THE ABNORMAL EXPRESSION OF CLONED REPEATED SEQUENCE DNA, L5B-4, IN RAT HEPATOMA BERH-2

A repeated sequence DNA fragment, L5B-4, was cloned from the 5 kb BamHI DNA fragments of rat genomic DNA. The expressions of the L5B-4 DNA fragment are different in liver and hepatoma cells. The amounts of transcripts in hepatoma cells are lower in nucleus and higher in cytoplasm, especially in polysomal RNA, as compared with that in liver cells. The alteration shown in polysomal RNA of hepatoma cells seems to be specific. These results are discussed with respect to the possible function of this repeated DNA and its variation in hepatoma cells.

Abnormal expression of hepatoma- derived γ-glutamyltransferase subtyping and its early alteration for carcinogenesis of hepatocytes

BACKGROUND: Although the hepatoma-specific band of gamma-glutamyltransferase ( GGT ) is a highly sensitive marker in diagnosis of hepatocellular carcinoma, the kine- tic expression and the early alterations of GGT in the deve- lopment of hepatoma remain unclear. In this study, we in- vestigated the expression and the alterations of GGT multi- ple molecular forms in hepatotumorigenesis. METHODS: The expression of GGT in a chemically in- duced hepatocarcinogenesis model was examined by giving 0. 05% of 2-fluoenylacetamide in diet for 12 weeks. The ex- pression levels of total RNA and GGT, and the changes of liver pathology, GGT multiple molecular forms and sugar- chain heterogeneity were investigated at the different stages of rat hepatoma development. RESULTS: Pathological examination and biochemical ana- lysis found that liver GGT was over-expressed and secreted into blood during canceration. Serum total GGT and liver GGT specific activities (IU/g) including soluble and mem- brane-combined GGT were significantly higher (P <0.05) in experimental groups than those in control group, respec- tively. A highly positive correlation was found between to- tal GGT activities and total RNA levels (r =0.90, P <0.05) of the liver. Both were higher six weeks later than before. Con A-non-reactive-GGT was increased consistantly dur- ing the development of rat hepatoma. GGT multiple mo- lecular forms in the liver and sera of experimental rats showed that fetal liver-type GGT bands were associated with the development of hepatoma. CONCLUSIONS: Fetal liver-type GGT in sera and the liver of rats is closely related to hepatotumorigenesis. It can be used as a sensitive enzymatic marker for the early diagnosis of liver cancer.

Efficacy of intra- tumor injection of Kang-Lai-Te in treating transplanted hepatoma in rats

BACKGROUND: Non-operative therapy takes an impor- tant position in comprehensive therapy of liver cancer. De- spite some effects by using ethanol, acetic acid and heat sa- line for intra-tumor injection in the treatment of liver canc- er, it is difficult to attain a complete cure but bring about injury to the liver to some extent. Hence, searching for other drugs for the local treatment of liver tumor is an im- portant option. This study was designed to set up rat mo- dels of transplanted liver cancer, intra-tumor injection of Kang-Lai-Te (KLT), and negative control (saline) and positive control (ethanol). The effect of intra-tumor injec- tion of KLT in treating transplanted hepatoma in rats and its advantages and disadvantages were assessed and the pos- sibility of its use in treating patients with liver cancer was evaluated. METHODS: Forty rats were divided into 4 groups ( G1, G2, G3 and G4, 10 rats in each group). Different drugs were injected into their implanted hepatoma (G1 with 0.2 ml saline as control, G2 with 10 mg KLT, G3 with 20 mg KLT, G4 with 0.2 ml ethanol). After 3 and 8 days, the hepa- toma volume (HV), the serum levels of albumin, alanine aminotransferase(ALT), aspartate aminotransferase alkaline phosphatase( ALP) and creatinine, as well as the expression of proliferation cell nuclear antigen (PCNA) in hepatoma were detected. RESULTS: After 3 days, the HVs were smaller in G3 and G4 than in G1 (P <0.05), the serum levels of albumin were higher in G2 and G3 than in Gl and G4 (P <0.05), the se- rum levels of ALT and AST were lower in G2 and G3 than in G4 (P<0.05), the serum levels of ALP was lower in G2 and G3 than in Gl and G4 (P <0. 05), the PCNA labeling indexes (PCNA LI) were lower in G2 and G3 than in Gl and GA (P <0.05). After 8 days, the HVs were smaller in G2, G3 and G4 than in Gl (P <0.05), and the differences of HVs among G2, G3 and G4 were not significant. The serum levels of ALP were lower in G1, G2 and G3 than in G4 (P <0.05), and the PCNA LI were lower in G3 than in Gl andG4 (P<0.05). CONCLUSION: Intra-tumor injection of KLT into implan- ted hepatoma is evidently effective, but it is less effective than ethanol. The effect of KLT on liver function is markedly lower than that of ethanol.

