Wnt通路相关因子在肝门部胆管癌细胞系FRH0201中的表达

目的 以体外培养肝门部胆管癌细胞系FRH0201为基础,研究Wnt经典通路主要因子在体外培养胆管癌细胞系中的表达,探讨Wnt信号通路在胆管癌发生、发展中的可能作用.方法 体外培养肝门部胆管癌细胞系FRH0201,利用RT-PCR技术、Western-Blot技术、免疫荧光染色技术分别从mRNA水平、蛋白水平检测Wnt经典通路相关因子及其靶基因的表达情况.结果 Wnt经典通路在体外培养肝门部胆管癌细胞系FRH0201有较高水平激活表达,包括Wnt2、Wnt3、TCF4、B-eatenin以及靶基因C-mye和cyclin D1.结论 体外培养胆管癌细胞系内存在Wnt经典通路的激活.     

HOXC8在人肝门部胆管癌中的表达及临床意义

目的 探讨HOXC8在人肝门部胆管癌组织中的表达及其对预后的影响。方法 采用免疫组化法检测49例肝门部胆管癌及对应癌旁正常组织中HOXC8蛋白的表达水平,并分析其与肝门部胆管癌患者临床病理特征及预后的关系。 结果 肝门部胆管癌组织中HOXC8蛋白的阳性表达率明显高于癌旁正常组织（57.1% vs 42.9%,P<0.001）。HOXC8表达与肝门部胆管癌患者肿瘤浸润深度、淋巴结转移、TNM分期有关（P<0.05）。单因素生存分析显示HOXC8阳性表达的肝门部胆管癌患者总生存期明显较HOXC8阴性表达患者差（12个月 vs 46个月,P<0.001）,多因素回归分析显示HOXC8是肝门部胆管癌患者的独立预后因素（P=0.039）。 结论　HOXC8在肝门部胆管癌组织中高表达,且与不良预后有关,可能作为判断肝门部胆管癌患者预后的标志物及潜在治疗靶点。     

胆管癌的蛋白质组学分析

背景与目的:胆管癌是目前常见的消化道恶性肿瘤,早期诊断困难,总体5年生存率极低,所以改善预后的关键在于早期诊断.近年来,蛋白质组学的迅猛发展,为检测肿瘤标志物提供了一种新技术.本研究通过比较胆管癌组织与正常胆管组织的蛋白质组表达差异,寻找胆管癌相关的蛋白质,选择敏感的分子标志物.方法:运用蛋白质组学技术,对16例胆管癌患者的胆管癌组织和正常胆管组织进行胶内差异双向凝胶电泳(2-DE),选择差异表达超过2倍的蛋白质进行MALDI-TOF质谱分析和生物信息学分析.结果:成功建立胆管癌组织和正常组织的双向凝胶电泳图谱,胆管癌组织和正常胆管组织平均蛋白质斑点数分别为1 087和1 048,其中表达差异超过2倍的斑点共有32个,质谱分析和数据库检索共鉴定出18种蛋白质,包括衔接蛋白成纤维细胞生长因子受体底物2(fibroblast growth factor recptor substrate 2,FRS2)、连环蛋白(catenin,beta 1)、细胞外信号调节激酶(extracellular response kinase)等.从功能上分析,这些差异蛋白质与癌细胞的发生、增殖、分化、转移等相关.结论:胆管癌组织与正常胆管组织间有18种差异蛋白质可能为研究胆管癌的生物学行为提供新的分子标记物.     

