制

用二乙基亚硝胺致大鼠肝癌前病变实验模型，研究从鲜柑皮提取的挥发性柑皮油对致癌作用的影响。于大鼠接受ＤＥＮ前１０天起，饲以含５％柑皮油饲料。经两次重复实验，结果显示：肝癌前病变Ｇａｍｍａ－谷氨酰转肽酶阳性肝细胞增生灶的发生类，用柑皮细胞明显少于于单给ＤＮＥ。     

柴胡皂甙-d对DEN致大鼠实验性肝癌形成的影响

目的:研究柴胡皂甙d(saikosaponin-d,SSd)对二乙基亚硝胺(diethylnitrosamine,DEN)致大鼠实验性肝癌形成的影响.方法:清洁级雄性SD大鼠90只,随机分为5组:模型组(n=20),对照组(n=10)与SSd大、中、小剂量治疗组(各组n=20).对照组给予等量生理盐水灌胃,其余各组大鼠均给予2g/L二乙基亚硝胺(DEN,10 mg/kg)灌胃,每周5次,同时各治疗组每天腹腔注射不同浓度SSd(2.0,1.5,1.0 mg/kg),至16 周停药.分别于第6、12、16周处死大鼠,自动生化分析仪测定血清总胆红素(total bilirubin,TBIL)、丙氨酸氨基转移酶(alanine aminotransferase,ALT)、天门冬氨酸氨基转移酶(aspartate amino transferase,AST)和白蛋白(albumin,ALB)含量的变化,HE染色观察实验大鼠各期肝组织病理学结构的改变.结果:SSd各治疗组与模型组相比,SSd能够改善大鼠血清各项肝功指标,增加体重,明显减轻大鼠肝细胞的变性坏死,降低死亡率,并且有统计学差异(P＜0.05).病理学检查发现SSd各治疗组大鼠癌结节数及灶的大小均小于模型组,镜下单纯造模组癌细胞呈多形性,异形性明显;相反,SSd各干预组癌细胞分化程度高,异形性较低.结论:SSd对实验性大鼠肝癌的形成有一定的抑制作用.     

突变型p53蛋白在二乙基亚硝胺诱发大鼠肝癌前病变中的表达

目的　探讨 p53基因与肝细胞癌变的关系。方法　用免疫组化方法 ,观察二乙基亚硝胺( DEN)诱发大鼠肝癌前病变组织及对照组肝组织中突变型 p53( mp53)蛋白、胎盘型谷胱甘肽 S转移酶 ( GST- P)的表达及其与细胞增殖状态的关系。结果　实验组第 5周时仅在 2 / 5例癌前病变肝组织中检测到 mp53蛋白的表达 ,此蛋白仅存在于部分较大的肝细胞增生结节 ( HN)或 GST- P阳性灶内 ;mp53蛋白阳性的 HN或 GST- P阳性灶 ,其细胞的溴化脱氧尿嘧啶标记指数 ( Brd U- LI)较阴性者高( P<0 .0 5)。结论　 p53基因的突变可发生于肝细胞癌变的早期阶段 ,与肝细胞的异常增殖和癌变有关 ,并有可能成为新的肝癌前肿瘤基因标记物。     

Preventive Effect of Hydrazinocurcumin on Carcinogenesis of Diethylnitrosamine-induced Hepatocarcinoma in Male SD Rats

The purpose of the present study was to evaluate the preventive effects of hydrazinocurcumin (HZC) ondiethylnitrosamine (DEN)-induced hepatocarcinogenesis in a male Sprague Dawley (SD) rat model. One hundredand twenty male SD rats used in this study were divided into six groups. Those receiving DEN with curcumin(CUR) or HZC were studied compared with the DEN-alone group. The study demonstrated that DEN inducedsevere histological and immunohistochemical changes in liver tissues, significantly increasing the levels of livermarker enzymes (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase(ALP), γ-glutamyltransferase (GGT) and total bilirubin level (TBL)). The hepatocarcinoma incidenceswere 100.0%, 36.7% and 20.0% in the DEN-alone, DEN-CUR and DEN-HZC groups, respectively. Althoughmacroscopic and microscopic features suggested that both CUR and HZC were effective in inhibiting DENinducedhepatocarcinogenesis, HZC was exerted a stronger influence. Immunohistochemical analysis with PCNAdemonstrated significantly differences among the groups (all P < 0.05). Taken together, the results suggestedapplication of CUR and HZC could prevent the occurrence of carcinogenesis and HZC may be a more potentcompound for prevention of DEN-induced hepatocarcinogenesis in rats than CUR.     

