AFP阴性与阳性肝细胞癌免疫组化和免疫电镜的比较性研究

目的比较性研究AFP阴性与阳性原发性肝细胞癌超微结构特征及AFP和Tn (Thomsen-Friedenreich-related antigen)蛋白表达及意义.方法 43例原发性肝细胞癌组织和5例正常肝组织分为三组:对照组(正常肝组织,5例);AFP阳性肝细胞癌组 (血清AFP>10ng/ml,22例);AFP阴性肝细胞癌组(血清AFP<10ng/ml,21例).应用透射电镜、免疫组织化学和细胞图象分析技术对AFP阴性与阳性肝癌细胞超微结构及AFP和Tn蛋白表达进行观察,并进行AFP和Tn蛋白免疫电镜标记.结果 1. 免疫组织化学结果显示:在AFP阴性肝细胞癌组癌细胞中(1)Tn蛋白表达强度(0.1498±0.0371)明显高于AFP阳性肝细胞癌组(0.0685±0.0156)(P<0.01);(2)AFP蛋白表达强度(0.1269±0.0347) 低于AFP阳性肝细胞癌组(0.1852±0.0234)(P<0.01).2.透射电镜观察:在AFP阴性组肝癌细胞中,癌细胞最突出的形态特征是胞质内细胞器大多十分简单,唯游离多聚核糖核蛋白体十分丰富.而在AFP阳性组肝癌细胞中,癌细胞胞质内细胞器相对较多,特别是粗面内质网尤为丰富.此外,线粒体及高尔基器也较明显.3.免疫电镜标记显示:AFP蛋白阳性标记主要位于粗面内质网,Tn蛋白阳性标记多位于游离多聚核糖核蛋白体,粗面内质网仅见有散在阳性分布.结论 (1)AFP和Tn蛋白在AFP阴性与阳性肝细胞癌组织中具有差异性分布特征,Tn蛋白有望成为AFP阴性肝细胞癌诊断辅助指标之一.(2)透射电镜和免疫电镜观察表明:AFP和Tn蛋白在肝癌细胞中的合成部位明显不同.     

人胚肝去组蛋白染色质抗原、甲胎蛋白及白蛋白在人肝癌和癌周组织中的定位关系

人胚肝去组蛋白染色质抗原(DHCAg)具有癌胚特异性,本文用免疫酶标法在肝癌连续切片上比较了DHCAg、甲胎蛋白(AFP)和白蛋白(ALB)彼此间的定位分布,得到的初步结果如下: 1.在肝癌标本中DHCAg和AFP无论在肝癌细胞、癌周细胞或幸存肝细胞中的分布有某种关系。提示DHCAg和AFP基因表达之间可能存在着某种联系。2.在DHCAg阳性的幸存肝细胞和癌周细胞中AFP和ALB通常同时表达,表明这二种细胞很可能是癌前期细胞。3.在DHCAg阳性的肝癌细胞中AFP阳性情况下ALB的合成明显下降,这就提示DHCAg和癌变后的某些特征相关。     

人胚肝去组蛋白染色质抗原、甲胎蛋白及白蛋白在人肝癌和癌周组织中的定位关系

人胚肝去组蛋白染色质抗原(DHCAg)具有癌胚特异性,本文用免疫酶标法在肝癌连续切片上比较了DHCAg、甲胎蛋白(AFP)和白蛋白(ALB)彼此间的定位分布,得到的初步结果如下:1.在肝癌标本中DHCAg 和AFP 无论在肝癌细胞、癌周细胞或幸存肝细胞中的分布有某种关系。提示DHCAg 和AFP 基因表达之间可能存在着某种联系。2.在DHCAg 阳性的幸存肝细胞和癌周细胞中AFP 和ALB 通常同时表达,表明这二种细胞很可能是癌前期细胞。3.在DHCAg 阳性的肝癌细胞中AFP 阳性情况下ALB 的合成明显下降,这就提示DHCAg 和癌变后的某些特征相关。     

