肝细胞癌组织中Cyr61、VEGF和HSP27的表达及意义

目的观察富半胱氨酸蛋白61（Cyr61）、血管内皮生长因子（VEGF）和热休克蛋白27（HSP27）在肝细胞癌（HCC）中的表达,分析其与HCC病理学特征和转移的关系。方法采用免疫组化SP法分别检测34例HCC患者肝癌组织和癌旁肝组织中的Cyr61、VEGF和HSP27表达情况,并与HCC患者的临床、病理学特征进行相关性分析。结果VEGF和HSP27在肝癌组织中的表达显著高于癌旁组织（P〈0．05）,Cry61在癌旁组织中的表达显著高于肝癌组织（P〈0．05）;HCC转移者的Cyr61、VEGF和HSP27阳性表达率明显高于无转移者（P〈0．06）。结论Cyr61、VEGF和HSP27的表达与HCC转移密切相关;联合检测Cyr61、VEGF和HSP27的表达情况对判断HCC转移和预后有—定的临床价值。     

肝细胞癌热休克蛋白27,70,90α的表达意义

目的探讨HSP27,HSP70和HSP90α在肝细胞癌(HCC)中的表达及其意义.方法采用免疫组化技术对44例HCC和癌旁组织中HSP27,HSP70和HSP90α的表达进行检测.结果 HCC和癌旁组织中HSP27阳性率分别为18%和30%,HSP70阳性率分别为68%和27%,HSP90α阳性率分别为63%和23%.HCC中HSP70和HSP90α阳性率明显高于癌旁组织(X12=7.3,X22=7.8,P＜0.01).而HSP27阳性率变化不大(X2=1.6,P＞0.05).HSP70和HSP90α在分化较好和分化不良的HCC中的阳性率分别为54%,85%和50%,80%,两者相比差异显著(X12=4.5,X22=4.2,P＜0.05),但与癌周淋巴细胞浸润(X12=3.2,X22=1.4,P＞0.05)和转移(X12=2.3,X22=2.7,P＞0.05)无关.结论 HCC是HSP70和HSP90α高表达肿瘤.HSP70和HSP90α与HCC分化有关,在HCC发生和发展中起重要作用.蒋业贵,王宇明,李奇芬.肝细胞癌热休克蛋白27,70,90α的表达意义.世界华人消化杂志,2001;9(7):755-758     

肝细胞癌组织中HSP27、VEGF的表达变化及意义

目的观察肝细胞癌（HCC）组织中热休克蛋白27（HSP27）和血管内皮生长因子（VEGF）的表达变化,并探讨其意义。方法采用免疫组化法检测68例HCC组织及癌旁组织中的HSP27、VEGF。结果 HCC组织中HSP27、VEGF阳性表达率分别为75.0%、72.0%,癌旁组织中分别为61.8%、36.8%,两者比较,P均〈0.05。HCC组织中,HSP27表达与乙肝表面抗原（HBsAg）阳性、淋巴结转移、肿瘤分化程度有关（P均〈0.05）,VEGF表达与肿瘤直径、淋巴结转移有关（P均〈0.05）;HCC组织中,HSP27与VEGF的表达呈正相关（r=0.248,P均〈0.05）。结论 HCC组织中HSP27、VEGF表达均升高;二者在HCC发生发展中具有重要作用,检测HSP27、VEGF对HCC的预后判断有一定参考价值。     

