P-糖蛋白在肝细胞癌中的表达及临床研究

目的：探讨Ｐ－糖蛋白在细胞癌组织中表达及临床意义。方法：采用免疫组化法对７２例肝细胞癌，９例正常肝组织进行Ｐ－糖蛋白测定，作相关临床因素分析。结果：肝细胞癌组织中Ｐ－糖蛋白的阳性表达率为４４．０％，正常肝组织表达率为２２．０％（Ｐ〈０．０５）。Ｐ－糖蛋白的表达与肝癌的组织分型、分级及预后无明显相关性，并且化疗并不影响Ｐ－糖蛋白阳性表达病人的预后。结论：Ｐ－糖蛋白在肝细胞癌中的过表达，提示其在肝细胞     

细胞粘附分子CD15在肝癌组织中的表达

目的：观察细胞粘附分子ＣＤ１５在肝癌组织中的表达情况及肝脏良恶性病变的鉴别价值，方法：应用免疫组化ＳＰ法观察细胞粘附人子ＣＤ１５在７例正常肝组织，１３例肝硬变（５例伴轻－中度不典型增生）和４９例肝细胞癌组织中的表达。结果：正常肝组织和肝硬变组织中均无ＣＤ１５表达，仅肝癌组织中出现ＣＤ１５阳性表达，阳性率为３０．６％（１５／４９），ＣＤ１５表达与患者性别，年龄，肝癌肉眼形态，组织分化程度无明显关系（     

增殖性玻璃体视网膜病变中IL-6及IL-8的水平变化

目的：产殖性玻璃体视网膜病变（PVR）患者IL－6、IL－8在玻璃体、视网膜下液及血浆中的水平。方法：用放射免疫分析方法检测34例PVR患者（实验组）和30例视网膜脱离患者（对照组）玻璃体、视网膜下液、血浆中IL－6、IL－8的水平。结果：PVR患者玻璃体、视网膜下液中IL－6、IL－8高于对照组，而两组血浆IL－6、IL－8水平无差别。结论：在PVR过程中，有免疫系统的参与，而且以局部反应为主。     

组织因子在肝细胞癌的发生及侵袭转移中的研究

目的：检测组织因子（TF）在肝细胞癌患者中的表达并探讨其意义。方法:采用酶联免疫法检测肝细胞癌患者50例及对照组30例的血浆TF水平，采用RT-PCR法检测其中27例肝癌、癌旁、正常肝组织TF mRNA表达。结果:①肝细胞癌患者血浆TF显著高于对照组（P<0.05）。血浆TF在分化高低、肿瘤大小及是否合并肝硬化组间表达有显著差异（P<0.05），在有淋巴转移、肝外脏器转移及门脉癌栓组高于无转移及癌栓组(P<0.05)，而在不同癌灶数目及包膜情况组间无显著差异（P＞0.05）。②TF mRNA在肝癌组织中阳性率及相对表达强度分别为62.96%（17/27）、0.567±0.268，均显著高于癌旁组织及正常组织。阳性患者相对表达强度在肿瘤大小及部分侵袭转移指标中表达有显著差异（P<0.05） 。结论：TF在肝癌患者血浆及组织中升高且与部分侵袭转移指标相关，提示其TF可能促进了肝癌的发生与侵袭转移。     

肝癌组织细胞角蛋白7、19表达的评估

目的： 分析细胞角蛋白7、19（CK7、CK19）和AFP在原发性肝癌组织中的表达，评估其在肝癌鉴别诊断中的价值。 方法： 采用免疫组化法检测原发性肝癌组织中CK7、CK19和AFP的表达，观察3种表达产物在肝癌不同组织类型中的表达。 结果： 106例肝癌组织中，CK7表达阳性率分别为：肝细胞癌29.4%(25/85)，胆管上皮癌58.3%(7/12)，混合型肝癌66.7%(6/9)。CK19表达阳性率为：肝细胞癌23.5%(20/85)、胆管上皮癌50.0%(6/12)、混合型肝癌55.6%(5/9)。CK7和CK19共同表达阳性率为：肝细胞癌9.4%（8/85），胆管细胞癌41.7%(5/12),混合型肝癌55.6%(5/9)。CK7、CK19、CK7/CK19在混合型肝癌和胆管上皮癌表达阳性率高于肝细胞癌。AFP表达的阳性率为：肝细胞癌56.4%(47/85)、胆管上皮癌25.0%(3/12)、混合型肝癌33.3%(3/9)，AFP在后两者的表达阳性率低于前者。 结论：CK7和CK19在混合型肝癌和胆管上皮癌表达阳性率高于肝细胞癌。而AFP在肝癌组织的表达与之相反。联合检测CK7、CK19和AFP可能对肝细胞癌和胆管上皮癌的鉴别诊断有参考价值。     

