论文标题:正肝方对大鼠肝癌前病变及人肝癌细胞分化和端粒酶的影响
论文作者
论文导师 杨大国,论文学位 博士,论文专业 中医内科学
论文单位 湖北中医学院,点击次数 88,论文页数 113页File Size14970K
2008-05-01论文网 http://www.lw23.com/lunwen_477776942/
Liver precancerous lesion;; Hepatoma carcinoma cell;; Chemopreventive agents;; Zhengganfang;; TCM therapy;; Cell cycle;; Cell differentiation
背景与目的 肝细胞癌(HCC)是我国常见的肿瘤之一,恶性程度极高,易于复发、转移。其死亡率在全部恶性肿瘤中列第二位,仅次于胃癌。肝癌一旦发生,治疗非常困难,预后极差,5年生存率仅2%。因此,积极预防肝癌,研究具有阻断或抑制肝癌发生的药物,不仅具有重要的学术价值,而且可以产生巨大社会经济效益。由于肝癌的发生、发展是一个多阶段的过程,在肝癌形成以前通常会经历一个较长的肝癌前病变过程。因此,如能延缓或阻断肝癌前病变的形成,就有可能阻断肝癌的发生。肝癌前病变在病理学上主要表现为肝细胞非典型增生,而细胞增殖失控和分化障碍是肿瘤的基本特征,所以寻找能抑制肝细胞恶性增殖和诱导其分化的药物,对肝癌的化学预防具有重要的价值。近些年来,中医药在预防肝癌发生方面进行了一些研究探索,并显现出较好的研究前景。已有较多研究发现一些中药具有一定程度的预防肝癌的作用。然而,由于研究周期长、作用因素复杂、对象不易控制等诸多原因,使得中医药在预防肝癌的临床和基础方面的研究较少,能够经得起实验重复和临床验证的中药复方更少。至今未见一种公认有效的肝癌预防制剂应用于临床。正肝方是针对肝癌的中医病机特点组方而成的中药复方,该方主要由黄芪、丹参、鳖甲、女贞子、半枝莲、白花蛇舌草等组成,具有活血软坚、益气养阴、清热解毒的功效。本研究采用组织化学、免疫组化、RT-PCR、PCR-ELISA等方法观察了正肝方对黄曲霉毒素B_1(AFB_1)诱发的大鼠肝癌前病变的影响,以及正肝方药物血清对人肝癌细胞分化和端粒酶的影响,以明确正肝方对肝癌的预防作用及其主要作用机制。本研究为将正肝方开发为肝癌预防新药,在临床上推广应用,提供一定的理论依据。 材料与方法 1.动物体内实验 Wister雄性大鼠100只,随机分为4组:模型对照组(A组)30只、正肝方小剂量预防组(B组)30只、正肝方大剂量预防组(C组)30只、正常大鼠对照组(D组)10只。除正常大鼠对照组外,先后用AFB_1和2-乙酰氨基芴(2-AAF)处理各组大鼠造模。正肝方大、小剂量预防组在造模期间,将正肝方水剂(单味免煎中药粉剂按比例称取,用无菌蒸馏水溶解,配成0.3g/ml和0.6g/ml的水剂)按10ml/kg分别灌喂大鼠;模型对照组(A组)用无菌蒸馏水按10ml/kg灌喂大鼠。8周后处死大鼠,采血,取肝组织标本。将肝组织标本分别HE染色和铀铅染色,光镜、电镜病理观察;采用组织化学法结合电脑图象分析检测肝组织γ-谷氨酰转移酶(GGT)阳性灶、免疫组织化学法(IHC)检测肝组织谷胱甘肽S-转移酶-π(GST-P)阳性灶、增殖细胞核抗原(PCNA)、甲胎蛋白(AFP)、胰岛素样生长因子Ⅱ(IGF-Ⅱ)和细胞周期蛋白D_1(Cyclin D_1)、细胞周期蛋白依赖性激酶4(CDK_4)蛋白表达,逆转录PCR(RT-PCR)法检测肝脏组织IGF-Ⅱ、Cyclin D_1和CDK_4基因表达;比色法检测血清丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、碱性磷酸酶(ALP)、GGT、总胆红素(TBIL)、白蛋白(ALB)生化指标。 2.体外血清药理实验 正肝方水煎剂(1.5g/ml),灌喂正常大鼠,每日2次,连续3d,于第4d,1次给予全日剂量后1h采血,制备药物血清,药物血清经56℃灭活30min。以正常鼠血清为阴性对照,维甲酸为阳性对照,将药物血清温育Bel-7402人肝癌细胞。噻唑蓝(MTT)比色法测定细胞增殖,图象分析系统测定细胞核质比,放射免疫法测定细胞分泌的AFP含量,动态比色法测定细胞内GGT和ALP活性,多聚酶链反应—酶联免疫吸附法(PCR-ELISA法)检测细胞端粒酶活性。 结果 1.动物体内实验 1.1各组大鼠一般情况 动物实验开始时,各组大鼠体重大小较一致,无统计学差异(P＞0.05)。在整个实验过程中,正常对照组的大鼠体重增长大于造模组(P＜0.01);模型组(A组)、正肝方小剂量组(B组)和大剂量组(C组)3组间大鼠生长情况相似,体重增长无差异显著性(P＞0.05)。表明正肝方无论是大剂量还是小剂量,对大鼠都是安全的,没有明显的毒副作用。实验结束时,A组死亡12只,B组15只,C组13只,D组无死亡。 1.2各组大鼠肝组织形态及细胞超微结构变化 光镜下:A、B、C组大鼠的肝组织切片(HE染色)可见到散在的,大小不等的增生肝细胞灶或结节;肝细胞增生灶多少不等,A组最多,C组最少;灶/结节内以嗜碱性肝细胞为主,个别灶内肝细胞浊肿变形、点状坏死;部分灶/结节内肝细胞异型,体积变大,见少量双核、核深染、巨核细胞。电镜下:增生灶内肝细胞可见细胞器排列集中,胞质内含大量线粒体,粗面内质网扩张。细胞核大,不规则,核仁突出,部分细胞双核,可见两个以上核仁,染色质疏松,边集。 1.3各组大鼠肝组织GGT和GST-P阳性灶的比较 正肝方小剂量组的每个GGT灶面积(mm~2/个)少于模型组(P＜0.05),但单位面积GGT灶个数(个/cm~2)、单位面积灶的总面积(mm~2/cm~2)无明显减少(P＞0.05)。正肝方大剂量组的单位面积GGT灶个数、单位面积灶的总面积和每个灶面积与模型组比均显著减少(P＜0.01)。与小剂量组比,正肝方大剂量组的GGT灶个数、灶的总面积和每个灶面积显著减少(P＜0.01),提示正肝方减少AFB_1诱发的大鼠肝组织GGT灶面积的作用有一定的量效关系。 与模型组比,正肝方小剂量组的每个GST-P灶面积(mm~2/个),单位面积GST-P灶个数(个/cm~2)、单位面积灶的总面积(mm~2/cm~2)无明显减少(P＞0.05)。正肝方大剂量组的单位面积GST-P灶个数、单位面积灶的总面积和每个灶面积与模型组比均显著减少(P＜0.05或P＜0.01),提示大剂量正肝方具有抑制癌前病变肝组织GST-P灶的作用。 1.4各组大鼠肝功能的比较与模型组比较,正肝方小剂量组和大剂量组的ALT、GGT、TBIL值显著降低(P＜0.01或P＜0.05),大剂量组的AST、ALP值也显著低于模型组(P＜0.01或P＜0.05)。与小剂量组比较,大剂量组的ALT值显著低于小剂量组(P＜0.01),表现出一定的量效关系。