细胞角质素19及消减基因P02在肝细胞癌组织及卵圆细胞中的表达

目的:观察细胞角质素19(CK19)及消减基因P02在肝细胞癌,肝硬化组织及卵圆细胞中的表达情况.方法:采用免疫组化SP法观察8例正常肝组织,27例肝硬化组织和43例肝细胞癌组织中CK19的表达.采用DIG-probe synthesis kit,聚合酶链反应方法制备P02探针,通过原位杂交方法检测P02在肝细胞癌,肝硬化组织及卵圆细胞中的表达.结果:CK19在肝硬化组与正常肝组织之间表达率无明显差异,但在肝细胞癌组与肝硬化组之间差异有显著性(69.77% vs 25.93%,P<0.01).肝硬化组织与肝细胞癌组织中CK19标记的卵圆细胞数量有显著性差异(5.74±1.05 vs 10.51±1.78,P<0.01).P02在肝硬化和肝细胞癌中的阳性表达率分别为26.7%和80%,二者之间表达有显著性差异(P<0.01).结论:CK19可能参与了肝硬化到肝细胞癌的癌变过程.卵圆细胞与肝脏损伤后再生及癌变有关,可能是P02通过促进卵圆细胞的增殖来介导肝细胞癌的发生.     

肝细胞癌中DcR3表达与卵圆细胞增殖的可能关系

目的研究DcR3（一种新的凋亡相关分子）在肝细胞癌（HCC）中表达情况及其与卵圆细胞增殖之间可能存在的关系,以探讨卵圆细胞增殖活化的分子机制。方法对52例HCC组织,用免疫组织化学EnVision法同时标记肝细胞分化标记物hepatocyte paraffinl（HEP）、胆管标记物CK7/CK19、细胞增殖标记物Ki-67以及DcR3,半定量分析卵圆细胞形态数量和凋亡细胞数量的关系。结果在所有HCC肿瘤旁硬化组织的反应性小胆管中,均可见CK7、CK19和DcR3的表达,而在52例HCC中有23例见部分肿瘤细胞表达CK7和/或CK19,36例部分肿瘤细胞表达DcR3。其中23例CK7和/或CK19阳性病例中,21例同时有DcR3阳性（91.3%）,而29例CK7/CK19阴性病例中仅有15例（51.7%）DcR3阳性表达,两组间差异有统计学意义（P=0.002）。结论HCC中存在CK7和/或CK19阳性表达的卵圆细胞,表明卵圆细胞在部分HCC的发病中起重要作用,而这些病例多数有DcR3高表达,同时可见DcR3高表达于癌旁硬化组织反应性小胆管中,这些均提示DcR3的抗凋亡作用可能与卵圆细胞的增殖分化有关。     

过渡型CK19阳性表达细胞的研究

目的探讨慢性乙型肝炎患者肝组织过渡型CK19阳性表达细胞与肝癌前病变的关系。方法用LSAB免疫组织化学染色方法观察慢性乙型肝炎患者肝组织CK19的表达，每3个月对患者检查B超和血清甲胎蛋白，随访至1年。结果肝组织出现卵圆细胞分化为肝细胞的过渡型CK19阳性表达细胞的患者，大多数出现血清甲胎蛋白的明显升高。随访至1年，有1例患者出现小肝癌，1例出现肝实质低回声结节。结论过渡型CK19阳性表达细胞有可能作为一种新的肝癌前病变的病理学标志。     

