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.     

补肾方对二乙基亚硝胺诱发大鼠肝癌的预防作用

目的:研究补肾方对二乙基亚硝胺(DEN)诱发大鼠肝癌的预防作用。方法:以100ppmDEN饮水法诱发大鼠肝癌为模型,同时中药组予补肾方水煎剂灌胃,分别于诱癌12周末和18周末分批处死大鼠,观察大鼠肝脏病理形态学改变、肝组织γ-GT和AFP阳性表达以及血清丙氨酸转氨酶(ALT)、碱性磷酸酶(ALP)和白蛋白(Alb)含量变化。结果:中药组肝癌发生率较低;肝细胞增生灶和癌灶形成数目、面积较小;肝组织γ-GT阳性灶和AFP阳性细胞均明显比模型组少(P<0.001);血清ALT、ALP含量有不同程度降低,Alb含量较高。结论:补肾方对DEN诱发大鼠肝癌前病变和肝癌形成具有一定的抑制和延缓作用,但并不能完全阻断肝癌的形成。     

扶正化淤方预防肝癌的实验研究

本文以二乙基亚硝胺（ＤＥＮ）诱发大鼠肝癌为模型，观察扶正化淤方预防肝癌的作用。发现用中药后在１４周肝细胞异型性增生灶明显减少，２４周时形成的肝癌结节小而少，多数细胞处于早期间变阶段。与此同时，肝脏ｒ－谷氨酰转酸酶（ＧＧＴ）与甲胎蛋白阳性灶明显较模型组少，肝组织ＧＧＴ与谷胱甘酞硫转移酶及血清碱性磷酸酶活性较模型组低。表明药物能抑制肝癌癌前期病变，延缓肝癌的形成。细胞流式仪检查结果，用药后ＤＮＡ合成期细胞比例明显降低，推测药物作用可能是抑制细胞ＤＮＡ的合成。     

正肝方影响大鼠癌前病变肝组织γ-谷氨酰转移酶和谷胱甘肽S-转移酶-π阳性灶的对比研究

[目的]探讨正肝方对黄曲霉毒素B1（AFB1）诱发的大鼠癌前病变肝组织γ-谷氨酰转移酶（GGT）、谷胱甘肽S-转移酶-π（GST-P）阳性灶的影响。[方法]Wistar大鼠100只随机分为4组：模型组30只、正肝方小剂量预防（小剂量）组30只、正肝方大剂量预防（大剂量）组30只、正常大鼠对照（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。大、小剂量组在造模期间,将正肝方水剂（0．6g／ml,0．3g／ml）按10ml／kg分别灌胃;模型组用无菌蒸馏水10ml／kg灌胃。8周后处死大鼠,肝组织取材。组织化学法做肝组织GGT染色、免疫组化法做肝组织GST-P染色,电脑图像分析系统测量GGT和GST-P阳性灶的数量、大小。[结果]大剂量组大鼠肝组织GGT阳性灶为（10．91±4．25）个／cm^2,灶总面积为（2．94±1．52）mm^2／cm^2;GST-P阳性灶为（24．75±10．99）个／cm^2,灶总面积为（4．80±2．75）mm^2／cm2,均显著低于模型组（P〈0．01）。[结论]正肝方能减少AFB1诱发的大鼠癌前病变肝组织GGT和GST-P阳性灶的数量和大小,对AFB1诱发的肝癌前病变具有一定的预防作用。     