以上提示:正肝方能保护肝功能,减轻AFB_1和2-AAF导致的肝损伤作用。 1.5各组大鼠肝组织PCNA、AFP免疫组化染色结果 正肝方大剂量组PCNA和AFP染色的阳性单位(PU)值较模型组均显著降低(P＜0.01或P＜0.05),大剂量组PCNA和AFP染色的标记指数(LI)值也低于模型组(P＜0.01)。提示大剂量正肝方能有效降低大鼠癌前病变肝组织PCNA和AFP的表达,减轻大鼠癌前病变肝细胞的异常增生。 1.6各组大鼠肝组织Cyelin D_1、CDK_4表达情况 正肝方小剂量组和大剂量组Cyclin D_1染色的LI和PU值较模型组均显著降低(P＜0.01),大剂量组Cyclin D_1染色的PU值也低于小剂量组(P＜0.05),呈现出一定的量效关系;正肝方小剂量组和大剂量组CDK_4染色的PU值均较模型组显著降低,大剂量组PU值也低于小剂量组。正肝方小剂量组和大剂量组的Cyclin D_1和CDK_4 mRNA表达水平较模型组表达水平均显著降低(P＜0.01)。提示大剂量正肝方能降低大鼠癌前病变肝组织Cyclin D_1、CDK_4蛋白和mRNA异常高表达水平。 1.7各组大鼠肝组织IGF-Ⅱ表达情况 与模型组比较,正肝方小剂量组和大剂量组的PU值显著降低(P＜0.01或P＜0.05),大剂量组的LI值也低于模型组(P＜0.05)。与模型组比较,对照组IGF-ⅡmRNA相对表达水平最低(P＜0.01),正肝方小剂量组和大剂量组的IGF-ⅡmRNA表达水平显著降低(P＜0.01)。与小剂量组比较,大剂量组的IGF-ⅡmRNA表达水平亦显著低于小剂量组(P＜0.01),呈现出一定的量效关系。提示正肝方能降低大鼠癌前病变肝组织IGF-Ⅱ蛋白和mRNA异常高表达水平。 2.体外血清药理实验 2.1药物血清对Bel-7402细胞增殖的影响(MTT比色法) 对照组、正肝方组、维甲酸组A_(490)值分别为1.704±0.102、1.483±0.077、1.510±0.104。与对照组比,正肝方药物血清组A值显著降低(P＜0.01)。提示正肝方可抑制Bel-7402细胞的增殖。 2.2药物血清对Bel-7402细胞核质比的影响 对照组、正肝方组、维甲酸组Bel-7402细胞核质比分别为1.04±0.09、0.71±0.07、0.69±0.08。与对照组比,正肝方药物血清组和维甲酸组核质比显著降低(P＜0.01)。提示正肝方可降低Bel-7402细胞核质比。 2.3药物血清对Bel-7402细胞分泌AFP的影响 对照组、正肝方组、维甲酸组Bel-7402细胞AFP分泌量(ng/10~6)分别为22.58±2.68、14.60±1.85、13.50±2.50。与对照组比,正肝方药物血清组和维甲酸组AFP分泌量显著低于对照组(P＜0.01),表明正肝方和维甲酸均可抑制Bel-7402细胞分泌AFP。 2.4药物血清对Bel-7402细胞GGT和ALP活性的影响 与对照组比,正肝方药物血清组和维甲酸组细胞内GGT活性明显降低(P＜0.05),ALP活性显著增高(P＜0.01)。提示正肝方可降低Bel-7402细胞内GGT活性,升高ALP活性。 2.5药物血清对Bd-7402细胞端粒酶活性的影响 Bel-7402对照组、正肝方组A_(450)值分别为0.929±0.066、0.360±0.105。两者比较,正肝方药物血清组端粒酶活性显著低于Bel-7402细胞对照组(P＜0.01),即正肝方可抑制Bel-7402细胞的端粒酶活性。 结论 正肝方具有预防化学致癌物引起的肝癌前病变作用,其机理可能是通过影响IGF-Ⅱ癌基因及蛋白的异常表达,经内源性调控和(或)外源性调控作用于细胞增殖周期,抑制肝细胞增殖活性,阻断肝细胞异常增生,起到延缓或阻断肝癌前病变发生、发展的作用。此外,正肝方具有诱导Bel-7402人肝癌细胞分化的作用,其机理可能是抑制了Bel-7402细胞端粒酶活性。
Background and Objectives Hepatic cellular cancer(HCC)is a kind of common malignant tumor in China with high degree of malignity,and high incidence of recurrence and metastasis.The death rate of HCC is the second in total malignant tumor,second only gastric cancer. Once HCC take place,the treatment is extremely difficult,and the prognosis is very bad.The survival rate of 5 years is only 2%.Therefore,preventing HCC and finding the drug which can block(or inhibit)hepatocarcinogenesis not only have significant academic value,but also can bring enormous economic benefit.Because of the multistage process of HCC development,there is a long process of hepatic precancerous lesion.Therefore,if hepatic precancerous lesion is blocked(or delayed),the genesis of HCC can be interrupted.The pathologic feature of hepatic precancerous lesion is atypical hyperplasia of hepatocyte,and abnormal proliferation and dysdifferentiation of body cells is the basic characteristic of tumor appears.It is important to find the drug which can depress abnormal proliferation of hepatocyte and induce the differentiation of hepatocyte.In recent years,some benefit studies of preventing HCC by traditional Chinese