应用SELDI-TOF-MS蛋白质芯片技术筛选肝细胞性肝癌血清标志物的研究

目的：应用表面增强激光解吸离子化飞行时间质谱（SELDI-TOF-MS）和蛋白质芯片检测肝细胞性肝癌患者血清蛋白质指纹图谱，筛选特异性候选标志物。方法：应用SELDI-TOF-MS蛋白质芯片技术检测50例肝细胞性肝癌患者和50例性别、年龄匹配的正常健康人血清，获得WCX2蛋白质芯片蛋白表达图谱。用Bio Marker Wizard软件分析差异蛋白并经数据库搜索初步鉴定。结果：应用弱阳离子交换芯片（WCX2类）在未经治疗肝细胞性肝癌患者、经介入治疗后的肝细胞性肝癌患者和正常人血清中检测到在不同质荷比处有7个差异蛋白质峰．其蛋白质含量差异有显著性（P〈0．01），经蛋白质数据库搜索，与这七种差异蛋白质分子量最接近的蛋白质分别为：Galanin相关肽、Pro-neuregulin-4蛋白、小诱导细胞因子A15前体、9kDa蛋白、CSL型锌指包涵蛋白1、线粒体铰链蛋白、Actin相关蛋白。结论：应用SELDI-TOF-MS筛选肝细胞性肝癌患者血清特异性生物标志物的方法快速、有效，操作自动化，检测到的这7个差异蛋白质可能是肝细胞性肝癌患者血清特异性生物标志物。     

黄曲霉毒素B1诱导的恶性转化肝细胞miRNA表达谱的变化

目的：检测黄曲霉毒素B1（aflatoxin B1,AFB1）诱导的恶性转化肝L02细胞（L02T）的miRNA表达谱,寻找差异表达的miRNA。方法：以含有AFB1的培养液多次间歇性染毒L02细胞获得转化细胞L02T,通过miRNA芯片技术检测和分析对照L02和转化L02T细胞的miRNA表达谱;用实时荧光定量PCR方法对芯片结果加以验证;采用TargetScan软件预测miRNA可能调控的靶基因。结果：获得2组细胞856个miRNA的表达谱,在25个表达差异显著的miRNA中,15个表达上调,10个表达下调;用定量RT-PCR对芯片结果中表达差异的miR-320a、miR-638和miR-98进行验证,并对其中上调显著的miR-638进行生物信息学分析,预测到4个与肝癌相关的潜在靶基因。结论：在AFB1诱导的恶性转化肝L02细胞中筛查出25个表达差异显著的miRNA,差异表达的miRNA可能在细胞恶性转化过程中起重要作用。     

多药耐药基因mdr-1在肝门部胆管癌组织中的表达及其意义

目的：检测肝门部胆管癌新鲜组织中多药耐药基因（ｍｄｒ－１）ｍＲＮＡ表达，探讨其与肝门部胆管癌临床病理间的关系。方法应用逆转录多聚酶链反应（ＲＴ－ＰＣＲ）对２６例术前未作治疗的肝门部胆管癌，１２例正常胆管组织的ｍｄｒ－１ ｍＲＮＡ表达进行了检测，并与癌组织的分化程度，病理类型、部位、浸润转移作对比研究。结果：２６例肝门部胆管癌组织中ｍｄｒ－１ ｍＲＮＡ的阳性表达率为６５．４％（１７／２６），正常胆管     

紫杉醇抗胆管癌细胞差异表达蛋白的分离、质谱鉴定及生物信息学分析

目的：筛选紫杉醇诱导胆管癌细胞凋亡的相关蛋白并鉴定差异表达的蛋白点以寻找抗胆管癌药物作用的新靶点,筛选胆管癌新的肿瘤标志物,为胆管癌的早期诊断、疗效观察提供基础理论依据。方法：应用比较蛋白质组学技术对紫杉醇诱导凋亡的胆管癌QBC939细胞进行蛋白分离和蛋白表达谱差异分析,筛选并鉴定其相关蛋白并测定其胶内酶解后的肽指纹图谱。结果：本研究发现对照组和实验组蛋白点匹配率分别为（90．1±0．14）％和（87．1±0．22）％（P〈0．05）,匹配后在干预组中共发现68个差异表达蛋白点,其中47个表达下调和21个上调;共获得28张PMF,初步质谱鉴定发现11个与凋亡相关的差异表达蛋白,其中6种蛋白表达上调,5种蛋白表达下调。结论：应用蛋白质组学技术分析所获得的68个差异蛋白质点可能在QBC939细胞发生凋亡过程中发挥重要作用;被鉴定出的11个与细胞凋亡相关的差异蛋白点提示紫杉醇是通过多个重要蛋白,发挥其诱导细胞凋亡的抗癌作用机制的。     