细胞外基质蛋白1在肝癌中的表达及意义

目的：探讨细胞外基质蛋白1（extracellular matrix protein1,ECMl）在肝癌中的表达及意义。方法：应用免疫组织化学EnVision法,检测肝脏细胞株（正常肝细胞株LO2、肝癌细胞株HepG2）和肝脏组织（正常肝组织和肝癌组织）中ECM1的表达,并比较正常肝组织和肝癌组织之间的ECM1表达差异。结果：ECM1的表达如下：正常肝细胞株LO2（-）,肝癌细胞株HepG2（＋）;正常肝组织阳性率20％（4／20）,肝癌组织阳性率85．4％（70／82）。统计分析表明,对ECM1的表达,肝癌组织明显高于正常组织（P〈0．01）。结论：ECM1在肝癌组织中过表达。     

大鼠肝癌发生过程中增殖细胞核抗原及原癌基因表达

为进一步探讨肥大细胞对实验性肝癌细胞生长速度的影响，及其与肝癌细胞原癌基因表达差异之间的内在联系，运用免疫组织化学方法及原位分子杂交技术，对化学致癌剂二乙基亚硝胺（Diethylnitrosamine，DEN）诱发的大鼠原发性肝细胞性肝癌细胞及癌前增生结节的增殖肝细胞的增殖细胞核抗原（PCNA）及ras家族癌基因（N－ras、（H－ras及Ki-rar。）表达进行了观察。结果表明：PCNA在癌细胞及癌前增殖肝细胞中均有阳性表达，PCNA阳性细胞数与癌巢及癌前增生结节周围浸润的肥大细胞数呈负相关关系。这一观察结果与本文癌基因表达结果基本一致。     

实验性大鼠肝脏癌变过程中生长抑素受体2表达的意义

目的：研究生长抑素受体2（SSTR2）在肝癌癌变过程中的表达和分布，探讨SSTR2在肝癌发生过程的作用。方法：二乙基亚硝胺溶液(DENA)诱发SD大鼠发生肝癌，RT-PCR和免疫组化检测肝硬化肝组织、肝癌中SSTR2的mRNA和蛋白表达，比较不同时期肝组织中SSTR2-mRNA和蛋白的表达。结果：DENA诱癌8周后，可见肝癌形成。诱癌8周时，癌旁肝组织SSTR2-mRNA明显高于正常肝脏（1.794±0.212 vs 0.950±0.138,P<0.05），随着诱癌时间的延长，SSTR2-mRNA的表达逐渐增强，到16周时到最高（2.053±0.169），随后逐步下降（22周时低至1.468±0.107），而肝癌组织SSTR2-mRNA表达只为1.219±0.249。免疫组化检测SSTR2蛋白表达发现，SSTR2主要位于胞浆和细胞膜，SSTR2蛋白表达的变化与SSTR2-mRNA的变化一致。结论：SSTR2在实验性肝硬化早期表达逐步升高，随着肝硬化的加重和肝细胞癌变的发生，SSTR2表达逐渐下降。SSTR2表达下降可能与肝细胞癌发生、发展密切相关。     