蛋白激酶CK2β在肝细胞癌中的表达及临床意义

目的 研究蛋白激酶CK2β在肝细胞癌组织中的表达及临床意义,并探讨其表达与患者预后的关系。方法 收集2012年1月-2013年6月于郑州大学第一附属医院肝胆胰外科确诊的127例肝细胞癌患者癌组织及癌旁组织,采用免疫组化法检测其CK2β的表达,分析癌组织中CK2β表达强度与肝癌患者临床特征的关系。另收集本院2018年3-6月期间手术切除的20例肝细胞癌癌组织、癌旁组织、肝硬化组织、正常肝组织标本并提取蛋白和mRNA,运用Westen Blot和real-time PCR检测其CK2β表达情况。多组间均数比较采用单因素方差分析,进一步两两比较采用Bonferroni检验,计数资料组间比较采用χ2检验。CK2β在肝细胞癌的表达差异与临床特征参数的关系采用Mann-Whitney U检验或Kruskal-Wallis H检验。生存分析采用Kaplan-Meier法,组间比较采用log-rank检验。结果 Westen Blot和real-time PCR结果证实CK2β在癌组织中表达率显著高于癌旁组织、肝硬化组织和正常肝组织(P值均0. 05);癌旁及肝硬化组织中CK2β表达无差异(P值均 0. 05),且均高于正常组织表达(P值均0. 05)。免疫组化染色结果发现127例肝细胞癌患者的癌组织和癌旁组织中CK2β阳性表达率分别为85. 8%和63. 0%,差异有统计学意义(P 0. 001)。肝癌组织中CK2β的表达分布在不同年龄(Z=-2. 277,P=0. 023)、肝硬化有无(Z=-2. 144,P=0. 032)、不同肿瘤大小(Z=-2. 289,P=0. 004)、不同Edmondson-Steiner病理分级(χ2=8. 210,P=0. 016)的肝细胞癌患者中差异均有统计学意义。Kaplan-Meier生存曲线发现,CK2β表达强阳性组术后生存期均明显低于中阳性组、弱阳性组及阴性组(P值均0. 001)。结论CK2β蛋白可能参与肝细胞癌的发生发展,其阳性表达与肝细胞癌患者预后有关。     

黄芪汤对肝硬化大鼠肝脏卵圆细胞肝向分化的作用

目的:研究黄芪汤对肝硬化大鼠肝脏卵圆细胞肝向分化的作用机制。方法:应用二甲基亚硝胺制备大鼠肝硬化模型,用黄芪汤治疗,进行肝功能、肝脏病理学检测;应用共聚焦免疫荧光技术检测肝脏卵圆细胞与肝细胞及胆管细胞共定位染色。结果:黄芪汤能显著降低模型大鼠血清AST、TBil水平和肝组织Hyp含量,减少肝组织α-SMA的表达,在模型大鼠肝纤维化逆转过程中,黄芪汤可使Thy1.1与CK19共定位细胞数量显著增加,使肝脏卵圆细胞(HOC)表型和功能发生改变。结论:黄芪汤通过诱导肝脏卵圆细胞肝向分化作用来促进肝硬化逆转。     

mTOR/P70S6K信号通路在肝细胞肝癌中的表达及临床意义

目的:研究mTOR/P70S6K信号通路在肝细胞肝癌(HCC)中的表达,探讨其在HCC发生发展中的作用及意义.方法:用逆转录聚合酶链反应(RT-PCR)技术检测120例HCC患者癌组织、癌旁肝组织以及10例正常肝组织中mTOR及P70S6K mRNA表达情况;并分析mTOR及P70S6K mRNA的表达与相关临床参数的关系.结果:mTOR及P70S6K mRNA在HCC组织中的表达水平显著高于在癌旁肝组织和正常肝组织中的表达水平(mTOR mRNA:0.594±0.218 vs 0.437±0.156.0.594±0.218 vs 0.383±0.081,均P＜0.05;P70S6K mRNA:0.610±0.147 vs 0.486±0.162.0.610±0.147 vs 0.440±0.141,均P＜0.05).mTOR mRNA和P70S6KmRNA在HCC组织中的表达呈正相关(r=0.548,P=0.012),且两者在癌旁肝组织及正常肝组织中的表达亦正相关性(r=0.607,0.737,P=0.005,0.015).mTOR及P70S6K mRNA在HCC组织中的表达水平与病理分期、门静脉癌栓等明显相关,而与肿瘤直径、血清AFP水平、性别等无明显关系.结论:mTOR/P70S6K信号通路在HCC中特异性激活.mTOR/P70S6K信号通路可能在肝细胞肝癌的发生、发展中起重要作用.     