二乙基亚硝胺诱导肝细胞癌癌前病变动物模型研究

目的:通过动态观察予二乙基亚硝胺(DEN)腹腔注射的大鼠在不同时间节点的肝脏大体形态及病理学特征,结合肝癌前特异性免疫组化指标,以明确DEN诱导肝细胞癌癌前病变形成的最佳时间点。方法:将56只大鼠随机分为正常组与模型组,模型组大鼠予50mg/kg DEN腹腔注射,每周1次,持续16周;正常组大鼠予生理盐水腹腔注射,每周1次,持续16周。实验分别于给药后第4、6、8、10、12、14及16周末取材,每次取材正常组2只、模型组4只,观察大鼠肝脏大体形态,并行HE、Masson染色及谷胱苷肽-S转移酶(GST-Pi)免疫组化检测。结果:随着DEN给药次数增加,病理观察显示,模型组大鼠在第4周表现为肝脏炎性反应,第6周可见纤维组织增生,第8周纤维间隔形成,第10周形成早期肝硬化,第12周形成肝硬化,第14周形成肝细胞癌前病变,第16周形成肝细胞癌。结论:予大鼠50mg/kg DEN腹腔注射,每周1次,可在第14周诱导肝细胞癌癌前病变模型形成,且安全性较好。     

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。     

细胞色素P450和谷胱甘肽S-转移酶在二甲基氨基偶氮苯诱发大鼠肝细胞癌中的变化

目的研究细胞色素P450和谷胱甘肽S-转移酶在大鼠肝细胞癌形成过程中的变化。方法以二甲基氨基偶氮苯诱发大鼠形成肝细胞癌,测定不同阶段细胞色素P450的含量和谷胱甘肽S-转移酶的活性。结果在诱癌过程中,大鼠肝脏细胞色素P450含量呈下降趋势;谷胱甘肽S-转移酶的活性在诱癌早期升高,并且此酶活性在血清中升高早于肝脏,到癌变阶段降至正常水平。结论细胞色素P450和谷胱甘肽S-转移酶在大鼠肝脏癌变过程中呈现不同的变化趋势。     

中药复方861对二乙基亚硝胺诱发大鼠肝癌过程的影响

目的:观察中药复方861对二乙基亚硝胺(diethylnitrosamine,DEN)诱癌过程的影响.方法:250只雄性SD大鼠随机分成模型组、干预组、正常对照组.模型组和干预组以1?N灌胃,干预组同时以中药复方861灌胃.实验开始后分别于第2、3、5、8、10、12、14、18周分批处死大鼠,观察肝脏外观,肝组织进行HE、Masson三色染色,并对炎症坏死、肝纤维化程度和卵圆细胞增生程度进行评分,应用免疫组化及Western-blot方法检测肝组织中胎盘型谷胱甘肽硫转移酶(GST-P)蛋白的表达,计算成癌率.结果:干预组比模型组肝脏炎症坏死程度(P＜0.01)、纤维化程度(P＜0.01)和卵圆细胞增生程度(P＜0.05)轻;GST-P蛋白表达量少.结论:中药复方861可减轻DEN所致肝损伤.     

养阴方对实验性肝癌诱癌过程的影响

以DEN与AFB1诱发大鼠肝癌为模型，通过病理形态与重化指标的观察，发现养阴方能养活模型鼠肝细胞异型性增生灶，降低肝组织GGT、GST与血清ALP活性升高的幅度，提示药物对癌前期病变形成有较明显的抑制作用。     

养阴解毒方与健脾理气方预防肝硬化伴癌前病变的实验研究

目的：观察养阴解毒、健脾理气法则对肝硬化及肝癌的防治作用。方法：采用0.7％DEN液对大鼠灌胃方法造模，设正常组、模型组、养阴组和健脾组，于造模前2周起分别灌以蒸馏水、养阴液与健脾液至实验结束。结果：9周时形成肝纤维化伴癌前期病变，18周后形成肝硬化，癌前期变加重。与模型组相比，两中药组肝纤维化、肝硬化与肝癌前期病变较轻；DNA含量明显降低；肝细胞GGT阳性灶与AFP阳性细胞数减少；肝组织GGT比活性及Hyp含量下降。两中药组相比似以养阴组为优。结论：养阴解毒方与健脾理气方均能减缓大鼠肝硬化的形成，在一定程度上抑制其癌变进程。     

Comparison of two chemical models to induce hepatic preneoplasia in male Wistar rats