抗炎治疗影响DEN诱导原发性肝癌进展的研究

目的 观察抗炎治疗对二乙基亚硝胺（diethylnitrosamine,DEN）诱导原发性肝癌进展的影响。 方法 选取42只大鼠建立DEN诱导肝癌模型,随机分为3组,观察诱癌不同时期各组肝组织结节个数及肝组织结构的变化,用免疫组化检测3组CD68、CD206和CD34的阳性细胞表达,用western blot检测3组肝组织中TNF-α、IL10、TGF-β、HGF及NFκB等蛋白的相对表达。 结果 与对照组及葡醛内酯组相比,氢化可的松组结节个数更少,肝癌出现时间更晚,肝组织中CD68、CD206和CD34的阳性细胞表达更少,在诱癌第8~12周TNF-α、IL10、TGFβ、HGF和NFκB等蛋白在肝组织中的相对表达更少。 结论 抑制肝脏微环境中单核-巨噬细胞介导的慢性非特异性炎症能在诱癌早期延缓DEN诱导原发性肝癌的进展。     

肝癌细胞中connexin32、connexin43的表达及其与间隙连接通讯功能的相关性

目的：研究肝细胞肝癌和正常肝细胞间隙连接蛋白connexin32（Cx32)、connexin43(Cx43）的表达，及其对间隙连接通讯功能（GJIC）的影响。方法：应用应用培养及流式细胞分析技术（FCM），研究肝癌细胞系HHCC、SMMC－7721和正常肝系QZG细胞中Cx32和Cx43的表达。结合Ｌucifer Yellow划痕标记荧光传输技术（SLDT），检测上述细胞的间隙连接通讯功能。结果：流式细胞仪分析证实，Cx32蛋白在肝癌细胞系HHCC、SMMC－7721和正常肝细胞系QZG细胞中表达的阳性率分别为1．9％、0．7％和99．0％；Cx43蛋白在HHCC、SMMC－7721和QZG细胞中表达的阳性率分别为7．3％、26．5％和99．1％。SLDT检测发现肝癌细胞HHCC，SMMC－7721的间隙连接通讯功能较正常肝细胞QZG明显减弱。结论；Cx3、Cx43蛋白在正常肝细胞中具有较高水平的表达，在肝癌细胞中表达水平显著降低，肝癌细胞的间隙连接通讯功能较正常肝细胞亦明显减弱。Cx32、Cx43表达调控异常引起的间隙连接通讯障碍可能与肝癌的发生密切相关。     

大鼠肝癌发生过程中胰岛素样生长因子-Ⅱ的表达及意义

目的:研究胰岛素样生长因子-Ⅱ(IGF-Ⅱ)在肝癌发生过程中的表达及意义.方法:采用二乙基亚硝胺(DEN)诱癌建立大鼠肝癌模型.应用放射免疫分析检测诱癌过程中血清IGF-Ⅱ浓度,应用免疫组织化学方法观察诱癌过程中大鼠肝组织IGF-Ⅱ的表达情况.结果:在大鼠肝癌的发生发展过程中,从肝细胞损伤期、增生-硬化期至癌变期3个阶段大鼠血清IGF-Ⅱ水平呈"高到低到高"的变化趋势;肝细胞损伤期嗜酸性变细胞中见IGF-Ⅱ阳性表达,癌变期癌灶中表达低于癌周增生灶、增生结节和非典型增生结节,而增生-硬化期阳性表达较少.结论:大鼠血清和肝中IGF-Ⅱ的高表达是肝癌发生发展过程中的早期及晚期事件,提示IGF-Ⅱ可能与肝细胞持续增殖及恶性转化有关.     

Overexpression of caspase-3 in hepatocellular carcinomas.

Caspase-3 is a downstream effector cysteine protease in the apoptotic pathway. It is ubiquitously expressed in normal human tissues including the liver. Overexpression and loss of expression of caspase-3 has been reported in diverse human malignancies. However, expression of caspase-3 in hepatocellular carcinoma (HCC) has not been studied. Therefore, we studied its expression in four hepatoma cell lines and 22 HCCs by Western blot, and correlated the findings with in vitro caspase-3 activity and apoptosis. In addition, 47 surgically resected HCCs and 29 metastatic colorectal carcinomas were evaluated for caspase-3 expression by immunohistochemistry on formalin-fixed, paraffin-embedded tissue sections, and the staining intensity was correlated with the clinicopathological features. Caspase-3 overexpression was present in all four hepatoma cell lines, and 68% (15/22) of HCCs in comparison to the non-neoplastic liver parenchyma by Western blot, and in 52% (36/69) of HCCs by immunohistochemistry. Caspase-3 overexpression in HCCs by Western blot correlated with caspase-3 overexpression by immunohistochemistry (P=0.002), and in vitro caspase-3 activity (P=0.01). Caspase-3 overexpression in HCCs by immunohistochemistry was associated with serum alpha-fetoprotein (AFP) levels (P=0.01). In conclusion, caspase-3 is frequently overexpressed in HCCs and is associated with high serum levels of AFP.     