Connexin43在肝门部胆管癌中的表达及其意义

目的 探讨肝门部胆管癌组织中Connexin43的表达及其意义。方法 应用免疫组化法检测Connexin43蛋白在49例肝门部胆管癌组织及癌旁组织中的表达情况,并分析其与肝门部胆管癌临床病理特征及总生存期（OS）和无病生存期（DFS）的关系。结果 Connexin43蛋白在49例胆管癌组织中的阳性表达率为57.1％（28／49）,而在癌旁黏膜组织中不表达或微弱表达。Connexin43蛋白表达与性别、年龄、淋巴结转移、术式和神经侵犯无关,而与TNM分期、组织分化和肿瘤大小相关。肝门部胆管癌组织中Connexin43阳性表达者的中位DFS和OS分别为11.5个月和20.7个月,阴性者分别为21.4个月和38.4个月,两者差异均有统计学意义（P＜0.05）。Cox多因素回归分析显示,Connexin43蛋白表达是预测肝门部胆管癌复发和生存的独立因素。结论 Connexin43蛋白与肝门部胆管癌的发生、发展密切相关,是判断胆管癌预后的重要指标。     

紫杉醇抗胆管癌细胞差异表达蛋白的分离、质谱鉴定及生物信息学分析

目的:筛选紫杉醇诱导胆管癌细胞凋亡的相关蛋白并鉴定差异表达的蛋白点以寻找抗胆管癌药物作用的新靶点,筛选胆管癌新的肿瘤标志物,为胆管癌的早期诊断、疗效观察提供基础理论依据.方法:应用比较蛋白质组学技术对紫杉醇诱导凋亡的胆管癌QBC939细胞进行蛋白分离和蛋白表达谱差异分析,筛选并鉴定其相关蛋白并测定其胶内酶解后的肽指纹图谱.结果:本研究发现对照组和实验组蛋白点匹配率分别为(90.1±0.14)%和(87.1±0.22)%(P＜0.05),匹配后在干预组中共发现68个差异表达蛋白点,其中47个表达下调和21个上调;共获得28张PMF,初步质谱鉴定发现11个与凋亡相关的差异表达蛋白,其中6种蛋白表达上调,5种蛋白表达下调.结论:应用蛋白质组学技术分析所获得的68个差异蛋白质点可能在QBC939细胞发生凋亡过程中发挥重要作用;被鉴定出的11个与细胞凋亡相关的差异蛋白点提示紫杉醇是通过多个重要蛋白,发挥其诱导细胞凋亡的抗癌作用机制的.     

Differential Proteomics in Malignant and Normal Liver Cell Lines

Objective： To detect differential protein expression in malignant and normal liver cell lines in vitro using the SELDI ProteinChip platform, for investigating the pathogenesis of liver cancer. Methods： Two cell lines, human normal liver cell line L02 and hepatoma cell line SMMC-7721 were cultured routinely, harvested in good condition and lysed. After quantification, the supernatant of the lysate was tested by IMAC3 （Immobilized Mental Affinity Capture） and WCX2 （Weak Cation Exchange） chips on the SELDI-TOF-MS ProteinChip reader. Results： Protein expression differed between the malignant and normal liver cell lines. A total of 20 differentially expressed proteins were found, among which, 7 were captured by the IMAC3 chip and 14 by the WCX2 chip. Peaks at 5,419, 7,979 and 11,265 Da were higher and at 8,103, 8,492, 10,160 and 11,304 Da lower in SMMC-7721 cells by the IMAC3 chip; peaks at 7,517, 7,945 and 7,979 Da were higher and at 5,061, 5,551, 5,818, 7,439, 9,401,10,100, 10,312, 11,621, 11,662, 11,830 and 12,772 Da lower in SMMC-7721 cells by the WCX2 chip. Interestingly, both chips captured the 7,979 Da peak. In addition, the 11,081 Da peak corresponded precisely with the molecular mass of the calcium binding protein S100A10, which may participate in the formation of liver cancer in association with p36. Conclusion： Detecting differential protein expression in malignant and normal liver cell lines using the SELDI ProteinChip platform was simple, sensitive and repeatable. The results we obtained can serve as a basis for investigating the pathogenesis of liver cancer and aid the discovery of new therapeutic targets.     