HBV所致终末期肝病患者肝组织胆管增生的研究

目的在乙型肝炎病毒（HBV）所致的终末期肝病患者肝组织内，常可见大量的胆管增生，但其发生机制及临床意义尚不清楚。为阐明该类患者胆管增生的发生机制及其与卵圆细胞增生及肝细胞再生的关系，我们对8例HBV相关的终末期肝病患者及2例正常人肝组织进行了免疫组化染色及图像分析。方法肝组织连续切片后进行免疫组化染色，观察指标为细胞角蛋白CK7、CK8、CK18、CK19、OV6、增殖细胞核抗原（PCNA）、谷胱甘肽S转移酶（GSTπ）、白蛋白（ALB）及甲胎蛋白（AFP）。结果在所有8例患者肝组织汇管区内均可见典型增生的胆管及非典型增生的胆管，且对CK7、CK8、CK9、CK19、OV6及PCNA染色呈阳性反应，但两种类型的胆管在染色强度上存在明显差异。一些非典型增生的胆管细胞表现出肝卵圆细胞的形态学及免疫组化特征。某些小型肝细胞样细胞在形态及免疫组化特征方面介于肝卵圆细胞及成熟肝细胞之间。结论在HBV相关的终末期肝病患者肝组织内，胆管增生可能存在不同起源。某些非典型增生的胆管细胞实际上就是活化的肝卵圆细胞。非典型增生的胆管细胞与肝细胞再生密切相关。小型肝细胞样细胞可能是肝卵圆细胞与成熟肝细胞之问的中间过渡细胞。     

核心蛋白聚糖在原发性肝癌中的表达

目的:研究核心蛋白聚糖(decorin,DCN)在原发性肝癌组织中的表达.方法:免疫组织化学(SP法)分别检测16例正常肝组织、20例肝硬化组织、30例肝癌及癌旁组织中DCN的表达,利用阳性表达率及吸光度进行半定量及定量分析.结果:DCN在肝硬化及癌旁组织中呈强阳性表达,阳性表达率、平均吸光度与正常肝组织及肝癌组织相比,有显著性差异(55%,73.3%vs 0%,10%; 0.2357±0.0396,0.2983±0.1990vs 0.1394±0.0072,0.1589±0.0115,均P<0.01).结论:DCN可能作为一种负性调控蛋白参与肝癌及肝纤维化的发生发展.     

原发性肝癌组织芯片中乙酰肝素酶的表达及意义

目的:探讨乙酰肝素酶(heparanase,HPA)蛋白在原发性肝细胞癌(HCC)组织芯片中的过度表达及临床意义.方法:125例HCC患者肝组织、48例肝癌患者癌旁组织、62例肝硬化患者肝组织及23例肝血管瘤患者相应正常肝组织构建组织微阵列.应用免疫组织化学检测HPA蛋白的表达水平,并分析其与HCC临床病理特征的关系.结果:HCC组织中的HPA蛋白的阳性率45.83%明显高于癌旁组织27.08%(x~2=2.23,P<0.05),肝硬化6.45%(x~2=5.262,P<0.05)和正常肝组织4.35%(x~2=3.895,P<0.05).癌旁组织中的HPA蛋白阳性率明显高于肝硬化(x~2=2.882,P<0.05)及正常肝组织(x~2=2.361,P<0.05);HCC中临床TNM分期ⅠⅡ期HPA阳性率明显低于ⅢⅣ期(29.41% vs 67.31%,x~2 =4.111,P<0.05);HCC中无转移组HPA阳性率明显低于转移组(14.71% vs 63.33%,x~2= 3.978,P<0.05);HPA表达率在AFP≥400μg/L和AFP<400μg/L组(52.05% vs 36.17%,x~2= 2.071,P<0.05)、有无门脉癌栓组(71.74% vs 29.73%,x~2=4.472,P<0.05)、多个和单个肿瘤结节组(73.91% vs 28.38%,x~2=4.847,P<0.05)以及肿瘤直径≥5 cm和<5 cm组(57.89% vs 25%,x~2=3.471,P<0.01)分别具有显著性意义.HPA表达与年龄、性别、分化程度、有无肝硬化及肿瘤包膜浸润无关.结论:HPA高表达在HCC的发生、发展及转移中起重要作用.检测HPA蛋白指标有助于HCC诊断和判断患者预后.     