KIF20A mRNA and its product MKlp2 are increased during hepatocyte proliferation and hepatocarcinogenesis

Mitotic kinesin-like protein 2 (MKlp2), a microtubule-associated motor, is required during mitosis exit for the final step of cytokinesis. It also contributes to retrograde vesicular trafficking from the Golgi apparatus to the endoplasmic reticulum in interphase. The KIF20A gene encoding MKlp2 is controlled by the E2F-retinoblastoma protein-p16 pathway, and its widely expressed mRNA is found in fetal and proliferating adult tissues. The expression pattern and function of MKlp2 in the adult liver, however, have not been investigated. We report herein that MKlp2 transiently accumulates in vivo during mouse liver regeneration after partial hepatectomy and is strongly overexpressed in preneoplastic and neoplastic mouse liver. In vitro in mitogen-stimulated primary hepatocytes, MKlp2 accumulated in the nucleus during the G2 phase of the cell cycle coincident with the mitotic kinase Aurora B. Human hepatoma cell lines exhibited high levels of MKlp2; however, it was undetectable in normal human hepatocytes. RNAi-mediated MKlp2 knockdown in hepatoma cells induced polyploidization consistent with its essential function in promoting cytokinesis and inhibited cell proliferation without inducing apoptosis. KIF20A mRNA was strongly accumulated in a large series of human hepatocellular carcinomas, with the highest expression observed in tumors with genomic instability. Accumulation of MKlp2 in normal proliferating, preneoplastic, and transformed hepatocytes suggests that MKlp2 contributes to both normal and pathologic hepatocyte proliferation and is linked to tumor aggressiveness in human hepatocellular carcinomas.     

转录因子EGR-3在肝细胞肝癌中的表达及意义

目的研究早期生长反应基因(EGR-3)在肝癌组织中的表达,并探讨其在肝癌发生发展中的作用。方法选取125例肝癌及癌旁组织、5例正常肝组织标本,采用免疫组织化学方法检测标本中EGR-3的表达分布,并分析其与肝癌临床病理因素的关系。另提取15例新鲜肝癌及相应癌旁组织标本和人肝癌HepG2、HepG2.2.15、SMMC7721、FHCC98细胞的总蛋白,采用蛋白质印迹法检测EGR-3的表达水平。结果①免疫组织化学检测结果显示,EGR-3在肝癌组织中的表达阳性率(30/125,24%)明显低于癌旁组织(100/125,80%)(χ2=72.5,P0.001);EGR-3主要表达于肝癌组织的细胞质中,而在癌旁组织的细胞质和细胞核均有表达;此外,EGR-3在肝癌组织中的表达强弱与患者年龄、性别以及肿瘤大小、肿瘤分化程度等均无明显相关性(P0.05);②蛋白质印迹技术检测结果也显示EGR-3在肝癌组织中的表达水平均明显低于相应癌旁组织(P0.05);EGR-3在4种肝癌细胞系中都有表达,并在HepG2.2.15细胞中表达水平最高。结论 EGR-3在肝癌组织中的表达明显低于癌旁组织,推测其与肝癌的发生密切相关,为肝癌的临床诊断提供了线索。     

Pleomorphism of cancer cells with the expression of plectin and concept of filament bundles in human hepatocellular carcinoma