Fiber confocal back-scattering micro-spectral analysis for single cell

A fiber confocal back scattering micro-spectrometer (FCBS) was established, which combined fiber confocal microscopy with light scattering spectroscopy (LSS) for early diagnosis of the cancer cell at cellular level. An adherent monolayer human normal gastric epithelium line GES-1 and a carcinoma cell line NCI-N87 as well as a normal liver cell line L02 and a high-metastatic-potential hepatocellular carcinoma cell line HCC-LM3 were measured respectively. The spectral results showed that micro-back-scattering intensity from GES-1 cell and L02 cell possessed interesting oscillations in contrast to NCI-N87 and HCC-LM3 cells. There was significant difference between the spectra of the normal and the cancer cells (p<0.001). This demonstrates that the FCBS system here is able to distinguish dysplastic cells from normal cells at cellular level.     

A case of diffuse type of combined hepatocellular and cholangiocellular carcinoma initially presented as a localized small nodule

A combined hepatocellular and cholangiocellular carcinoma of diffuse type in a Japanese man is described. A small localized solitary tumor apparently grew rapidly into a diffuse-type carcinoma, and the liver weight increased about 4-fold during the last two months. The clinical course of this case was as expected for a diffuse type of hepatocellular carcinoma except that unusually high levels of serum carcinoembryonic antigen were found. The patient died of hepatic failure with systemic bleeding five months later. At autopsy, multiple small nodules were suspected to be intrahepatic metastatic foci because portal tumor thrombus was observed in the right antero-superior segment where the initial tumor was localized. Histologically, the tumor had components of both hepatocellular and mucin-producing cholangiocellular carcinoma. This is believed to be the first report on a diffuse type of combined hepatocellular and cholangiocellular carcinoma initially presented as a localized small nodule.     

应用SELDI-TOF-MS技术分析南通地区食管癌血清差异表达蛋白

目的:应用SELDI-TOF-MS技术寻找食管鳞癌血清中相关差异表达蛋白。方法:应用表面增强激光解析离子化飞行时间质谱(SELDI-TOF-MS)技术,用WCX2芯片分析了24对食管鳞癌和正常对照的血清蛋白表达谱。结果:与正常对照相比,发现其中6个血清蛋白在食管鳞癌和正常对照组的差异有统计学意义(P<0.05),其中在食管鳞组低表达的有M/Z为4623,5012,5809和9422,而高表达的有5910,11681。结论:SELDI-TOF-MS技术是一种快速、简便易行且高通量的分析方法,不仅能直接筛选出食管癌患者血清中差异表达的潜在标记物,而且可能具有较好的临床应用前景。     

Combined hepatocellular-cholangiocarcinoma. A histologic and immunohistochemical study

Combined hepatocellular-cholangiocarcinoma is a rare form of primary liver cancer showing features of both hepatocellular and biliary epithelial differentiation. In a review of 24 cases of this tumor, three histologic types were encountered. Four cases were Type I or "collision tumors," apparently a coincidental occurrence of both hepatocellular carcinoma and cholangiocarcinoma in the same patient. Twelve cases were Type II or "transitional tumors," in which there were areas of intermediate differentiation and an identifiable transition between hepatocellular carcinoma and cholangiocarcinoma. Eight cases were Type III or "fibrolamellar tumors" which resembled the fibrolamellar variant of hepatocellular carcinoma but which also contained mucin-producing pseudoglands. Type III tumors differ from other combined tumors, occurring at a younger age, in the absence of cirrhosis, and having a slightly longer survival. Immunohistochemical (immunoperoxidase) staining for intracellular antigens showed that alpha-fetoprotein is a fairly specific, although insensitive, marker of hepatocellular differentiation in primary liver cancers, being present in 50% of typical hepatocellular carcinomas and in hepatocellular areas in 29% of combined tumors, but in no cholangiocarcinomas or cholangiocellular areas of combined tumors. Keratin is a good marker of biliary epithelial differentiation, being found in 90% of cholangiocarcinomas and in 52% of combined hepatocellular cholangiocarcinomas, but in no hepatocellular carcinomas. Alpha-1-antitrypsin, fibrinogen, IgG, and carcinoembryonic antigen may be found in both hepatocellular carcinoma, cholangiocarcinoma, and in combined tumors; these antigens are therefore of limited use in differential diagnosis.     