线粒体促凋亡因子Omi/HtrA2在肝癌组织中表达的研究

目的探讨丝氨酸蛋白酶Omi/HtrA2在肝癌组织、癌旁组织与正常肝组织中的表达及意义。方法应用免疫组化SABC法检测43例肝癌、30例癌旁组织及10例正常肝组织中Omi/HtrA2的表达。结果29例(67·44%)肝癌中Omi/HtrA2蛋白表达阳性,30例癌旁组织和10正常肝组织没有或只有少量很弱的表达。肝癌高分化组中Omi/HtrA2蛋白的表达明显高于中、低分化组(P<0·01)。另外,Omi/HtrA2表达与肿瘤大小及临床分期相关,但Omi/HtrA2表达与肝硬化、有无癌栓、HBsAg和AFP无关。结论肝细胞癌可能需要Omi/HtrA2的表达来促进凋亡,Omi/HtrA2的表达对肝癌的发展有重要作用。     

大鼠肝癌发生过程中NF-κB的表达

目的探讨核转录因子-κB(NF-κB)在大鼠肝癌发生发展中的作用和意义。方法应用免疫组织化学SP法,对二乙基亚硝胺(DEN)诱发的大鼠肝癌发生过程中NF-κB的动态表达进行了检测。结果 DEN诱发的肝癌为肝细胞癌,诱癌率为100%,大鼠肝癌癌变过程大致经过肝细胞损伤期、肝细胞增生-硬化期和肝细胞癌变期等三个阶段。在正常大鼠肝组织,偶见少量肝细胞呈阳性表达,随着肝癌发生发展,NF-κB阳性表达细胞逐渐增多,至诱癌晚期,可见大量NF-κB阳性表达细胞,均比正常肝组织表达高(P<0.05)。结论本研究表明肝细胞NF-κB的过度表达与肝癌的发生和发展密切有关。     

miR-375 在HBV相关性肝细胞癌肿瘤组织与癌旁组织中的表达

目的:比较HBV相关性肝细胞癌患者的肝癌组织与癌旁组织中miR-375的表达。方法:以西京医院进行肝癌切除术治疗的HBs Ag阳性的25例患者为研究对象,收集肝癌组织、癌旁组织及术前外周血标本,并采集相关临床信息。采用反转录和实时荧光定量PCR的方法检测组织中miR-375的表达水平。结果:本研究共收集的25例肝癌患者HBs Ag均为阳性,在肝癌组织中miR-375的表达水平为2.51×10-3±3.61×10-3,显著低于在癌旁组织中的表达水平(15.23×10-3±20.85×10-3;t=3.12,P0.05)。结论:miR-375在HBV相关性肝细胞癌肝癌组织中表达显著下调。     

大鼠肝癌发生过程中p53的突变和甲胎蛋白的表达

采用免疫组织化学ＡＢＣ和ＰＡＰ法,对二乙基亚硝胺（ＤＥＮ）诱发大鼠肝癌发生过程中突变型ｐ５３蛋白（ｍｐ５３）和甲胎蛋白（ＡＦＰ）在肝细胞中的表达进行了系统观察。结果显示：（１）ＤＥＮ诱发大鼠肝癌发生率为１００％;（２）正常大鼠及诱癌第４周大鼠的肝细胞均不表达ｍｐ５３,至诱癌第８周,可见少量肝细胞表达ｍｐ５３,诱癌晚期的癌结节内大部分肝癌细胞呈ｍｐ５３阳性表达,ｍｐ５３免疫反应阳性产物为胞核内棕褐色颗粒;（３）正常大鼠肝细胞不表达ＡＦＰ,诱癌早期（４～８周）的大鼠肝小叶内可见少量ＡＦＰ阳性肝细胞,多为小肝细胞,呈散在分布,此后ＡＦＰ阳性肝细胞逐渐增多,晚期的癌结节内大部分癌细胞呈ＡＦＰ阳性,ＡＦＰ免疫反应阳性产物为胞浆内棕褐色颗粒。结果提示,ｍｐ５３和ＡＦＰ可作为分析肝癌进展的病理学指标     