Intermediate filaments are important in building the architecture of liver cells and are proposed to interact with other cellular components. Among intermediate filament associated proteins, plectin is a versatile cytoskeletal linkage protein which has been shown to interact with a variety of cytoskeletal structures. Intermediate filament and plectin might play some roles in tumorigenesis of human hepatocellular carcinoma since cells of hepatocellular carcinoma were morphologically different from normal liver. Plectin exhibited wide distribution spectrum among various tissues, however, it was poorly investigated in human liver and hepatoma tissues. In this paper, we studied the plectin expression in 18 cases of human hepatocellular carcinoma and normal hepatocytes by immunohistochemistry. The results revealed that plectin expression was deficient in human hepatocellular carcinoma and was probably through post-translational modification. Many 0.4 to 0.8 microm-thick keratin bundles were found in intermediate filament extracts of liver and hepatoma tissues. These bundles were greater in diameter about 40 to 80 times of single intermediate filament. We speculated that intermediate filament organized into "filament bundles" to maintain the shape of normal cells. In cancer cells, plectin was deficient and the irregularly loosened filament bundles could cause pleomorphism of cancer cells.     

Anterior gradient-2 is overexpressed by fibrolamellar carcinomas

Anterior gradient-2 expression is critical in normal embryonic development. Aberrant expression of anterior gradient-2 in adult tissues has been linked to breast, prostate, esophageal, and pancreatic carcinoma. To define the role of anterior gradient-2 in primary hepatocellular neoplasms, we used tissue microarrays and examined protein expression in typical hepatocellular carcinomas (n = 44), fibrolamellar carcinomas (n = 12), and hepatic adenomas (n = 9). In nonneoplastic liver tissues, anterior gradient-2 was expressed in the septal-sized bile ducts and weakly in zone 3 hepatocytes in 11 (18%) of 61 cases. In tumors, anterior gradient-2 was overexpressed by only 1 (2%) of 44 hepatocellular carcinomas. In contrast, 6 (75%) of 8 fibrolamellar and 3 (75%) of 4 metastatic fibrolamellar carcinomas were positive. All 9 hepatic adenomas were negative. Further analysis of mRNA in fibrolamellar carcinomas identified 2 novel splice variants, but expression levels were very low. Sequencing of the anterior gradient-2 gene in fibrolamellar carcinomas identified several polymorphisms (refSNP Ids: rs6842, rs8071, rs1051905) but no mutations. In conclusion, anterior gradient-2 is overexpressed in the majority of fibrolamellar carcinomas but is only rarely overexpressed in hepatocellular carcinomas.     

Potential interaction between CCR1 and its ligand,CCL3, induced by endogenously produced interleukin-1 in human hepatomas

Hepatoma cell lines can produce a massive amount of chemokines in response to various stimuli including hepatitis viruses and their products. However, it remains elusive on the types of chemokine receptor(s) expressed in the hepatoma tissues and its roles in hepatoma development. To clarify these points, we examined the chemokine receptor expression in six human hepatoma cell lines. All of the hepatoma cell lines constitutively and exclusively expressed CCR1 mRNA and its protein on their cell surface. CCR1 expression was also detected on hepatoma cells and to a lesser degree, on endothelial cells in hepatoma tissues but not in normal liver tissues. Furthermore, CCL3 expression was detected in hepatoma cells, endothelial cells, and to a lesser degree, fibroblast-like cells in hepatoma tissue, whereas only occasional vascular endothelial cells and inflammatory cells in normal liver tissues were weakly positive for CCL3. Moreover, the forskolin-mediated increases in intracellular cAMP concentrations were inhibited by the ligands for CCR1, CCL3, CCL4, and CCL5, suggesting that the expressed CCR1 was functional. Four hepatoma cell lines produced CCL3 only in response to interleukin (IL)-1 alpha and IL-1 beta. Finally, IL-1 alpha and IL-1 beta were detected abundantly in hepatoma tissues but not in normal liver tissues. Thus, IL-1 may enhance the local production of CCL3, which may interact with CCR1 expressed on hepatoma cells, in an autocrine and/or paracrine manner.     