NDRG1在原发性肝细胞癌及胎肝中的表达及意义

目的:探讨NDRG1在原发性肝细胞癌(HCC)及胎肝组织中的表达及其意义.方法:收集2002-01/2008-12广州市第一人民医院手术切除的肝细胞癌标本81例. 所有患者术前未行放疗和化疗; 25例胎肝组织, 取自不同月份流产或引产的胎儿(4、5、6、7、8 mo胎儿各5例); 另选43例癌旁组织, 10例肝硬化组织, 9例正常肝组织(移植肝), 8例原发癌转移灶组织作为对照. 观察肝脏组织病理形态特征, 并用免疫组织化学EnVison法检测NDRG1的表达.结果:NDRG1在正常肝组织中呈强阳性表达, 平均吸光度值为0.206±0.056, 随着肿瘤的发生, 在癌旁组织中有减弱(0.176±0.083),在HCC中表达明显减弱(0.128±0.096), 在转移灶中表达最低(0.059±0.051), 而在胎肝组织中表达亦较低(0.059±0.074). 各组总体差异均有统计学意义(F = 33.669, P <0.05). HCC与患者年龄、性别、肝炎病史、肝硬化、肿瘤大小、AFP值、HbsAg、淋巴结转移及有无远处转移、肿瘤分型、Child-Pugh分级、TNM分期、CLIP分期均无关(P >0.05), 但与肿瘤的Edmondson分级有关(F = 2.881, P <0.05).结论:NDRG1在HCC中低表达, 并且随着肿瘤的发生发展, 表达量逐渐降低. NDRG1可能对HCC起着抑制作用, 提示该基因可望成为早期预测肝癌转移的分子生物学标志物之一.     

Expression of cytokeratin 19 during human liver organogenesis

ABSTRACT— Immunohistochemistry with monoclonal anti-cytokeratin antibodies has revealed the presence of cytokeratin 19 in embryonic and early fetal hepatocytes. With the differentiation of bile ducts at about the 10th week, cytokeratin 19 disappears from liver cells but remains in bile duct cells. This marks an important step in the organogenesis of the liver.     

Expression of cytokeratin confers multiple drug resistance.

The cytokeratin network is an extensive filamentous structure in the cytoplasm whose biological function(s) is unknown. Based upon previous data showing the modification of cytokeratin by mitoxantrone, we investigated the ability of cytokeratin networks to influence the survival response of cells to chemotherapeutic agents. We have compared the survival of mouse L fibroblasts lacking cytokeratins with that of L cells transfected with cytokeratins 8 and 18 in the presence of chemotherapeutic drugs. The expression of cytokeratins 8 and 18 conferred a multiple drug resistance phenotype on cells exposed to mitoxantrone, doxorubicin, methotrexate, melphalan, Colcemid, and vincristine. The degree of drug resistance was 5-454 times that of parental cells, depending upon the agent used. Drug resistance could not be attributed to altered growth characteristics, altered drug accumulation, or an altered drug efflux in the transfected cells. Cytokeratin does not confer resistance to ionizing radiation, which damages DNA independently of intracellular transport mechanisms. These data suggest a role for cytokeratin networks in conferring a drug resistance phenotype.     