medicine have been done.Many studies have proved that some Chinese herbs may prevent liver cancer.But some factors,such as long cycle,complex influencing factor,difficulty of controlling object,cause that less studies in clinic and foundation for preventing HCC by traditional Chinese medicine have been thoroughly done.There is no effective preventive agent has been accepted generally in clinic practice.Zhengganfang(ZGF)is a compound recipe of Chinese herbs which is organized according to the HCC pathogenesis of traditional Chinese medicine.Zhengganfang composed of Radix Astragali(30g),Radix Salviae Miltiorrhizae(30g),Carapax Trionycis(15g),Fructus Ligustri Lucidi(15g),Herba Scutellariae Barbatae(15g),Herba Hedyotis Diffusae(3Og),et al,has three functions of activating blood and dissipating a mass,benefiting qi and nourishing yin,clearing away heat and toxic materials.In this study,histochemical,immunohistochemical, RT-PCR,PCR-ELISA methods and technique of computer imageanalysis were used to observe the influence of Zhengganfang on AFB_1-induced precancerous lesion of rat liver,and to investigate the effect on differentiation of BEL-7402 human hepatocarcinoma cell(HHC)induced by Zhengganfang drug serum and the influence of Zhengganfang drug serum on the telomerase activity of HHC.The purposes of the study are to identify the function of Zhengganfang for preventing HCC,to find the main mechanism of action,to offer academic foundation for further clinic study,and to spread out its application. Methods 1.Animal experiment in vivo One hundred Wister rats were randomly divided into four groups:control group of model(group A,30 rats),group of Zhengganfang(ZGF)low dosage(group B,30 rats),group of ZGF high dosage(group C,30 rats)and control group of normal rats(group D,10 rats).Except the control group of normal rats,all the rats in each group were treated with AFB_1 and 2-AAF successively for making model.During the period of making model,the rats in group of ZGF low dosage and group of ZGF high dosage were fed with ZGF aqua(dusts of free-decoction dissolved in water at the concentration of 0.3g/ml,0.6g/ml)according to 10ml/kg.The rats in control group of model were fed with sterile distilled water according to 10ml/kg.After eight weeks,the rats were killed,and blood and hepatic tissues of each rat were taken.After stained with HE staining and uranium- lead staining,the specimens of hepatic tissue were observed by light microscope and electron microscope,the number and size of gama-glutamyltranspeptidase-positive hyperplastic liver cell foci(GGT foci)were checked with histochemical method and technique of computer imageanalysis.The expression of GST-P,PCNA,AFP,IGF-Ⅱ,Cyclin D_1,CDK_4 in hepatic tissue were tested with immunohistochemistry(IHC)method.The genetic expression of Cyclin D_1, CDK_4,IGF-Ⅱwere detected with RT-PCR.The biochemical indicators of ALT,AST, ALP,GGT,TBIL,ALB were tested with colorimetric method. 