AFP表达调控载体对肝癌模型的靶向治疗作用

目的观察甲胎蛋白(AFP)表达调控载体pAFP-P53-EGFP对AFP表达阳性肝癌模型靶向治疗作用。方法以人肝癌HepG2(AFP阳性)、人肝癌SMMC7721(AFP阴性)细胞于BALB/c-nu裸小鼠右腋皮下荷瘤,14d成瘤,免疫组化检测AFP。将构建好的pAFP-EGFP和pAFP-P53-EGFP重组质粒于肿瘤内注射,观察肿瘤体积变化,通过免疫组化观察p53在HepG2肿瘤中的特异性表达及对肿瘤细胞的杀伤作用。结果荷HepG2、SMMC7721细胞株裸鼠14d皮下肿瘤生长良好,且肿瘤体积均为500mm3左右,经HE染色证实造模成功。同时,AFP免疫组化结果显示接种HepG2细胞的肿瘤组织AFP表达阳性,SMMC7721细胞的肿瘤组织AFP表达为阴性。经pAFP-EGFP和pAFP-P53-EGFP重组质粒治疗后,p53的免疫组化分析结果显示接种HepG2细胞的裸鼠pAFP-P53-EGFP治疗组p53表达量显著高于其他各组,可见明显细胞凋亡现象,且肿瘤体积较对照组减小。结论含AFP基因调控序列的pAFP-P53-EGFP载体可专一性地作用于AFP阳性肝癌细胞,引起肝癌细胞周期阻滞和凋亡。     

凋亡诱导因子AIF在肝癌组织中的表达及临床意义*

目的:探讨凋亡诱导因子(AIF)在肝细胞癌组织中的表达及其临床意义。方法:采用免疫组织化学Envision法检测75例肝细胞癌组织及其相应癌旁肝组织、30例正常肝组织中AIF的表达,并分析其表达与肝细胞癌临床病理因素的相关性。结果:肝癌组织中AIF阳性表达率明显高于癌旁组织及正常肝组织,差异均具有统计学意义(P<0.01)。AIF在肝癌组织中的表达仅与病理分级密切相关(P<0.01),而与年龄、性别、肿瘤大小、临床分期、肿瘤数目、有无肿瘤包膜和有无淋巴结转移、有无门静脉癌栓均无关。结论:AIF表达可能参与了肝癌的发生和发展过程。     

凋亡诱导因子AIF在肝癌组织中的表达及临床意义

目的：探讨凋亡诱导因子（AIF）在肝细胞癌组织中的表达及其临床意义。方法：采用免疫组织化学Envision法检测75例肝细胞癌组织及其相应癌旁肝组织、30例正常肝组织中AIF的表达,并分析其表达与肝细胞癌临床病理因素的相关性。结果：肝癌组织中AIF阳性表达率明显高于癌旁组织及正常肝组织,差异均具有统计学意义（P〈0.01）。AIF在肝癌组织中的表达仅与病理分级密切相关（P〈0.01）,而与年龄、性别、肿瘤大小、临床分期、肿瘤数目、有无肿瘤包膜和有无淋巴结转移、有无门静脉癌栓均无关。结论：AIF表达可能参与了肝癌的发生和发展过程。     

Immunocytochemical localization of alpha-fetoprotein in the developing and carbon tetrachloride-treated rat liver.