蛋白翻译起始因子C_2在原发性肝细胞肝癌中表达的意义

目的研究C_2mRNA及其蛋白在原发性肝细胞肝癌(HCC)中的表达及意义。方法用原位杂交检测C_2mRNA住21例HCC及其癌旁组织中的表达,ABC免疫组化法检测C_2蛋白在60例HCC及其42例癌旁组织中的表达,Western blot法检测C_2蛋白在HCC及其癌旁组织中的表达。结果 21例HCC及其癌旁组织中,C_2mRNA阳性率分别占23.8％(5/21)和85.7％(18/21),60例HCC及42例癌旁组织中,C_2蛋白阳性分别占27.3％(17/60)和83.3％(35/42),27例肝硬变中,C_2蛋白阳性率为77.8％(21/27)。χ~2检验:C_2mRNA及其蛋白在HCC癌旁组织中表达明显高于癌组织(P<0.001);C_2蛋白在肝硬变中的表达明显高于HCC癌组织(P<0.01);C_2的表达与患者年龄、HBsAg及AFP有明显关系(P<0.05);而与癌组织的分化程度、肿瘤大小、淋巴转移无关;Western blot与免疫组化结果一致。结论 C_2基因表达下调可能与HCC的发生、发展及早期诊断有关。     

转录组测序结合蛋白组学技术筛选肝癌转移基因

目的:通过高通量转录组测序和相对定量血清蛋白组学技术筛选肝癌转移相关的关键基因.方法:利用Ion Proton.测序仪比较人肝癌细胞株(Smmc-7721)和正常人肝细胞株(L-02)的差异表达基因,对差异表达基因进行聚类分析、GO功能注释和富集分析,获得肝癌细胞转移相关基因;收集10例肝细胞癌(hepatocellular carcinoma,HCC)患者血清及匹配10例正常人血清,通过相对和绝对定量的同位素标记(isobaric tags for relative and absolute quantitation,iTRAQ)联合基质辅助激光解吸电离串联飞行时间质谱(ma t r ixassisted laser desorption/ionization tandemtime of flight mass spectrometry,MALDITOF/MS)检测肝癌与正常对照组血清差异表达蛋白;将两组学结果进行交集分析,确定肝癌转移关键基因,并在76例HCC和癌旁组织中进行免疫组织化学验证.结果:对肝癌细胞及正常肝细胞进行转录组测序分析,共得到肝癌细胞差异表达基因618个,生物信息学分析差异表达基因主要聚集在转移相关、转录因子相关、氧化还原过程等14个分子功能,其中转移相关功能基因所占比例最大,达15.05%(93/618);血清蛋白组学分析共得到69个肝癌血清差异表达蛋白,其中在肝癌组上调33个,下调36个;将转录组测序筛选的与转移功能相关基因与蛋白质组学进行交集分析,发现有3个共同交集的差异因子,其中差异最明显的是肝癌中表达上调的热休克蛋白90 AA1(heat shock protein 90 AA1,HSP90AA1);免疫组织化学验证结果显示,HSP90α表达强阳性在门脉转移和无门脉转移HCC组织中的比例分别为66.7%(16/24)和25%(13/52)(P0.005),HSP90α在有门脉转移的肝癌组织表达明显增高.结论:利用转录组结合血清蛋白组策略,发现HSP90AA1可能是在肝癌转移重要基因.     

p27、Cyclin A在肝细胞癌中的表达及其意义

细胞周期调节因子p27及Cyclin A蛋白水平升高或基因改变与多种肿瘤的发生有关。用免疫组织化学SABC法，联合检测45例肝细胞癌(HCC)组织及30例癌旁肝组织中p27及Cyclin A的表达，以探讨其与HCC发生、发展及侵袭转移的关系。     

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.     

肝细胞癌中磷脂酰肌醇蛋白聚糖3、热休克蛋白和谷氨酰胺合成酶的表达及其临床意义

目的探讨磷脂酰肌醇蛋白聚糖3(GPC3)、热休克蛋白70(HSP70)和谷氨酰胺合成酶(GS)在肝细胞癌中的表达及临床意义。方法运用组织芯片技术,通过免疫组织化学Envision法检测182例肝细胞癌和92例癌旁肝组织中GPC3、HSP70和GS的表达情况,并结合临床病理因素进行分析。结果 GPC3、HSP70和GS在肝细胞癌中表达均明显高于癌旁肝组织(P<0.05),GPC3表达与肝细胞癌分化程度、脉管内瘤栓、TNM分期与转移显著相关(P<0.05);HSP70和GS表达与肝细胞癌TNM分期、转移和术后复发显著相关(P<0.05)。结论 GPC3、HSP70和GS在肝细胞癌的发生、发展中起着不同程度的作用。联合检测GPC3、HSP70和GS可能有助于判断肝细胞癌的恶性程度、转移潜能及预后分析。     