Effects of La^(3+) and Gd^(3+) on Ca^(2+) influx in rat hepatoma H-35 cells

Effects of La93+) and Gd3+ on Ca2+ influx were investigated in rat hepatoma H-35 cells by measuring the initial rate of45Ca2+ uptake. It was found that the maximum initial rate of Ca2+ uptake was increased six- to ten-fold at low concentrations of La3+ and Gd3+. Kinetic analyses by measuring the initial rate of Ca2+ influx at different external Ca2+ concentrations indicated the existence of two intracellular exchangeable components in the basal Ca2+ system, with low and high affinities for Ca2+,and only one class of Ca2+ binding sites was observed in the La3+- or Gd3+-treated cells. For high affinity, La3+ and Gd3+ increased both kinetic parameters Km and V(max of basal Ca2+ influx. La3+ and Gd3+ compete directly with Ca2+ for Ca2+ binding site for low affinity. The kinetics is competitive.

Therapeutic Effects of All Trans-retinoitc Acid Combined with Transarterial Chemoembolization on Walker-256 Hepatoma in Rats

In order to investigate the inhibitory effects of all trans-retinoitc acid(ATRA)on differentiation and apoptosis of Walker-256 hepatocellular carcinoma cells and the therapeutic effects of ATRA combined with transarterial chemoembolization(TACE)on rat Walker-256 transplanted hepatocarcinoma,Walker-256 hepatocarcinoma cell lines were treated with ATRA at different concentrations.After culture for 48h,the inhibitory rate of cell proliferation was determined by MTT assay;the changes of Fas and Bcl-2mRNA expres...

EFFECTS OF VARIOUS VASOACTIVE AGENTS ON TUMOR BLOOD FLOW IN RAT

Effects of angiotensin Ⅱ and other six vasoactive agents on tissue blood flow of Yoahida rat ascites hepatoma AH109A and normal liver were measured by the hydrogen clearance method. The mean blood flow in the tumor peripheral part under normal tension was 11. 9±8. 2ml/ min/100g tissue and was not influenced by tumor size. Tumor blood flow was more significantly increased in infusion angiotensin Ⅱthan 0.5mg/ ml methoxamine, however, normal liver blood flow of tumor-bearing rats was unchanged in contrast to an Increase seen in the tumor. A pronounced reduction of tumor blood flow was found after administration of epinephrine, norepinephrin and ethylphenylephrine. In addition, metaraminol and phenyleprine as well as 1. 0 and 2. 5mg/ ml methoxamine were not found to significantly change blood flow of the tumor.

苦参碱和氧化苦参碱对二乙基亚硝胺诱发大鼠肝癌作用的影响

目的 研究苦参碱和氧化苦参碱对二乙基亚硝胺诱发大鼠肝癌作用的影响。方法 采用二乙基亚硝胺法诱发大鼠肝癌 ,观察腹腔注射苦参碱 2 5mg·kg-1和氧化苦参碱10 5mg·kg-130d后 ,大鼠肝表面癌结节数、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 氧化苦参碱组大鼠肝表面癌结节数、肝 /体重比和血清ALT、γ GT明显低于模型组 (P<0 0 5 ) ;苦参碱组大鼠肝表面癌结节数和血清γ GT明显低于模型组 (P <0 0 5 )。结论 苦参碱和氧化苦参碱 ,尤其是氧化苦参碱 ,不仅能保护肝细胞免受损伤 。