2-乙酰氨基芴诱发大鼠肝癌变过程中rasp21、AFP的原位表达

目的探讨了rasp21、AFP在2-乙酸氨基芴（2－FAA）诱发的大鼠肝癌变过程中的分布情况及关系。方法rasp21和AFP采用免疫组化S－P法被检测及分析。结果诱癌早期增生的肝细胞及变异肝细胞灶中即显示rasp21和AFP的过度表达,并随增生肝细胞结节的形成和演变二者过度表达细胞增多且相伴而存,由此提示,两者与大鼠肝癌发生有密切关系,rasp21过度表达直接参与了大鼠肝癌的启动和演进。结论同时表达AFP,rasp21的细胞和病变是较具特异的癌前病变,p21与AFP的联合表达可为人类肝癌提供一个参考诊断标志。     

Inhibitory Effects of Benzyl Isothiocyanate and Benzyl Thiocyanate on Diethylnitrosamine-induced Hepatocarcinogenesis in Rats

The effects of two aromatic thiocyanates, benzyl isothiocyanate (BITC) and benzyl thiocyanate (BTC), on diethylnitrosamine (DEN)-induced hepatocarcinogenesis were examined in rats. A total of 108 male ACI/N rats, 5 weeks old, were divided into 6 groups (18 rats in each). Group 1 was given a single i.p, injection of DEN (200 mg/kg body weight) one week after the start of the experiment and then kept on the basal diet until the end of the experiment (1 year). Groups 2 and 3 were treated with DEN and received dietary BITC (100 ppm) or BTC (100 ppm), respectively, throughout the experimental duration. Groups 4 and 5 were not given the carcinogen and were fed the diet containing BITC or BTC, respectively. Group 6 was kept on the basal diet alone and served as a control. Liver neoplasms were seen in Groups 1, 2 and 3. Incidence and average number of liver neoplasms in Group 2 were significantly smaller than in Group 1 ( P <0.0005 and P <0.001, respectively). The incidence of liver neoplasms in Group 3 was slightly lower than in Group 1, although the difference was not statistically significant. The numbers of glutathione S-transferase placental form (GST-P)-positive foci in Group 2 and γ-glutarnyltranspepridase (GGT)-positive foci in Groups 2 and 3 were significantly smaller than those in Group 1 ( P <0.001). The average and unit areas of GST-P- or GGT-positive foci in Group 2 or 3 were also significantly smaller than those in Group 1 ( P <0.05). These results suggest that BITC and BTC are chemopreventive agents for DEN-induced liver tumorigenesis.     

Decreased Expression of Bcl-x Protein during Hepatocarcinogenesis Induced Exogenously and Endogenously in Rats

Dysregulations of apoptosis have been widely recognized as important events in multi‐stage carcinogenesis. Bcl‐x, a member of the Bcl‐2 family, is known to act as a regulator of apoptosis. The present study was conducted to assess the role of altered Bcl‐x protein expression in exogenous and endogenous hepatocarcinogenesis in rats. In the short‐term exogenous models, male Fischer 344 rats, 6 weeks old, were given a single intraperitoneal injection of diethylnitrosamine (DEN) at a dose of 200 mg/kg body weight, partially hepatectomized at the end of week 3, administered phenobarbital at a concentration of 0.05% from the end of week 2 for 6 weeks, and sacrificed. In the livers, glutathione S‐transferase (GST‐P)‐positive, putative preneoplastic lesions were induced, and Bcl‐x protein expression was decreased in 24.7% of such lesions. The incidence of GST‐P‐positive lesions with decreased Bcl‐x increased depending on the size of the lesions; 18.9%, 32.4% and 86.5% in the lesions smaller than 0.03, between 0.03 and 0.3, and larger than 0.3 mm², respectively. In GST‐P‐positive lesions larger than 0.3 mm², both apoptosis induction and cell proliferation activity were enhanced when Bcl‐x protein expression was decreased. In the long‐term exogenous models, rats were given 10 mg/kg of DEN, partially hepatectomized 4 h after treatment, administered 0.5 mg/kg of colchicine at the end of days 1 and 3, subjected to a selection procedure, and sacrificed at the end of week 45. Hepatocellular carcinomas were induced with the decreased Bcl‐x protein expression. In the endogenous model, rats were fed a choline‐deficient, l ‐amino acid‐defined diet for 16 or 80 weeks and sacrificed. Bcl‐x protein expression was decreased both in GST‐P‐positive lesions and hepatocellular carcinoma. These results suggest that this decrease of Bcl‐x protein might serve as an indicator of the advanced form of preneoplastic lesions, and that this decrease could also be associated with a potential to progress into carcinoma in both exogenous and endogenous hepatocarcinogenesis of rats.     