CDK5在肝细胞癌中的异常表达

目的:研究周期素依赖性激酶5(cyclin-dependent kinase5.CDK5)在肝细胞癌(hepatocellular carcinoma,HCC)发生发展中的异常表达.方法:应用反转录聚合酶链式反应(RT-PCR)检测CDK5 mRNA的表达水平;免疫组织化学技术检测CDK5蛋白在正常肝组织、肝硬化和HCC组织中的表达:分析CDK5与临床病理学特征的关系.结果:CDK5 mRNA在HCC中呈上调表达,阳性表达率在正常肝组织、肝硬化和HCC组织中分别为35.3%、64.3%、89.7%;正常肝组织与HCC组织有显著差异(P<0.05),肝硬化组织与HCC组织也有显著差异(P<0.01).CDK5蛋白在HCC中高表达,阳性表达率在人正常肝组织、肝硬化和HCC组织之间分别为29.4%.64.3%,89.7%,三组之间差异有统计学意义(x2=58.095,P<0.01).HCC组织中CDK5蛋白表达强度与肿瘤的病理学分级显著相关(x2=19.330,P<0.01).结论:CDK5在HCC中上调表达,他的异常表达在HCC的发生发展过程中发挥重要的作用,且与肿瘤的分化程度相关.     

HCC患者癌组织VDR mRNA和VDR蛋白表达水平研究*

目的 研究肝细胞癌（HCC）患者肝癌组织维生素D受体（VDR）mRNA和VDR蛋白表达水平的变化。方法 2014年3月～2016年3月我院诊治的乙型肝炎病毒相关性HCC患者49例,同期健康肝移植供体者50例,分别检测肝组织VDR mRNA和VDR蛋白水平。结果 HCC组织VDR mRNA水平为（2.8±0.3）,VDR蛋白水平为（1.2±0.5）,分别显著高于健康人的（1.6±0.2）和（0.4±0.1）,差异有统计学意义（P＜0.05）。结论 HCC患者癌组织VDR mRNA及其蛋白表达水平显著高于正常人,其意义还有待于进一步研究。     

Proteomic analysis on metastasis-associated proteins of human hepatocellular carcinoma tissues

Purpose: A comparative proteomic approach was used to identify and analyze proteins related to metastasis of hepatocellular carcinoma (HCC). Methods: Proteins extracted from 12 HCC tissue specimens (six with metastases and six without) were separated by two-dimensional gel electrophoresis (2-DE). The protein spots exhibiting statistical alternations between the two groups through computerized image analysis were then identified by mass spectrometry. In addition immunohistochemistry (IHC), Western blotting and RT-PCR were performed to verify the expression of certain candidate proteins. Results: 16 proteins including HSP27, S100A11, CK18 were annotated by mass spectrometry, relevant to chaperone function, cell mobility, cytoskeletal architecture, respectively. Most were previously unconnected with metastasis of HCC. Of these HSP27 was found overexpressed consistently in 2-DE patterns of all metastatic HCC tissues compared with nonmetastatic ones. IHC and Western blotting of HCC tissues confirmed this difference while RT-PCR did not. Conclusion: There are various proteins joined together in HCC metastasis. The overexpression of HSP27 may serve as a biomarker for early detection and therapeutic targets unique to the metastatic phenotype of HCC.     