Background. One established model to induce hepatic preneoplasia (HP) (DEN 150) uses diethylnitrosamine (DEN) as initiator agent and 2-acetylaminofluorene (2-AAF) as a promoter drug. In addition, both chemicals cause liver cholestasis and fibrosis. Aim. We compared DEN 150 model with another adapted by us, DEN 200 to simplify the first one and to evaluate the effectiveness of both treatments to induce HP in rats.Material and methods. Male Wistar rats were divided in 3 groups: controls; DEN 150 (rats received 2 doses of DEN, 150 mg/kg body weight, 2 weeks apart, and then 2-AAF, 20 mg/kg body weight, 4 doses per week during 3 weeks); and DEN 200 (rats received a single dose of DEN 200 mg/kg body weight, and 2 weeks apart 2-AAF, 20 mg/kg body weight, 2 doses per week during 3 weeks). Four hepatic enzymes, prothrombin time percentage, the number of bile ductules, total collagen amount, the number of altered hepatic foci (AHF) per liver and the percentage of liver occupied by foci were analyzed.Results. There were no differences in the number of AHF per liver between treated groups. Rats from DEN 200 group showed a significant diminution in the volume of liver occupied by foci. DEN 200 group had no fibrosis and better hemostatic conditions than DEN 150 group. Both groups developed cholestasis. Conclusion. In conclusion, both protocols are good alternatives to induce HP in rats and the new protocol proposed is an effective and a simple methodology to provide subclinic states of liver cancer.     

The in vivo apoptotic effect of interferon alfa-2b on rat preneoplastic liver involves Bax protein

To determine whether interferon alfa (IFN-alpha) prevents in vivo oncogenesis in very-early-stage cancer cells, we evaluated the action of IFN-alpha2b over preneoplastic foci in rats. Animals were divided into 6 groups: subjected to a 2-phase model (diethylnitrosamine [DEN] plus 2-acetylaminofluorene [2-AAF]) of preneoplasia development (group 1), treated with IFN-alpha2b during the 2 phases (group 2), only during initiation with DEN (group 3), only during administration of 2-AAF (group 4), subjected only to an initiation stage (group 5), and treated with IFN-alpha2b during this period (group 6). The numbers of placental form of rat glutathione S-transferase (rGST-P)-positive foci per liver and the foci as percentage of liver were significantly reduced in groups 2, 3, and 6 but not in group 4. Rats treated with IFN-alpha2b showed a higher apoptotic index (AI) in altered hepatic foci (AHF). Levels of p53 and Bax protein in liver lysates were significantly increased in those animals. Similarly, levels of antiapoptotic proteins Bcl-2 and Bcl-x(L) in mitochondrial fraction were decreased. Finally, increased levels of Bax protein were localized in the mitochondria of rats that received IFN-alpha2b, at least during the DEN phase (groups 2, 3, and 6), whereas mitochondrial Bax expression was not increased in group 4. In conclusion, the preneoplastic hepatocytes in rats that received IFN-alpha2b during the initiation stage undergo programmed cell death as a primary result of a significant increase in the amount and translocation to the mitochondria of Bax protein.     

Preventive effect of Ganfujian granule on experimental hepatocarcinoma in rats

AIM: To investigate the inhibitory effect of dietary and medicinal formula Ganfujian granule on diethylnitrosamine (DEN)-induced hepatocarcinoma in rats. METHODS: Male SD rats had free access to water containing 0.1 g/L DEN for 16 weeks, during which the rats fed with standard diet or administration of Ganfujian granule (30.4 g/Kg in diet). At weeks 4, 8, 12 and 16 of hepatocarcinogenesis 5 rats of each group were sacrificed, and at week 20 another 30 rats were sacrificed from each group. The end point for survival observation was at week 28.Immunochemistry methods were used to examine the effect of Ganfujian granule on the process of hepatocarcinogenesis including proliferation of hepatocytes and cell cycle modulation. RESULTS: Ganfujian granule could reduce and delay the incidence of hepatocarcinoma in rats and prolong the survival of animals. In addition, Ganfujian granule had a marked inhibitory effect on high expression of cyclin dependent kinase (CDK4) during the whole process of hepatocarcinogenesis and cyclin D1 at week 16 and the number of proliferating cell nuclear antigen (PCNA) positive cells in different stages of hepatocarcinogenesis. CONCLUSION: Ganfujian granule can reduce and delay the incidence of hepatocarcinoma in rats by exerting direct or indirect effects on cell cycle and inhibiting uncontrolled proliferation of hepatocytes.     