Hepatocyte expressions in hepatocellular carcinomas, gastrointestinal neoplasms, and non-neoplastic gastrointestinal mucosa: its role as a diagnostic marker

We performed immunohistochemical staining against Hepatocyte (Hep) and CD10 antibodies in 75 hepatocellular carcinoma (HCC), 50 cholangiocarcinomas, 49 colorectal adenocarcinomas, and 308 gastric adenocarcinomas by tissue array method. We also evaluated the various non-neoplastic adult tissues and fetal digestive organs. Hep was expressed in 80% of HCCs, and HCCs without Hep expression were more likely to have a higher Edmondson & Steiner grade than HCCs with Hep expression (p=0.004). In non-HCCs, 16% of cholangiocarcinomas, 8.2% of colorectal carcinomas, and 44.2% of gastric carcinomas expressed Hep. Gastric carcinomas with Hep expression were significantly associated with early gastric carcinomas (p<0.001). In non-neoplastic tissues, Hep was found expressed in normal hepatocytes, small intestinal mucosa, and intestinal metaplasia of the stomach. Fetal hepatocytes expressed Hep after 19 weeks of gestation. CD10 was detected in 46.7% (35/75) of HCCs, and canalicular staining pattern was predominant in HCCs. In conclusion, the expression of Hep and CD10 may help to distinguish HCCs from non-HCCs.     

Overexpression of insulin receptor substrate-1 emerges early in hepatocarcinogenesis and elicits preneoplastic hepatic glycogenosis.

Insulin receptor substrate-1 (IRS-1) is a multisite docking protein occupying a central position in signaling cascades stimulated by a number of growth factors including insulin. Using Western blotting and immunohistochemistry, we investigated the expression of IRS-1 in more than 400 preneoplastic foci of altered hepatocytes and in 12 hepatocellular carcinomas induced in rats by oral administration of N-nitrosomorpholine. In both N-nitrosomorpholine-treated and untreated rat livers, IRS-1 was demonstrable by Western blotting, but with the exception of a few single hepatocytes it was not detectable in the normal parenchyma by immunohistochemistry. In contrast, immunohistochemistry revealed that IRS-1 was strongly expressed in the majority of foci of altered hepatocytes particularly in approximately 97% of the clear/acidophilic and mixed cell foci showing excessive storage of glycogen (glycogenosis). In glycogen-poor basophilic foci of altered hepatocytes and hepatocellular carcinomas, IRS-1 was not detected by immunohistochemistry, but a weak expression was observed in small subpopulations of three hepatocellular carcinomas containing remnants of glycogen. These results indicate that the focal overexpression of IRS-1 is an early event in hepatocarcinogenesis, which is closely correlated with preneoplastic hepatic glycogenosis. During progression from glycogenotic foci to hepatocellular carcinomas, IRS-1-overexpression is gradually down-regulated, and this late event is associated with a fundamental metabolic shift leading to the malignant neoplastic phenotype.     

Expression of transforming growth factor-beta1 and transforming growth factor-beta receptors in hepatocellular carcinoma and dysplastic nodules.

In this study we analyzed by immunohistochemistry the expression of TGF-beta1 protein and TGF-beta receptors I and II in 4 low-grade dysplastic nodules, 2 high-grade dysplastic nodules, 6 early, 22 small, and 62 advanced hepatocellular carcinomas. The expression of TGF-beta1 protein by hepatocytes was decreased in advanced hepatocellular carcinoma compared with small or early hepatocellular carcinoma(P < .05). Frequent and intense staining of TGF-beta1 protein was noted in the sinusoidal endothelium of advanced hepatocellular carcinomas despite of its decreased staining in hepatocellular carcinoma cells. Reduced expression of TGF-beta receptors I and II compared with surrounding nontumorous tissue were noted from the early hepatocellular carcinoma stage suggesting that down-regulation of TGF-beta receptors is correlated with progression from premalignant to malignant phenotype. Reduced expression of both TGF-beta1 and TGF-beta receptor II in neoplastic hepatocytes were also significantly correlated with increased tumor size and increased proliferative activity(P < .05). These findings suggest that during hepatocarcinogenesis, the inhibitory effects of TGF-beta1 protein on hepatocellular carcinoma cells is outweighed by its effects on stromal elements, which, overall, contributes indirectly to a tumor growth stimulatory environment. Also, the growth-inhibitory effects of TGF-beta1 may have been further negated by reduced TGF-beta receptors on hepatocellular carcinoma cells.