Differential proteomic analysis of a highly metastatic variant of human breast cancer cells using two-dimensional differential gel electrophoresis

Distant metastasis represents the major lethal cause of breast cancer. To understand the molecular mechanisms of breast cancer metastasis and identify markers with metastatic potential, we established a highly metastatic variant of parental MDA-MB-231 cells (MDA-MB-231HM). Using two-dimensional electrophoresis (2-DE), we performed a proteomic comparison of the two kinds of cells. As much as 51 protein spots were differentially expressed between the selected variant and its parental counterpart in at least 3 experiments. Ten unique proteins were identified using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS), liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), and database searching software. Among them, nine proteins were up-regulated in MDA-MB-231HM cells, including Macrophage-capping protein (CapG), Galectin-1, Chloride intracellular channel protein 1, Endoplasmic reticulum protein ERp29 precursor, Stathmin-1 (STMN1), Isoform 1 of uridine–cytidine kinase 2(UCK2), Rho GDP-dissociation inhibitor 2 (ARHGDIB), isocitrate dehydrogenase [NADP] cytoplasmic (IDH1), and N-myc downstream regulated gene 1 (NDRG1) protein. Only transgelin-2 was down-regulated. Differential expression was confirmed for three proteins including CapG, STMN1, and transgelin-2 by Western blotting analysis. Transgelin-2 was chosen for further verification by immunohistochemistry. The results suggested that 2-DE would be an efficient way to screen the proteins responsible for specific biological function. Furthermore, the findings imply that different proteins may be involved in the metastatic process in breast carcinomas.     

肝内转移和多中心发生的多结节肝癌蛋白质表达谱的差异分析

目的 比较分析多结节肝癌的肝内转移癌灶和多中心发生癌灶的蛋白质表达谱,为更加准确的分子分型方法提供实验依据. 方法对5例肝内转移(IM)和6例多中心发生(MO)的多结节肝癌按照结节大小分为IM1、IM2、MO1、MO2四组,联合双向凝胶电泳与质谱技术分析多结节肝癌的蛋白质表达谱;并用Westem blot验证质谱结果.结果 双向凝胶电泳联合质谱技术鉴定IM1、IM2、MO1、MO2四组共30个差异蛋白质点,确认为25种差异表达的蛋白质;Gene Ontology 分类显示其与细胞运动、信号转导、氧化还原、脂类代谢、氨基酸代谢等有关. 结论多结节肝癌肝内转移和多中心发生的蛋白质表达不同;双向凝胶电泳联合质谱技术可作为二者分子分型的方法,有利于临床医师对肝癌患者实施治疗和判断预后.     

特发性间质性肺炎蛋白质组学研究及Annexin Ⅱ表达验证

目的 应用蛋白质组学方法筛选出特发性间质性肺炎(idiopathic interstitial pneumonia,IIP)相关蛋白质,为阐明IIP的发病机制和预后提供理论依据.方法 收集8例通过局部麻醉小切口开胸肺活检获取的新鲜肺组织及5例远离肺部良性病变的肺组织,采用双向凝胶电泳(2-DE)分离组织总蛋白,运用Imagemaster 2D 5.0图像分析软件识别差异表达的蛋白质点,应用基质辅助电离解析飞行时间质谱获取肽质量指纹图谱,检索数据库鉴定差异表达的蛋白质点,明确其生物学功能.利用Western blot和RT-PCR技术检测差异蛋白在IIP患者肺组织和正常肺组织的表达.结果 建立双向电泳图谱,鉴定出7个表达量有明显差异的蛋白质点,包括热休克蛋白70、AnnexinⅡ和Haptoglobin等.Western blot和RT-PCR技术证实IIP组AnnexinⅡ表达下调,与2-DE结果一致.结论 建立重复性较好的IIP患者肺组织与正常肺组织双向电泳图谱,并鉴定出一些与IIP发病机制相关的蛋白质.本实验所证实的AnnexinⅡ差异表达蛋白质可能通过引起纤溶系统的失衡来参与IIP的发生和发展,从而为进一步阐明IIP的发病机制提供重要方法和新线索.     