牵牛子对二乙基亚硝胺诱发大鼠肝癌的抑制作用

目的研究牵牛子在二乙基亚硝胺(NDEA)诱发大鼠肝癌中的抑制作用。方法将大鼠分为正常对照组、模型对照组和牵牛子组,采用0.01%NDEA诱发模型对照组和牵牛子组大鼠肝癌90 d,牵牛子组在诱发癌的同时,用含6%牵牛子饲料按40 g·kg-1·d-1剂量喂养。停止诱发,继续喂养30 d后处死大鼠,观察大鼠肝脏的病理改变、肝表面癌结节数、肝质量、肝/体质量比以及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。利用单因素方差分析LSD法检验进行统计分析。结果正常对照组大鼠肝表面癌结节数为(0.0±0.0)个,肝质量为(9.87±1.30)g,肝/体质量比为(2.62±0.24)%,血清ALT为(64.10±12.71)U·L-1,γ-GT为(0.80±0.42)U·L-1,ALP为(121.20±37.57)U·L-1。模型对照组大鼠肝表面癌结节数为(27.4±9.5)个,肝质量为(21.38±7.29)g,肝/体质量比为(5.82±2.31)%,血清ALT为(175.70±48.75)U·L-1,γ-GT为(41.80±15.38)U·L-1,ALP为(200.50±35.78)U·L-1。牵牛子组大鼠肝表面癌结节数为(8.6±5.3)个,肝质量为(13.91±3.55)g,肝/体质量比为(3.86±0.76)%,血清ALT为(113.10±45.35)U·L-1,γ-GT为(13.40±6.15)U·L-1,ALP为(155.80±30.26)U·L-1。结果显示,牵牛子组各项指标高于正常对照组,低于模型对照组,差异有统计学意义(P<0.01或P<0.05)。结论牵牛子能减轻NDEA对肝细胞的损伤,抑制NDEA诱发大鼠肝癌的过度生长。

肝癌特异性 GGT 的发生及其定量分析的临床价值

目的探讨肝癌特异性ＧＧＴ的动态变化及其定量分析的临床价值。方法以化学致癌剂２－ＦＡＡ（００５％）制备鼠肝癌模型，观察肝癌ＧＧＴ的产生及其肝组织内分布，并对不同肝病患者血清肝癌特异性ＧＧＴ区带（ＨＳＢ）进行定量分析研究。结果在实验性鼠的诱癌过程中，鼠肝脏癌变组织中ＧＧＴ大量表达，出现早并具有胎肝ＧＧＴ的特点；人血清中ＨＳＢ的定量检测发现：肝癌组明显高于其他肝病组，肝外肿瘤及正常对照组（Ｐ＜０００１）；ＨＳＢ在小肝癌中的阳性率为７５％，在ＡＦＰ阴性肝癌中为７８３％。结论研究资料提示ＨＳＢ的定量检测其临床价值优于ＨＳＢ的定性分析，且有助于小肝癌和ＡＦＰ阴性肝癌的早期诊断。

高温合并化疗对耐药性大鼠肝癌CRBH-7919细胞系的协同作用

目的 探讨高温合并阿霉素对耐药性大鼠 CRBH-7919细胞株的作用 .方法 4 2 .5℃持续 1h、阿霉素 32 mg·L- 1 ,单独或联合作用于耐药性大鼠 CRBH- 7919细胞 ,MTT法检测不同处理后细胞的存活率 ;免疫组化检测不同处理后细胞膜 P-糖蛋白 (P- glycoprotein,P- gp)的表达 .结果 加温可增加阿霉素对耐药性 CRBH- 7919细胞的杀伤作用 ;免疫组化提示 ,正常培养下该细胞 P- gp染色强阳性 ,单独用药和单独加热作用下 P- gp染色为阳性 ,加温合并阿霉素作用下 ,P- gp染色为极弱阳性 .结论 高温合并化疗 (热化疗 )可以显著提高耐药性 CRBH- 7919细胞对阿霉素的敏感性 .