Combined hepatocellular and cholangiocarcinoma: a clinicopathologic study of 26 resected cases

BACKGROUND: Combined hepatocellular and cholangiocarcinoma (cHCC-CC) is an uncommon subtype of primary liver cancer, the clinicopathological features of which have rarely been reported in detail. The aim of this study was to clarify the characteristics of cHCC-CC in comparison with hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC). METHODS: The clinicopathological features of 26 cHCC-CC patients, who were surgically treated, were reviewed by comparing them with the features of patients suffering from ordinary hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC). RESULTS: The cHCC-CC patients showed greater similarity with HCC patients than with CC patients with regard to male/female ratio, status of hepatitis viral infection, serum alpha-fetoprotein (AFP) level, and non-tumor liver histology. The disease stage of the cHCC-CC patients was more advanced than that of either the HCC or CC patients. The cHCC-CC tumors were significantly more invasive to the portal vein than the HCC tumors and were comparable to the CC tumors. The overall 3-, 5-, and 10-year survival rates and the median survival times (95% confidence interval) were 34.6%, 23.1%, 11.5% and 1.8 (0.7-3.0) years for cHCC-CC patients, 86.7%, 66.2%, 46.8% and 4.6 (4.3-5.0) years for HCC patients, and 68.5%, 32.3%, 23.9% and 1.9 (1.1-2.7) years for CC patients, respectively. Survival of patients with cHCC-CC was significantly poorer than that of HCC or CC patients. Among the 26 patients, six survived for >5 years. CONCLUSIONS: In most cases, cHCC-CC seems to be a variant of ordinary HCC with cholangiocellular features, rather than a true intermediate disease entity between HCC and CC. The surgical approach is recommended for selected patients with cHCC-CC.     

FOXK1对肝癌高转移细胞系Huh7侵袭和迁移能力的影响

目的 探讨叉头框转录因子1(FOXK1)对肝癌高转移细胞Huh7侵袭和迁移能力的影响。方法 采用实时定量逆转录聚合酶链式反应(qRT-PCR)检测40例肝癌组织及其癌旁正常组织FOXK1 mRNA的表达,qRT-PCR和Western blot检测正常肝细胞HL7702、肝癌细胞BEL7402和肝癌高转移细胞Huh7中FOXK1 mRNA和蛋白的表达。小RNA干扰技术下调FOXK1表达后,qRT-PCR和Western blot检测肝癌高转移细胞Huh7 FOXK1 mRNA和蛋白的表达,Transwell实验检测其侵袭和迁移能力。 结果 qRT-PCR检测结果显示,肝癌组织中FOXK1 mRNA的表达高于癌旁正常组织(2.87±0.21 vs 1.35±0.11,P=0.004);qRT-PCR和Western blot检测结果显示,相比正常肝细胞HL7702,肝癌细胞BEL7402和肝癌高转移细胞Huh7中FOXK1 mRNA和蛋白表达升高(P<0.001)。下调FOXK1表达后,肝癌高转移细胞Huh7中FOXK1 mRNA和蛋白表达显著下调(P<0.01),侵袭和迁移细胞数目明显减少(P<0.01)。结论 下调FOXK1表达能抑制肝癌高转移细胞Huh7侵袭和迁移能力。     

黏着斑激酶对肝癌细胞生物学行为的影响

目的：探讨降低黏着斑激酶（FAK）的表达对FAK高表达的人肝癌细胞株SMMC－7721恶性生物学行为影响。方法用Western杂交法比较不同肝癌细胞株FAK含量的差别,构建FAK反义质粒,转染至FAK高表达的细胞株,研究其多种细胞生物学行为的变化。结果SMMC－7721FAK含量比正常人肝细胞L02明显增高,转染FAK反义质粒后、SMMC－7721细胞生长受到抑制,软琼脂培养克隆形成率下降;细胞周期分析,S期细胞下降15％;黏附能力下降;细胞表面整连蛋白含量不变。结论在SMMC－7721中存在FAK过表达,降低FAK表达能部分逆转该肝癌细胞株的恶性表型。