The Therapeutic Effect of Myrrh (Commiphora molmol) and Doxorubicin on Diethylnitrosamine Induced Hepatocarcinogenesis in Male Albino Rats

Background: This study was conducted to assess the therapeutic effect of Myrrh on Diethylnitrosamine (DEN)-induced hepatocarcinogenesis (HCC) in male albino rats. Methods: Fifty male albino rats were divided into five groups (10 rats each).  Group 1 (control group) received distilled water. Group 2 (positive control) was injected intraperitoneally with DEN (55 mg/kg b.w) twice a week for two weeks, while group 3 (DOX) received doxorubicin i.p (10 mg/ kg b.w) after concomitant with DEN twice a week for four weeks.  Groups 4 and 5 received a low dose of Myrrh (250 mg/kg b.w) and a high dose of Myrrh (500 mg/kg b.w) respectively daily for four weeks after the induction with DEN. The sera were used to estimate the liver enzymes (ALT, AST, and ALP), Alpha-fetoprotein (AFP), Total antioxidant capacity (TAC), and Tumor necrosis factor-ἁ (TNF-ἁ). Also, the liver tissues were collected to determine the oxidative stress markers in addition to the histopathological and immunohistochemical investigations. Results: The results showed that the induction of DEN causes a significant increase in the level of liver enzymes (ALT, AST, and ALP), AFP and TNF-ἁ as well as produce oxidative stress indicated by increasing of malondialdehyde (MDA) with the reduction in TAC and glutathione (GSH). Meanwhile, there are noticeable histopathological lesions with loss of hepatic architecture. This was accompanied by a significant increase of immunohistochemical markers; Caspase-3, vascular endothelial growth factor (VEGF), transforming growth factor β1(TGF- β1), and carcinoembryonic antigen (CEA) percentage area. The treatment of DEN rats with DOX reduced the alterations in most parameters. A marked amelioration of all parameters in a dose-dependent manner of Myrrh to the values almost near to those of the control group. Conclusion: Our data revealed that Water extract of Myrrh (C. molmol) has a potential therapeutic effect in attenuation of HCC induced DEN.     

Diethylnitrosamine-induced Hepatic Lesions are Greater in Rats Maintained under a Light-dark Cycle than under Constant Light,Related to the Locomotor Activity Rhythm

Environmental lighting conditions affect circadian rhythm and carcinogenesis. The effect ofdiethylnitrosamine (DEN, i.p., 200 mg/kg) on carcinogenesis and circadian rhythmicity under a light-dark (LD)cycle, constant dark (DD) and constant light (LL) was analyzed in rats. After the recognition of entrainment inlocomotor activity rhythm to LD cycle, animals remained under the LD cycle or were released into DD or LL.Liver carcinogenicity, measured by GST-P immunostaining, was higher under the LD cycle than under DD andLL. Two weeks after DEN injection, locomotor activity in 24 hr had increased under the LD. Circadianrhythmicity might be coupled with the carcinogenicity of DEN.     

Cytochrome P-450 Isozyme Pattern Is Related to Individual Susceptibility to Diethylnitrosamine-induced Liver Cancer in Rats

Differences in susceptibility to chemical carcinogenesis between rodent strains and species have been linked to variations in genetically-determined mixed function oxidase activities. In order to verify whether such variations also determine the susceptibility of individual animals of the same strain to a chemical carcinogen, outbred male Wistar rats were administered diethylnitrosamine (DEN) (1, 2, or 3 nig/kg) five times a week for 20 weeks. The relationship was examined between the outcome (i.e. presence or absence of liver tumors, and latency period) and the hepatic activities of mixed function oxidases and conjugating enzymes, as well as of O⁶-methylguanine-DNA-methyltransferase, measured before the carcinogen treatment. In addition, the metabolic profiles of two model drugs, antipyrine and disopyramide, in the urine were analyzed and correlated with the carcinogen susceptibility. The length of the latency period of hepatocellular tumors in individual rats was negatively related to the activities of hepatic dimethylnitrosamine N -demethylase, aryl hydrocarbon hydroxylase and epoxide hydrolase and positively related to the amount of microsomal protein. Consistent relationships between the other 10 measured parameters and the susceptibility to DEN-induced carcinogenesis were not detected. Long-term treatment with DEN slightly decreased the proportion of metabolism of antipyrine into norantipyrine, and increased the share of 4-hydroxyantipyrine; a decrease in the metabolism of disopyramide to N-deisopropyldisopyramide was also detected. It is concluded that the pattern of cytochrome P-450 isoenzymes is related to differences in individual susceptibility to nitrosamineinduced carcinogenesis. The relationship was most marked at low dose levels, which are the levels at which nitrosamine exposures of humans are known to occur.     