Clinical equivalence of two cytokeratin markers in mon-small cell lung cancer: a study of tissue polypeptide antigen and cytokeratin 19 fragments

STUDY OBJECTIVES: We have longstanding experience with tissue polypeptide antigen (TPA), a tumor marker of the cytokeratin (CK) family. In the mid-1990s, a new CK marker, CK 19 fragments (CYFRA 21-1), became popular and widely accepted. This is the first study specifically designed to compare the two markers. DESIGN: Analysis of a single institution database over a 3-year period (ie, 1998 to 2000). SETTING: Community-based hospital and second referral level institution for a province of 500,000 people. PATIENTS: The study included 180 new consecutive patients (143 men) with pathologically documented non-small cell lung cancer (NSCLC), who were observed during and after treatment, and eventually were assessed for status. INTERVENTIONS: Anthropometric, clinical, and laboratory data, including TPA and CYFRA 21-1 serum levels, were recorded prospectively. Standard nonparametric tests, Kaplan-Meyer survival analyses, Cox proportional hazards models, receiver-operating characteristic (ROC) curves, and estimates were used for statistical analysis. MEASUREMENTS AND RESULTS: A total of 1,299 twin TPA and CYFRA 21-1 serum assays (180 performed at diagnosis and 1,119 performed during or after treatment) were obtained. Intermarker correlation tests revealed incredibly high Spearman rho indexes, ranging from 0.935 at diagnosis to 0.813 to 0.921 at the different follow-up times. The substantial equivalence of the two tests explained all the other results, as follows: their similar profile of correlation with the other variables (objective treatment response: TPA rho, 0.456; CYFRA 21-1 rho, 0.463; follow-up performance status: rho range, 0.424 to 0.435); their superimposable capability to predict important clinical situations (eg, recognizing a metastatic disease at diagnosis with areas under the ROC curve of 0.742 and 0.706, respectively); their nearly identical prognostic significance (the D statistic of the goodness-of-fit of a multivariate survival model: TPA, 851.0; CYFRA 21-1, 851.6). CONCLUSIONS: In most of their traditional clinical applications the two serum tests are equivalent because of their virtual identity. We strongly recommend using a CK test in the evaluation of each NSCLC patient. The choice between TPA and CYFRA 21-1 can be based on nonclinical factors, such as the laboratory experience or preference, and the cost of the two kits.     

Expression of cytokeratin 19 during human liver organogenesis.

Immunohistochemistry with monoclonal anti-cytokeratin antibodies has revealed the presence of cytokeratin 19 in embryonic and early fetal hepatocytes. With the differentiation of bile ducts at about the 10th week, cytokeratin 19 disappears from liver cells but remains in bile duct cells. This marks an important step in the organogenesis of the liver.     

Mismatch repair protein MSH2, cytokeratin 18 and cytokeratin 20 expression: clinicopathological correlation and prognostic value in colorectal cancer patients

BACKGROUND/AIMS: The survival of patients with colorectal cancer has not varied appreciably in recent years. The knowledge that genetic factors and disruption in apoptosis could play a role in the etiology and prognosis of patients with sporadic colorectal cancer has opened up new lines of research. We have studied a group of patients with colorectal cancer and the possible influence on the prognosis of immunohistochemical MSH2, M30 cytodeath and cytokeratin 20 expression. METHODOLOGY: Forty-nine consecutive patients with unselected colorectal cancer treated by resection and with a minimum follow-up period of 5 years. Tumor specimens were evaluated by an inmunohistochemical method for MSH2, cytokeratin 18 (M30 cytodeath) and cytokeratin 20 expression and correlated with epidemiological, clinicopathological and survival data. RESULTS: Thirty-four patients were resected with curative intention. At the end of the follow-up period, 25 (51%) had died, the majority (21) in relation to tumor progression, the overall median survival period being 47.9 months (95% CI = 27-86.6). Only vascular invasion, (lower median values), (p = 0.04) was related to MSH2 expression and tumor stage (p = 0.02) with cytokeratin 20. Patients' survival was related to tumoral stage (p = 0.04) and vascular invasion (p = 0.002). MSH2 expression, apoptosis (M30 cytodeath) and cytokeratin 20 staining did not influence the prognosis of patients. CONCLUSIONS: A change in the percentage of tumoral staining cells for MSH2, M30 cytodeath and cytokeratin 20 is frequent in patients with colorectal cancer. Only vascular invasion was correlated with MSH2 expression and stage of disease with cytokeratyn 20. Survival was related to TNM stage and vascular invasion, but not to MSH2, M30 cytodeath or cytokeratin 20 expressions.     