p16真核表达载体的构建及其对肝癌细胞的作用

研究p16基因对肝癌细胞的作用;构建pcDNA3.0/p16真核表达质粒转导到大鼠肝癌细胞CBRH-7919中,对其p16基因的表达、细胞的生长抑制及机制进行分析;转染细胞p16蛋白免疫组化阳性,MTY法结果显示,50×103/cm2细胞经培养24h～96h后,每组细胞数均有不同程度的增加,但经pcDNA3.0/p16转染的CBRH-7919细胞数比对照明显减少。与空白对照比,细胞在24h,48h,72h和96h的生长抑制率分别为29％,29％,40％和52％,流式细胞仪检测细胞周期显示经pcDNA3.0/p16转染的CBRH-7919细胞有显著的细胞凋亡现象和G0/G1期阻滞。pcD-NA3.0/p16真核表达质粒转导到大鼠肝癌细胞CBRH-7919中能够抑制细胞的增殖活性,p16基因可能通过诱导肿瘤细胞凋亡及G1期阻滞在肿瘤的基因治疗方面发挥作用。

Overexpression of insulin-like growth factor-Ⅰ receptor as a pertinent biomarker for hepatocytes malignant transformation

AIM: To investigate the dynamic features of insulin-like growth factor-I receptor (IGF-IR) expression in rat hepatocarcinogenesis, and the relationship between IGF-IR and hepatocytes malignant transformation at mRNA or protein level.METHODS: Hepatoma models were made by inducing with 2-fluorenylacetamide (2-FAA) on male Sprague-Dawley rats. Morphological changes of hepatocytes were observed by pathological Hematoxylin and eosin staining, the dynamic expressions of liver and serum IGF-IR were quantitatively analyzed by an enzyme-linked immunosorbent assay. The distribution of hepatic IGF-IR was located by immunohistochemistry. The fragments of IGF-IR gene were amplified by reverse transcription-polymerase chain reaction, and confirmed by sequencing.RESULTS: Rat hepatocytes after induced by 2-FAA were changed dynamically from granule-like degeneration, precancerous to hepatoma formation with the progressing increasing of hepatic mRNA or IGF-IR expression. The incidences of liver IGF-IR, IGF-IR mRNA, specific IGF-IR concentration (ng/mg wet liver), and serum IGF-IR level (ng/mL) were 0.0%, 0.0%, 0.63 ± 0.17, and 1.33 ± 0.47 in the control; 50.0%, 61.1%, 0.65 ± 0.2, and 1.51 ± 0.46 in the degeneration; 88.9%, 100%, 0.66 ± 0.14, and 1.92 ± 0.29 in the precancerosis; and 100%, 100%, 0.96 ± 0.09, and 2.43 ± 0.57 in the cancerous group, respectively. IGF-IR expression in the cancerous group was significantly higher (P < 0.01) than that in any of other groups at mRNA or protein level. The closely positive IGF-IR relationship was found between livers and sera (r = 0.91, t = 14.222, P < 0.01), respectively.CONCLUSION: IGF-IR expression may participate in rat hepatocarcinogenesis and its abnormality should be an early marker for hepatocytes malignant transformation.

AFP-L3、HSP70及AFP水平在大鼠肝癌发展中的动态变化

目的:通过动态检测大鼠肝癌模型造模各阶段血清中AFP-L3、HSP70和AFP水平的变化,对其在肝癌早期诊断中的价值做综合评价。方法:SD大鼠CCl4橄榄油溶液腹壁皮下注射,食用白酒溶液代替饮用水,建立大鼠肝硬化基础上的肝癌模型。从造模第6周起,每2周心脏采血1次,ELISA法检测大鼠血清中AFP-L3、HSP70和AFP水平,并取部分肝组织行病理学检验造模进度。结果:造模第8周发生肝硬化,第10周发生不典型增生及早期肝癌结节。AFP-L3和HSP70在造模第10周即显著升高,其阳性率分别为60%和70%,两者联合检测的阳性率为90%,HSP70在肝硬化阶段也有升高,AFP-L3没有显著变化。AFP水平在造模第12周才有显著升高,阳性率为60%,肝硬化阶段也略有升高,肝癌早期无显著变化。AFP-L3/AFP比值在造模第10周即显著升高(35.5%),其敏感性和特异性分别达70%和100%。结论:成功建立了大鼠肝硬化基础上的肝癌模型,传统指标AFP对极早期的肝癌不敏感,特异性不高,而AFP-L3和HSP70均较AFP敏感,两者联合可提高检测的敏感性,AFP-L3和AFP-L3/AFP比值均较AFP特异。