The immunocytochemical localization of alpha-fetoprotein (AFP)-producing cells was observed in pre- and postnatal and carbon tetrachloride (CCl4)-treated rat livers in comparison with that of albumin (ALB)-producing cells. According to immunoblotting data, considerable numbers of AFP-positive hepatocytes were observed in the differentiating liver between prenatal day 19 and postnatal day 0 (6 h after birth). Analyses by serial section profiles of these cells revealed that certain AFP-positive hepatocytes are also stained with ALB antiserum. Immunoelectron microscopy of the AFP-producing cells revealed that immunoreactive gold particles are preferentially localized in rough endoplasmic cisternae, Golgi apparatus and Golgi-derived vesicles near the cell surface. In addition, the release of the content of the Golgi-derived vesicles into the differentiating bile canaliculi as well as into the space of Disse by exocytosis is apparent. In CCl4-treated rat liver, immunoreactions to AFP are localized exclusively in newly formed hepatocytes of the regenerative tissue. These AFP-positive cells have not established the hepatic cell cords, and the adjacent ones are conjugated to each other mainly by simple attachment devices as in the case of those in pre- and postnatal rat liver.     

Immunohistochemical study of alpha-fetoprotein in the postnatal ontogeny of mice with a varying sensitivity to liver carcinogens]

The ontogenesis of alpha-fetoprotein (AFP) was studied in the C57BL, CC57BR, C3H/He, A/He and DD mice during the first 4 weeks of life. The AFP hepatocytes were determined by indirect immunofluorescent staining of liver cells isolated with sodium tetraphenyl borate; the content of AFP in blood was determined by the method of rado immunodiffusion. The methods utilized allowed to obtain the quantitative characteristics of the dynamics of AFP-positive cells in the liver and the content of AFP in the blood. In the newborn mice over 90% hepatocytes contain AFP, the intensity of luminescence being heterogenous. The number of bright-liminescent cells equals to 50% during the first day of life rapidly decreases and none of them are found by 9--11 days. The number of average- and weak-luminescent hepatocytes does not decrease during the first 10 days, but then gradually decreases and none of them are found by 23 days in the CC57BR and by 27 days in the DD and A/He mice. A comparison of the dynamics of AFP-positive cells in the liver and the content of AFP in the blood has shown that the bright-luminescent hepatocytes are the main producent of this protein in the early postnatal ontogenesis.     

Mechanisms of alpha-fetoprotein synthesis in hepatoma cells. Combined electron microscopic immunocytochemistry and autoradiography

Immunoreaction of alpha-fetoprotein (AFP) was detected not only in well-differentiated hepatocellular carcinoma but also in hepatocytes forming foci in livers with hyperplastic nodules during 3'-methyl-4-dimethylaminoazobenzene hepatocarcinogenesis. The subcellular location of AFP in hepatoma cells was in the rough endoplasmic reticulum, perinuclear space and well-developed Golgi apparatus around the nucleus. In livers with hyperplastic nodules it was also in some parts of the smooth endoplasmic reticulum and Golgi regions in hepatocytes in the vicinity of submembranous areas or bile canaliculi. These findings suggest that the Golgi apparatus in hepatoma cells acts mainly as an organelle for glycosylation of AFP and that the Golgi complexes in the hepatocytes in livers with hyperplastic nodules are organelles for secretion of AFP. Combined light microscopic immunoperoxidase study and autoradiography with 3H-thymidine revealed a higher cumulative labeling index in AFP-positive hepatoma cells than in non-tumorous areas. Combined electron microscopic immunoperoxidase study and autoradiography showed that hepatoma cells with AFP immunoreactivity only in the rough endoplasmic reticulum had a significantly higher labeling index than did cells with AFP immunoreactivity in both rough endoplasmic reticulum and Golgi apparatus. These findings suggest that AFP is synthesized in hepatoma cells before or during the stage of their DNA synthesis and is then transported to the Golgi apparatus.     