Identification of cytokeratin antigens in Novikoff ascites hepatoma.

Further characterization of three polypeptide antigens present in Novikoff ascites hepatoma (molecular weights 39,000, 49,000, and 56,000--p39, p49, and p56, respectively), has established the identity of these proteins as cytokeratins. Two-dimensional gel electrophoresis revealed that each of the three proteins exists in several isoelectric forms, all characteristic of the keratins. The antigens p39, and p56 had amino acid compositions compatible with this family of proteins, and immunofluorescence microscopy localized the antigens on elaborate filament arrays indicative of the cytokeratins. Antibodies monospecific for the p39 antigen, purified by using affinity chromatography, were used to demonstrate that protein p39 was specific for Novikoff hepatoma cells. These results suggest that the cytokeratins contain cell-specific that can be useful as markers for differentiation and neoplasia.     

细胞角蛋白20检测在大肠癌微转移中的临床意义

近年来随着微转移在肿瘤研究领域中的广泛开展,人们开始研究大肠癌微转移,以期获得更准确,更早期的转移信息,为临床分期及预后判断、辅助治疗提供更多的依据．随着分子生物学、分子免疫学的迅速发展．使大肠癌的淋巴结、血液、骨髓及各脏器等用常规组织学难以诊断的癌细胞微转移灶的检测成为可能．细胞角蛋白20(cytokeratin-20,CK20)是新近发现的一种多肽,局限在胃肠上皮细胞,具有严格的组织特异性,几乎所有大肠癌都明显表达,优于其他标志物,尤其实用于检测大肠癌微转移．通过检测大肠癌患者淋巴结、血液、骨髓中CK20 mRNA的表达来诊断微转移,对指导临床大肠癌的分期、判断预后复发、指导治疗显示出较高的临床应用价值．微转移是一项独立的预后指标．其价值优于 Dukes分期和肿瘤分级．现综述细胞角蛋白20 检测在大肠癌微转移中的临床应用及意义研究进展．     

Aberrant cytokeratin expression and high susceptibility to apoptosis in autoimmune hepatitis

Immunopathological differences between autoimmune hepatitis (AIH) and chronic hepatitis C (CH-C) have not been well investigated. Therefore, we immunohistochemically examined the expression of various cytokeratins (CKs) not only in liver tissues of AIH but also in those of CH-C at the active stage. Furthermore, to evaluate the immune surveillance system and the susceptibility to apoptosis, immunohistochemical staining of human leukocyte antigen (HLA)-DRα, cathepsin D, B cell leukemia-2 (bcl-2), bcl-2-associated X protein (bax) and caspase 3 was also performed. Heterogeneous expression of CK 8 and CK 18 was observed in hepatocytes of AIH, while homogeneous expression was observed in hepatocytes of CH-C. Aberrant expression of CK 7 and CK 19 was observed in hepatocytes of AIH, while it was not in hepatocytes of CH-C. Expression of HLA-DRα was observed in hepatocytes of AIH but not in those of CH-C. Furthermore, expression of cathepsin D, bax and caspase 3 was much stronger in hepatocytes of AIH than in those of CH-C. These results indicate that cytoskeletal alterations of hepatocytes in AIH may increase the susceptibility to apoptosis and induce hepatocyte destruction.