Expression of heat shock proteins （HSP27, HSP60, HSP70, HSP90,GRP78, GRP94） in hepatitis B virus-related hepatocellular carcinomas and dysplastic nodules

AIM: Expression of heat shock proteins (HSPs) is frequently up-regulated in hepatocellular carcinoma (HCC), which evolves from dysplastic nodule (DN) and early HCC to advanced HCC. However, little is known about the differential expression of HSPs in multistep hepatocarcinogenesis. It was the purpose of this study to monitor the expression of HSPs in multistep hepatocarcinogenesis and to evaluate their prognostic significance in hepatitis B virus (HBV)-related HCC. METHODS: Thirty-eight HCC and 19 DN samples were obtained from 52 hepatitis B surface antigen-positive Korean patients. Immunohistochemical and dot immunoblot analyses of HSP27, HSP60, HSP70, HSP90, glucose regulated protein (GRP)78, and GRP94 were performed and their expression at different stages of HCC development was statistically analyzed. RESULTS: Expression of HSP27, HSP70, HSP90, GRP78, and GRP94 increased along with the stepwise progression of hepatocarcinogenesis. Strong correlation was found only in GRP78 (Spearman's r= 0.802). There was a positive correlation between the expressions of GRP78, GRP94, HSP90, or HSP70 and prognostic factors of HCC. Specifically, the expression of GRP78, GRP94, or HSP90 was associated significantly with vascular invasion and intrahepatic metastasis. CONCLUSION: The expressions of HSPs are commonly up-regulated in HBV-related HCCs and GRP78 might play an important role in the stepwise progression of HBV-related hepatocarcinogenesis. GRP78, GRP94, and HSP90 may be important prognostic markers of HBV-related HCC, strongly suggesting vascular invasion and intrahepatic metastasis.     

黏附分子CD44的表达及其糖基化与肝癌转移的相关性

目的 探讨CD44S及其变异体CD44v分子的表达和其糖基化与肝癌细胞侵袭转移的关系. 方法 用免疫组织化学法、量子点、RT-PCR、Western blot、细胞免疫荧光染色、甲基化特异性聚合酶链反应等技术检测转移与非转移性肝癌组织、不同转移潜能人肝癌细胞株中CD44S及其变异体CD44v的定位与表达;并应用多重凝集素印迹法检测这些细胞株中CD44v6的糖基化差异.组间均数比较应用方差分析及t检验,多组间等级资料的比较采用Wilcoxon秩和检验,各组间率的比较采用x2检验.结果 免疫组织化学结果显示,CD44S蛋白定位以细胞质为主; CD44v3、CD44v4/5蛋白定位于细胞膜与细胞质;而CD44v6主要定位于细胞膜.组织芯片结果显示,相对于CD44S及其他CD44v,CD44v6在转移组的表达水平高于非转移组(阳性率为75％与46％),差异具有统计学意义(x2=8.828,P＜0.05);量子点检测(t=2.392,P＜0.05)与血清学检测(t=2.56,P＜0.05)也证实这一结果.Western blot结果显示,CD44v6的表达与肝癌细胞转移潜能呈正相关.此外,在MHCC97L、MHCC97H肝癌细胞中CD44v6基因启动子发生不完全甲基化,而在Hep3B细胞中则发生完全甲基化.而且,相对于Hep3B细胞,MHCC97L及MHCC97H细胞中CD44v6蛋白对朝鲜槐凝集素、黑接骨木凝集素及麦胚凝激素的亲和力较高. 结论 在CD44S及其变异体CD44v中,CD44v6蛋白的高表达与肝癌转移潜能的增强呈正相关;其高表达可能与基因启动子低甲基化有关.此外,CD44v6蛋白唾液酸寡糖链的增加可能与肝癌细胞转移潜能增高有关.     