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。     

粒细胞集落刺激因子和干细胞因子对急性心肌梗死后心室重构的影响

目的研究粒细胞集落刺激因子(G-CSF)、干细胞因子(SCF)对急性心肌梗死大鼠心室重构的影响。方法78只大鼠结扎前降支,术后6h尚存活60只。将此60只大鼠随机分为治疗组(30只)和对照组(30只),再将每组分为1,2,4周3个亚组。治疗组皮下注射G-CSF和SCF,对照组皮下注射等体积的生理盐水,计算心室肌质量/体重的比值、心肌梗死面积与左心室肌总面积的百分比,行心肌病理和免疫组化检查。结果(1)心室肌质量/体重的比值:第4周治疗组的比值明显小于对照组(P<0.01);对照组中第4周的比值大于第1周(P<0.01)和第2周(P<0.05);治疗组中第4周的比值大于第1周(P<0·01)。(2)心肌梗死面积与左心室肌总面积的百分比:第2,4周治疗组的面积百分比小于对照组(P<0.05,P<0.01);两组各自组内比较,第4周的面积百分比小于第1,2周(P<0.01)。(3)第4周时HE染色显示治疗组心肌梗死区的心肌较多,而对照组几乎为透壁的疤痕。1~4周治疗组均可见Brdu与TroponinT-I双染阳性的心肌细胞,以第2周时最多,对照组双染阳性细胞少见或偶见。结论G-CSF、SCF治疗大鼠急性心肌梗死可以促使心肌细胞分化,减轻心室重塑。     

肝癥口服液对大鼠肝癌前病变的预防作用

目的 :观察肝疒徵口服液对大鼠肝癌前病变的预防作用。方法 :以自研中药肝疒徵口服液对二乙基亚硝胺诱导大鼠肝癌前病变进行预防 ,并以乙氧三甲基喹啉、全反式维甲酸作对照。结果 :肝疒徵口服液能明显减少肝细胞不典型增生及 GGT阳性灶的数量和面积 ,效果显著优于乙氧三甲基喹啉和全反式维甲酸 ,而维甲酸未表现出阻断癌前病变的作用。结论 :肝疒徵 口服液对大鼠肝癌前病变有明显的阻断作用。     

MT1E在肝癌形成过程中的表达及其在肝癌细胞中的作用

目的: 探讨MT1E基因在肝癌形成过程中不同阶段的表达及其在肝癌细胞中的生物学功能.方法: DEN诱发大鼠肝癌形成模型,分别观察造模4、8、16、20 wk后肝脏组织病理形态学的改变以及MT1E基因表达的差异;针对MT1E基因mRNA序列设计2个siRNA靶点,分别经质粒重组后电转入SMMC-7721肝癌细胞株中,实时荧光定量PCR检测MT1E基因的表达,MTT法检测细胞生长增殖.结果: 大鼠肝癌造模4、8 wk时肝组织主要表现为炎性病变,而到16 wk后呈现典型的增生病变,20 wk后已全部发展为肝细胞癌. MT1E基因在造模16 wk后表达明显增加,与对照组比较差异显著(芯片读数: 11524 vs 5462). 成功筛选到MT1E基因RNA干扰的一个有效靶序列,电转染72 h后该基因的表达量较空白对照组与阴性对照组明显降低(0.38 vs 1.00,0.93,P<0.01). 与阴性对照组比较,有效干扰靶点电转染144 h后细胞的生长增殖得到了明显抑制(0.1700±0.0313 vs 0.5748±0.0480,P<0.01).结论: 成功应用D E N诱发大鼠肝癌形成,MT1E基因在肝癌形成的后期表达明显升高,其高表达与肿瘤细胞的恶性增殖有关.     