生物免疫刺激剂对肝癌大鼠枯否细胞和单核细胞抑人肝癌细胞增殖作用的影响

以ＤＥＮ制成实验性肝癌大鼠，同时连续１２周分别给予肝癌大鼠干扰素α－２ｂ（ＩＦＮ）、卡介苗（ＢＣＧ）或两者联用，对照组给予生理盐水。于第１２周和１６周分离培养各组动物肝枯否细胞（ＫＣ）和血单核细胞（ＭＣ），并分别与人肝癌细胞联合培养，用￣３Ｈ－ＴｄＲ掺入法测定ＫＣ和ＭＣ对肝癌细胞的抑增殖作用。分离培养第１８周和２２周对照组肝癌大鼠的ＫＣ经体外ＩＦＮ、ＢＣＧ或两者联用后，同上测定ＫＣ对肝癌细胞的抑增殖作用。结果显示：１．肝癌大鼠的ＫＣ对肝癌细胞增殖仍具有一定的自然抑制作用，ＫＣ的作用明显强于ＭＣ；２．上述免疫刺激剂的在体应用或体外对ＫＣ的直接作用，均以ＩＦＮ和ＢＣＧ联用增强ＫＣ的抑肝癌细胞作用最强，最强者可增强５倍以上；３．单用免疫刺激剂者，ＢＣＧ的效果较ＩＦＮ强。讨论分析了联用免疫刺激剂增强ＫＣ抗肝癌最佳效果的机制及其实用意义。

EGFR-STAT3信号通路在大鼠实验性肝癌发生中的作用

背景与目的:许多研究表明,EGFR-STAT3信号传导通路参与肿瘤的形成。本试验目的在于探讨EG-FR-STAT3信号通路在大鼠实验性肝癌发生中的作用。方法:3’-甲基-4-二甲基偶氮苯(3'M e-DAB)诱发大鼠肝癌,免疫组化法检测EGFR、TGFα、STAT3、p-STAT3在诱癌各阶段的表达,分别用蛋白印记方法检测TGFα、STAT3、p-STAT3,原位杂交法检测STAT3在肝癌及正常肝组织中的表达,进行图像定量分析。结果:EGFR、TGFα、STAT3在肝细胞坏死和修复期表达有升高趋势。EGFR、TGFα、STAT3、p-STAT3在肝癌中100%表达,与正常组织比较,肝癌中EGFR、TGFα表达明显升高(P<0.05)。肝癌中STAT3在mRNA、蛋白水平的表达都较正常肝组织明显升高(P<0.05)。且其活化形式(p-STAT3)也显著增高(P<0.05)。肝癌中TGFα、p-STAT3表达具有相关性。结论:EG-FR-STAT3信号转导通路参与大鼠肝癌的发生,高表达的TGFα可激活EGFR信号转导通路形成自分泌环,并导致STAT3的持续活化。

大鼠肝癌模型CBRH-3的建立及其生物学特性

用ＤＥＮＡ诱发近交系Ｗｉｓｔａｒ大鼠，经三个月后得原发性肝癌，移植于同品系幼鼠，从而建立了 一株染色体众数正常，ＡＥＰ阳性的大鼠移植性肝癌模型，命名为ＣＢＲＨ－３，病理鉴定为肝细胞型肝 癌。至今已传至６０余代，目前生长稳定。