Differential proteomic analysis of human colorectal carcinoma cell lines metastasis-associated proteins

Purpose Metastasis is a common phenomenon and the major lethal cause of colorectal carcinoma (CRC). To better comprehend the mechanism underlying CRC metastasis and to search for potential markers for predicting CRC metastasis, two CRC cell lines with different metastatic potentials, SW480 and SW620, were investigated by phenotypic analyses and proteomics technologies. Methods The surgical orthotopic implantation (SOI) technique was originally used to develop a reproducible colorectal cancer model in nude mice with stable tumor growth and metastasizing course. Furthermore, differential proteome analysis of two CRC cell lines was conducted using two-dimensional (2-D) gel electrophoresis combined with matrix-assisted laser desorption/time of flight (MALDI-TOF) mass spectrometry. Results Among the differential spots, 12 protein spots (11 proteins) were further identified using in-gel tryptic digestion and peptide mass fingerprinting (PMF). Interestingly, most of these proteins we identified have been reported to be associated with distinct aspect of tumor metastasis to some extent. Our immunohistochemistry assays of colorectal cancer revealed that heat shock protein (HSP) 27 overexpression relates to metastatic behavior of CRC cell. Conclusions The protein profile between two colorectal cell lines with different potential metastasis displayed obvious differences. The results imply that various different proteins may lead to CRC metastasis together. HSP27 overexpression played an important role in metastasis and progression of CRC.     

人肝癌细胞转移相关蛋白膜联蛋白Ⅱ的筛查与分析

目的应用糖组学方法筛查肝癌转移相关的异常核心岩藻糖基化蛋白,分析膜联蛋白Ⅱ（annexinⅡ）与肝癌细胞转移的关系。方法双向电泳（2-DE）、凝集素亲和印迹及沉淀联合质谱筛查并验证肝癌转移相关核心岩藻糖基化蛋白；实时荧光定量PCR、细胞免疫荧光和Western blot测定annexinⅡ基因和蛋白表达。结果不同转移潜能人肝癌细胞有核心岩藻糖基化蛋白差异表达,鉴定并验证annexinⅡ岩藻糖基化与肝癌转移相关；它分布于细胞质,在MHCC97-L和MHCC97-H中基因和蛋白质表达较Hep3B高。结论AnnexinⅡ转录、翻译水平和核心岩藻糖基化增加可能与肝癌转移潜能相关。     

Establishment and characteristics of human hepatocellular carcinoma cells with metastasis to lymph nodes

BACKGROUND/AIMS: Although the mechanism of cancer metastasis has been gradually elucidated, less is known concerning the characteristics of human hepatocellular carcinomas (HCCs) with metastatic potential. We examined the expression of molecules that mediate cell-cell or cell-substrate interaction, nm23-H1 expression, and ultrastructural features of several human HCC cell lines. METHODOLOGY: Expression of E-cadherin, integrin (alpha3beta1), intracellular adhesion molecule-1 (ICAM-1), and nm23-H1 protein was analyzed by immunocytochemistry or Western blotting, and ultrastructural features were further studied by electron microscopy in 4 human HCC cell lines, PLC/PRF/5, HuH-7, OCUH-16, and Nuk-1 which were originally established from metastatic cells in lymph nodes at our institute. RESULTS: Neither E-cadherin, integrin, nor ICAM-1 was immunocytochemically detected in any of the 4 cell lines. Expression of nm23-H1 protein was weakly detected in OCUH-16, Nuk-1, and Huh-7 cells by Western blotting, but was clearly detected in PLC/PRF/5 cells by Western blotting. Ultrastructurally, metastatic Nuk-1 cells exhibited the intracytoplasmic canaliculus-like structures found in fibrolamellar carcinoma and the intracytoplasmic glandular lumina found in bile-duct carcinoma, while the other 3 cell lines did not. In addition, Nuk-1 cells expressed neither cytokeratin 8 nor cytokeratin 19. CONCLUSIONS: Nuk-1 cells, which are human HCC cells with metastasis to lymph nodes, alone exhibited intracytoplasmic canaliculus-like structures and glandular lumina, as well as a marked reduction of nm23-H1 protein, but did not express E-cadherin, integrin, or ICAM-1. Formation of both intracytoplasmic canaliculus-like structures and intracytoplasmic glandular lumina is one of the characteristics that may be involved in metastasis of HCC cells to lymph nodes, as is reduction of nm23-H1 protein.