2.Experiment of serum pharmacology in vitro The normal rats were fed with with ZGF decoction(1.5g/ml),two times a day,for three days.On the forth day,the rats took full day"s dose.After one hour,the blood was extracted and centrifugated to get sera.The sera were inactivated at 56℃.Serum of normal rat was taken as negative control and retinoic acid(RA)as the positive control.The drug-serum was used for Bel-7402 HHC culture.Cell proliferation was detected by MTT colorimetric assy,nucleocytoplasm ratio by image assay,α-fetoprotein(AFP)by RIA,alkaline phosphatase(ALP)activity and gama-glutamy transpeptidase(GGT)activity by dynamic colorimetric assy,and telomerase activity of Bel-7402 HHC by PCR-ELISA. Results 1.Animal experiment in vivo 1.1 General state of rat health in each group At initiation of the experiment,body weight of rats in each group had no difference in statistics(P＞0.05).In whole lab proc,body weight of rats in control group increased faster than that of model group(P＜0.01).Body weight of rats in model group(group A),group of ZGF low dosage(group B)and group of ZGF high dosage(group C)had similar condition(P＞0.05).This indicated that both low dosage and high dosage of ZGF are safe to rats.At the end of the experiment,the number of death rats in each group were 12 in group A,15 in group B,13 in group C, respectively.There was no death in group D. 1.2 Appearance of hepatic tissue and hepatocyte ultrastructure of rats in each group Under light microscope:Sporadic,different size hyperplastic liver cell foci(or tubercle)could be seen in the histological section of rat liver in group A,B,C(HE staining).The number and size of hyperplastic liver cell foci in group A were the most, and that in group C were the least.The cells in hyperplastic foci were mainly basophilia hepatocyte,and little cells displayed cloudy swelling,disfiguration and punctiform necrosis.Some cell showed allotype,great volum,conjugate nuclei, anachromasis nuclus,megakaryocyte.Under electron microscope:There were concentrated arrangement of organelle,many mitochondrions and extending rough endoplasmic reticulurn in liver cells of hyperplastic foci.Some abnormal phenomena, such as:big anomal- nucleus,outstanding nucleolus,conjugate nuclei,conjugate nucleoli,chromatin porosity,also could be seen. 1.3 Comparison of positive GGT foci and positive GST-P foci among each group The areas of per positive GGT foci(mm~2/entries)of group B were less than that of group A(P＜0.05).But the amount of positive GGT foci per square centimeter (entries/cm~2)and the total areas of the positive GGT foci per square centimeter (mm~2/cm~2)had no significant deviation between tow group(P＞0.05).The amount of positive GGT foci per square centimeter,the total areas of the positive GGT foci