Differential Changes in Expression of Gap Junction Proteins Connexin 26 and 32 during Hepatocarcinogenesis in Rats

We examined expressions of the gap junction proteins, connexin 26 (C×26) and 32 (C×32), in preneoplastic and neoplastic lesions during rat hepatocarcinogenesis. A marked reduction in the numher of C×32-positive gap junctions was observed in 17% of the glutathione S-transferase placental form-positive foci, whereas 44% of the foci showed increased expression of C×26. Most hyperplastic nodules exhibited decreased expression of C×32, whereas 16% of the nodules showed increased expression of C×26. In hepatocellular carcinomas, expressions of both C×32 and C×26 were significantly reduced. These results suggest that the expressions of C×32 and 26 are differentially regulated during hepatocarcinogenesis, and that the decrease in C×32 expression occurs earlier, whereas reduction in C×26 expression occurs later in association with promotion and progression of carcinogenesis.     

Cytochrome 4Z1 Expression Is Correlated with Poor Prognosis in Patients with Cervical Cancer

Background: cervical cancer is one of the most common malignancies in women worldwide and its management remains challenging and complex. As Cytochrome4Z1 (CYP4Z1) is overexpressed in many tumours, its expression in cervical cancer is unknown. Therefore, the present study aimed to evaluate CYP4Z1 expression in cervical cancers. Methods: CYP4Z1 expression was immunohistochemically assessed in 100 cases of cervical cancers along with ten normal cervix tissues, and the enzyme’s relationship to several clinicopathological features and survival was explored. Results: CYP4Z1 was strongly expressed in 55% of cervical cancer patients. Normal cervix samples were negative for CYP4Z1 expression. Importantly, this expression was significantly found in patients with the late stage of the disease, lymph node metastasis, and high tumour invasion (p < 0.05). Interestingly, CYP4Z1 expression was significantly correlated with shorter survival times of cervical cancer patients. Univariate analysis showed that CYP4Z1 expression, tumour stage, lymph node metastasis, and tumour invasion were significantly correlated with patient survival (p < 0.05). The multivariate analysis revealed that only CYP4Z1 expression and tumour stage were significantly correlated with patient survival (p < 0.05). Conclusions: CYP4Z1 expression is associated with cervical cancer patients’ survival and may serve as an independent predictor of poor prognosis in cervical cancer patients.     

黄曲霉毒素诱导大鼠肝细胞癌变中JNK1的变化

目的 观察黄曲霉毒素诱导大鼠肝细胞癌变过程中JNK的变化,进一步了解JNK1信号转导通路在肝癌（Hepatocellular carcinoma,HCC）发生发展过程中的作用。方法 取雄性4周龄SD大鼠77只为研究对象,随机分为实验组66只,空白对照组11只。实验组应用黄曲霉毒素B1（Aflatoxin B1,AFB1）诱发大鼠肝细胞癌,空白对照组大鼠,按正常饲养方法饲养,两组大鼠分别于实验第12、20、36、46周进行肝组织活检,58周时处死取肝。所有标本均进行常规病理组织学检测,并应用RT-PCR、Western blot分别检测癌组织及肝组织JNK1 mRNA及JNK1活性蛋白质水平。结果 实验组大鼠46周时发现首例肝癌,存活至实验结束并发生肝癌的大鼠共24只,6只无任何肿瘤发生,空白对照组大鼠11例均未发生肿瘤。实验组及空白对照组大鼠均可检出不同程度的JNK1 mRNA表达,其中肝癌组织较癌旁组织及正常肝组织明显增强（P<0.05）。Western blot结果显示,实验组中无癌大鼠20周、出癌大鼠36周始即有不同程度p-JNK1活性表达,并随着AFB1作用时间延长阳性率明显增加,且无癌大鼠p-JNK1检出的时间较出癌大鼠早;半定量分析表明无癌大鼠肝组织p-JNK1活性较同期出癌大鼠肝组织及肝癌组织增强,在实验第46周及58周时尤其明显（P<0.01）。结论 在AFB1诱导肝细胞癌的过程中JNK信号通路处于激活状态,JNK信号通路的激活可能起到抑制HCC的作用,如JNK信号通路激活时间较早、强度较大可阻止HCC的发生。