Effect of millimeter wave radiation on the expression of proliferating cell nuclear antigen,CDK4 and P16 in rat hepatocellular carcinoma

OBJECTIVE: To study the effects of millimeter wave (MMW) radiation on rat hepatocellular carcinoma. METHODS: Forty male Wistar rats were randomly divided into four groups, and the liver region of the rats was directly radiated by MMW (35.8 GHz, 100 mW/cm²) for 20 min twice per week. Rats in groups 1 to 3 were fed with diethlnitrosamine (DEN). Group 1 was a tumor control group with sham radiation. Group 2 was given radiation for 10 weeks starting from week 5 and group 3 was radiated for 5 weeks starting from week 10. Group 4 was a normal control group radiated for 5 weeks and given distilled water. At week 14, all rats were sacrificed. A serological test for γ‐glutamyltransferase (γ‐GT) and immunohistochemical staining of proliferating cell nuclear antigen (PCNA), CDK4 and P16 in liver tissue were performed. RESULTS: The serum level of γ‐GT in group 1 (18.44 ± 4.88 U/L) was higher than that in group 2 (13.75 ± 2.41 U/L, P < 0.05), and the level of γ‐GT in group 3 (16.43 ± 2.12 U/L) was lower than that in group 1, but the difference was not significant. Based on histological examination of the livers, adenocarcinoma only developed in group 1. In the other DEN‐induced tumor groups, only eosinophilic and basophilic nodules were formed in the liver; no carcinoma cells were found. Expression of proliferating cell nuclear antigen (PCNA) and CDK4 staining in liver tissue in groups 2 and 3 were significantly lower than those in group 1, but the expression of P16 in the former was higher than that in the latter. CONCLUSIONS: Millimeter wave radiation partially inhibits cell proliferation and suppresses DEN‐induced hepatocellular carcinoma in rats.     

Effect of Boschniakia rossica on expression of GST-P, p53 and p21(ras)proteins in early stage of chemical hepatocarcinogenesis and its anti-inflammatory activities in rats

AIM:To investigate the effect of Boschniakia rossica (BR) extract on expression of GST-P, p53 and p21(ras) proteins in early stage of chemical hepatocarcinogenesis in rats and its anti-inflammatory activities.METHODS:The expression of tumor marker-placental form glutathione S-transferase (GST-P), p53 and p21(ras) proteins were investigated by immunohisto-chemical techniques and ABC method. Anti-inflammatory activities of BR were studied by xylene and croton oil-induced mouse ear edema, carrageenin, histamine and hot scald-induced rat pow edema, adjuvant-induced rat arthritis and cotton pellet induced mouse granuloma formation methods.RESULTS:The 500mg/kg of BR-H2O extract frac-tionated from BR-Methanol extract had inhibitory effect on the formation of DEN-induced GST-P-positive foci in rat liver (GST-P staining was 78% positive in DEN+AAF group vs 20% positive in DEN+AAF+BR group, P<0.05) and the expression of mutant p53 and p21(ras) protein was lower than that of hepatic preneoplastic lesions (33% and 22% positive respectively in DEN+AAF group vs negative in DEN+AAF+BR group). Both CH(2)Cl(2) and H(2)O extracts from BR had anti-inflamatory effect in xylene and crotonoil induced mouse ear edema (inhibitory rates were 26%-29% and 35%-59%, respectively). BR H(2)O extract exhibited inhibitory effect in carrageenin, histamine and hot scald-induced hind paw edema and adjuvant-induced arthritis in rats and cotton pellet-induced granuloma formation in mice.CONCLUSION:BR extract exhibited inhibitory effect on formation of preneoplastic hepatic foci in early stage of rat chemical hepato-carcinogenesis.Both CH(2)Cl(2) and H(2)O extracts from BR exerted anti-inflammatory effect in rats and mice.