Cellular immunolocalization of alpha-fetoprotein in rat liver

Increased synthesis of alpha-fetoprotein (AFP) was induced in rat liver by the administration of 3'-methyl-4-dimethyl-aminoazobenzene. The indirect immunoperoxidase technique was used to detect AFP. Cellular localization of AFP was studied using a number of different fixation procedures. Serial sections stained with immunoglobulin served to determine the extent of diffusion of serum proteins into liver cells during fixation. Background staining was minimized when Lillie's neutral buffered formalin plus acetic acid was used as the fixative. After 3'-methyl-4-dimethylaminoazobenzene ingestion, bile duct cell proliferation occurred. The serum AFP was positive in all rats after 17 days on the diet. In rats with AFP-positive sera the immunohistochemical reaction in mature hepatocytes was positive while bile duct cells and small hepatocytes were negative for AFP.     

Inhibition of alpha-fetoprotein synthesis in hepatocytes of adult mice by dextran sulfate in vivo and in vitro

Embryospecific serum protein alpha-fetoprotein (AFP) is known to be synthesized in the adult liver only during regeneration and development of hepatocellular carcinomas. It was shown that collagenase digestion of hepatic tissue followed by monolayer cell cultivation was a powerful inducer of AFP synthesis, more potent than the liver regeneration in vivo. The treatment of hepatocytes in culture with 50-100 micrograms/ml of dextran sulphate caused a remarkable inhibition of cell proliferation, formation of cord-like multicellular structures and reduction of AFP synthesis. Mouse liver regeneration after CCL4 poisoning was accompanied by a 1000-fold increase in blood AFP levels. Blood AFP levels and the content of AFP-positive cells in the liver tissue were maximum on the 3rd-4th day after poisoning. Injections of 50 micrograms of dextran sulphate per g body weight 3-5 h after poisoning and 24 and 48 h later caused nearly tenfold reduction in AFP blood level and a decrease in the content of AFP-positive cells in the liver on the 3rd day of regeneration.     

alpha 1-Fetoprotein production during the hepatocyte growth cycle of developing rat liver.

The relation of AFP production to DNA synthesis was investigated in newborn rat liver and in primary cultures of fetal rat hepatocytes, by combining immunoperoxidase AFP localization and autoradiography after ³H-thymidine labelling. The vast majority of AFP-positive hepatocytes did not incorporate ³H-thymidine after ≤4-h isotope pulses, suggesting that in the developing liver, essentially no production of AFP occurs in S, G₂ or M phases of the hepatocyte cell life cycle. Serial or continuous thymidine labelling experiments further indicated that post-mitotic hepatocytes constitute a sizable fraction of AFP-producing cells.     

Mechanisms of α-fetoprotein synthesis in hepatoma cells

Summary Immunoreaction of -fetoprotein (AFP) was detected not only in well-differentiated hepatocellular carcinoma but also in hepatocytes forming foci in livers with hyperplastic nodules during 3-methyl-4-dimethylaminoazobenzene hepatocarcinogenesis. The subcellular location of AFP in hepatoma cells was in the rough endoplasmic reticulum, perinuclear space and well-developed Golgi apparatus around the nucleus. In livers with hyperplastic nodules it was also in some parts of the smooth endoplasmic reticulum and Golgi regions in hepatocytes in the vicinity of submembranous areas or bile canaliculi. These findings suggest that the Golgi apparatus in hepatoma cells acts mainly as an organelle for glycosylation of AFP and that the Golgi complexes in the hepatocytes in livers with hyperplastic nodules are organelles for secretion of AFP.Combined light microscopic immunoperoxidase study and autoradiography with ³H-thymidine revealed a higher cumulative labeling index in AFP-positive hepatoma cells than in non-tumorous areas. Combined electron microscopic immunoperoxidase study and autoradiography showed that hepatoma cells with AFP immunoreactivity only in the rough endoplasmic reticulum had a significantly higher labeling index than did cells with AFP immunoreactivity in both rough endoplasmic reticulum and Golgi apparatus. These findings suggest that AFP is synthesized in hepatoma cells before or during the stage of their DNA synthesis and is then transported to the Golgi apparatus.