两种方式接种肝癌模型的比较

目的观察直接注入法和瘤块种植法制作Wistar大鼠肝癌模型的差异。方法采用大鼠含有Walker-256肿瘤细胞的腹水离心洗涤后接种于另一组大鼠的后腿后外侧皮下，待肿瘤长到直径约为1．0em，取下肿瘤并切成1．0-2．0mm^3大小。然后将瘤块接种于15只大鼠的肝叶上；另一组（15只）按上述方式将癌性腹水接种于正常大鼠的肝叶上；两组均在第7天后采用CT和开腹后游标卡尺分别测量种植性肝癌的直径。结果腹水直接注射法与瘤块种植法的肿瘤成瘤率分别为：86．7％和80％，两者并不具有统计学意义（P〉0．05）。第7天时，直接注射法所形成肿瘤的直径大于瘤块种植法的肿瘤（P〈0．05）。但瘤块法所引起的腹腔转移的可能性要少于腹水直接种法。结论直接注射法和瘤块种植法制作大鼠肝癌模型均可以满足临床实验研究的需要，但直接注射法的成瘤时间短，腹腔转移可能性也大；瘤块种植法成瘤时间略长，但腹腔转移可能性少于直接法。

大鼠肝癌发生过程中转化生长因子-α在肝组织中的表达

以二乙基亚硝胺诱发大鼠肝癌,用免疫组织化学法检测肝组织中转化生长因子-α的表达变化.结果显示:正常大鼠肝的枯否细胞呈转化生长因子-α阳性表达;至诱癌第8周,除少量枯否细胞仍呈转化生长因子-α阳性外,部分肝细胞亦呈转化生长因子-α阳性,且呈两种反应类型,即部分肝细胞胞质和胞膜均呈转化生长因子-α阳性.有的肝细胞仅胞膜呈转化生长因子-α阳性.随着肝癌病变发展,转化生长因子-α阳性肝细胞数渐增多,转化生长因子-α反应也明显增强.晚期肝癌的癌结节内大部分癌细胞呈转化生长因子-α阳性,癌周相对正常肝组织为阴性.结果提示,肝内转化生长因子-α表达变化与肝癌发生和发展有显著相关性.

木霉菌醇对肝癌细胞CBRH7919凋亡及形态变化的影响

目的探讨木霉菌醇(trichomerol)对大鼠肝癌细胞CBRH7919的作用。方法采用SRB细胞活性检测法检测木霉菌醇对CBRH7919细胞活性的影响;用流式细胞仪检测CBRH7919对细胞周期和凋亡的影响;激光共聚焦显微镜观察吖啶橙荧光染色细胞形态变化。结果 SRB检测显示木霉菌醇对CBRH7919细胞作用具有浓度和时间依赖性;木霉菌醇作用后CBRH7919细胞周期S期比例上调,而G1期比例下调;凋亡率具有浓度依赖性;吖啶橙荧光染色发现,细胞有明显凋亡。结论木霉菌醇对肝癌细胞CBRH7919具有良好的抑制作用,并可诱导细胞凋亡。

三种大鼠移植性肝癌原位模型制作的对比研究

目的常用大鼠移植性肝癌模型制作方法存在诸多不足,通过对直接注射法的改良以解决其缺陷。方法将90只Wistar大鼠随机分成3组,每组30只,分别以直接注射法、瘤块种植法和改良注射法进行模型制作,观察动物模型的肿瘤生长情况和动物模型的生存情况。结果术后第8天对3组大鼠进行比较,改良注射法模型组(C组)的肿瘤最大直径、体积和质量与直接注射法模型组(A组)、瘤块种植法模型组(B组)间差异并不显著(P>0.05),具有可行性,在肿瘤移植的成功率方面以改良注射法模型组最高;在腹水和腹壁瘤发生率方面C组明显低于A、B两组(P<0.01);生存时间长于A、B两组(P<0.01)。结论基于易行性和重复性考虑而产生的改良注射法,优化了直接注射法的实验操作方法、弥补了直接注射法的缺陷,很好地控制了实验条件,既拥有直接注射法的易行性,又提升了其可重复性,同时保证了动物模型的适用性和可靠性。