per square centimeter and the areas of per positive GGT foci of group C were less than that of group A(P＜0.01).Comparing with group B,the amount of positive foci per square centimeter,the total areas of the positive foci per square centimeter and the areas of per positive foci of group C decreased.This indicated that there was dose-effect relationship about the function of ZGF. The amount of positive GST-P foci per square centimeter(entries/cm~2),the total areas of the positive GST-P foci per square centimeter(mm~2/cm~2)and the areas of per positive GST-P(mm~2/entries)had no significant deviation between group A and group B(P＞0.05).But the amount of positive GST-P foci per square centimeter,the total areas of the positive GST-P foci per square centimeter and the areas of per positive GST-P foci of group C were less than that of group A(P＜0.01).This indicated that ZGF could inhibite positive GST-P foci of liver precancerous lesion. 1.4 Comparison of Liver function among each group Comparing with group A,ALT,GGT,TBIL of group B and groupC reduced markedly(P＜0.01 or P＜0.05).AST and ALP of group C were less than that of group A(P＜0.01 or P＜0.05).This indicated that ZGF could protect liver founction and reduce hepatic injury dueto AFB_1 and 2-AAF. 1.5 PCNA and AFP immunohistochemistry result of rat hepatic tissue about each group The numeral PU of PCNA and AFP in group C and the numeral LI of PCNA and AFP in group C were less than that of group A(P＜0.01 or P＜0.05).This indicated that ZGF could reduce the PCNA and AFP expression of rat liver precancerous lesion, and inhibit the hepatocellular paraplasm of rat liver precancerous lesion. 1.6 Cyclin D_1 and CDK_4 expression of rat hepatic tissue in each group Both numeral PU and numeral LI of Cyclin D_1 in group B and group C were less than that in group A(P＜0.01).The numeral PU of Cyclin D_1 in group C were also less than that in group B.This presented dose-effect relationship.The numeral PU of CDK_4 in group B and group C were less than that in group A(P＜0.01),and the numeral PU of CDK_4 in group C were also less than that in group B.Comparing with group A,Cyclin D_1 and CDK_4 mRNA expression of both group B and group C reduced markedly(P＜0.01).This results suggested that ZGF could depress the abnomal expression of Cyclin D_1 and CDK_4 in hepatic tissue of rat hepatic precancerous lesion at protein and mRNA level. 1.7 IGF-Ⅱexpression of rat hepatic tissue about each group The numeral PU of IGF-Ⅱin group B and group C were less than that in group A(P＜0.01 or P＜0.05),and the numeral LI of CDK_4 in d group C were also less than that in group A(P＜0.05).Among four group,IGF-ⅡmRNA expression of group D was the lowest.Comparing with group A,IGF-ⅡmRNA expression of group B and group C reduced markedly(P＜0.01).Comparing with group B,IGF-ⅡmRNA expression of group C decreased markedly(P＜0.01),which showed dose-effect relationship.This results suggested that ZGF could depress the abnomal expression of IGF-Ⅱin hepatic tissue of rat hepatic precancerous lesion at protein and mRNA level. 2.Experiment of serum pharmacology in vitro 2.1 The effect of ZGF drug serum on the proliferation of Bel-7402 cell(MTT colorimetric method) The numeral A of the group of negative control,ZGF drug-serum and RA were 1.704±0.102,1.483±0.077,1.510±0.104,respectively.Comparing with the group of negative control,numeral A of the group of ZGF drug-serum reduced markedly(P＜0.01).This meanted ZGF could inhibit the proliferation of Bel-7402 cell. 2.2 The effect of ZGF drug serum on the nucleocytoplasm ratio of Bel-7402 cell The nucleocytoplasm ratio of the group of negative control,ZGF drug-serum and RA were 1.04±0.09,0.71±0.07,0.69±0.08,respectively.Comparing with the group of negative control,nucleocytoplasm ratio of the group of ZGF drug serum decreased markedly(P＜0.01).This indicated that ZGF could reduce nucleocytoplasm ratio of Bel-7402 cell. 2.3 The effect of ZGF drug serum on the AFP secretion of Bel-7402 cell The secretory volume of AFP(ng/10~6)in the group of negative control,ZGF drug-serum and RA were 22.58±2.68,14.60±1.85,13.50±2.50,respectively.The secretory volume of AFP in the group of ZGF drug serum and RA were markedly lower than that in the group of negative control,which indicated that both ZGF and RA could inhibit the secretion of AFP of Bel-7402 cell. 2.4 The effect of ZGF drug serum on GGT and ALP of Bel-7402 cell The GGT of the group of ZGF drug serum and RA were markedly lower than that of the group of negative control(P＜0.05),while the ALP of the groups were markedly higher than that of the group of negative control.(P＜0.01).This indicated that ZGF could decrease GGT of Bel-7402 cell and raise ALP of Bel-7402 cell. 2.5 The effect of ZGF drug serum on telomerase activity of Bel-7402 cell The numeral A of the group of Bel-7402 cell control and ZGF drug-serum were 0.929±0.066、0.360±0.105,respectively.The telomerase activity of ZGF drug serum was markedly lower than that of the group of Bel-7402 cell control,which indicated ZGF could inhibit the telomerase activity of Bel-7402 cell. Conclusion ZGF has inhibitive effect on the process of hepatic precancerous lesion induced by chemical carcinogen.The mechanism of inhibitive effect of ZGF may involve that the agent can regulate abnormal expression of IGF-Ⅱoncogenes and oncoproceins, then affect cell-cycle process by endogenous regulation and(or)exogenous regulation, inhibit proliferative activity of hepatocytes and retard the malignant proliferation and cancerization of hepatocytes.In addition,ZGF can induce the differentiation of Bel-7402 HHC,the main mechanism maybe through the supression of the telomerase activity.
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