肝癌及癌旁肝组织的高尔基体差异蛋白质分析

目的 筛选并鉴定肝癌及癌旁肝组织高尔基体蛋白质组中差异表达的蛋白质,从高尔基体层面解释肝癌的发生和发展机制,为早期诊断和抗癌药物的研发提供线索.方法 应用亚细胞比较蛋白质组学研究方法,比较分析肝癌及癌旁肝组织高尔基体蛋白质组.收集肝癌患者手术切除的癌组织和癌旁肝组织标本,蔗糖密度梯度离心法分离高尔基体.建立并优化两种组织高尔基体的双向电泳方法,用PD-Quest软件分析差异表达的蛋白质点,然后用质谱仪获取相应蛋白点的肽质指纹图谱,联网到Swiss-Prot蛋白质组数据库鉴定获得的差异蛋白质点,最后用Western blot验证双向电泳结果.蛋白质相对表达量的比较采用配对t检验.结果 获得了分辨率和重复性均较好的双向电泳银染图谱.与癌旁肝组织相比,肝癌高尔基体蛋白质组有包括膜联蛋白5在内的27个蛋白质位点表达上调,包括染色体修饰蛋白2b在内的20个蛋白质位点表达下调,初步鉴定出了其中17个蛋白质,并用Western blot从蛋白质水平上验证了双向凝胶电泳结果.结论 肝癌及癌旁肝组织高尔基体蛋白质组具有不同的蛋白质位点,这些差异表达的蛋白质涉及到细胞的能量代谢、肿瘤的侵袭和转移,细胞周期调控等方面,为进一步阐释高尔基体在肿瘤的发生和发展中所起的作用提供了有价值的信息.     

Coronin-1 C蛋白表达与肝癌侵袭和转移相关

目的 采用膜蛋白质组学技术筛选与原发性肝癌侵袭和转移相关的分子,并加以验证.方法 以不同转移潜能的人肝癌细胞HCCLM9和MHCC97L为研究对象,采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和电喷雾串联质谱技术比较和鉴定差异表达的膜蛋白,进一步行Westernblot、动物模型标本和临床标本验证. 结果电喷雾串联质谱鉴定表达差异条带中蛋白质14个,其中Western blot验证coronin-1 C在人肝癌细胞HCCLM9中灰度值coronin-1 C/整合素α3比值为7.31±0.73,在MHCC97L中为2.84±0.99.coronin-1C在HCCLM9细胞中的表达水平显著性升高(t=6.27,P<0.05).在裸鼠肝癌组织中免疫组织化学提示coronin-1C表达也明显升高.115例临床病理标本免疫组织化学显示,coronin 1C表达越强,肿瘤的侵袭程度越强、临床病期越晚.结论 Coronin-1C与肝癌的侵袭和转移相关.     

人胎儿肝组织形态学及蛋白质组学分析

目的 探讨不同发育时期胎儿肝组织形态学及蛋白质表达谱的差异.方法 胎儿肝组织常规切片、HE染色、光学显微镜下观察肝组织形态.采用基于双向电泳-基质辅助激光解吸电离飞行时间质谱的蛋白质组学分析方法,分析早,晚期胎儿肝脏蛋白质表达谱的差异,并与肝癌组织蛋白质表达谱比较;用Mascot软件对差异表达的蛋白质在蛋白质数据库中进行检索并鉴定.结果 早、晚期胎儿肝脏不仅组织形态学上存在明显差异,而且蛋白质表达谱也存在明显差异.与肝癌组织蛋白质表达谱比较,对有差异的和相似的32个蛋白质点进行分析,初步鉴定出26个蛋白质.它们涉及细胞的增殖、凋亡及各种物质代谢等方面.结论 提示肝癌的发生是原始胎儿肝细胞发育分化的重现,在肝癌的发生过程中由于癌细胞遗传特性的紊乱获得了某些胎儿肝细胞的特性.     

H22荷瘤小鼠组织蛋白质的双向电泳比较

目的建立H22荷瘤小鼠组织蛋白质双向凝胶电泳图谱,初步分析肝癌小鼠组织蛋白质的差异表达。方法分别取对照组小鼠正常肝组织和H22荷瘤小鼠瘤组织,利用双向电泳技术分离对照组正常肝组织及荷瘤组瘤组织的蛋白质,获得图谱后通过Image Master 5.0软件分析两组蛋白表达的差异特性。结果组织分析结果表明,差异明显的蛋白点有22个,其中在荷瘤组有16个点高表达,6个低表达;有19个点仅在对照组出现,新增27个点在荷瘤组表达。结论异常蛋白表达与细胞增殖异常、代谢紊乱、肿瘤发生密切相关。     

转乙型肝炎病毒表面抗原基因小鼠肝细胞质膜蛋白质组学研究

目的 分析转HBsAg基因小鼠与正常小鼠肝细咆质膜的差异蛋白质组,为了解乙型肝炎发病机制,寻找药物作用靶标提供指导.方法 构建6月龄转HBsAg基因C57小鼠模型,检测转基因组小鼠和正常对照C57小鼠肝脏病理变化.以转基因组和正常对照C57小鼠肝脏为材料,蔗糖密度梯度离心结合磁珠纯化法纯化肝细胞质膜,Western印迹验证纯化的质膜组分纯度,二维凝胶电泳结合ImageMaster软件图像分析质膜蛋白质,获得的差异蛋白质点经胰酶酶切后,进行差异蛋白质的液相色谱串联质谱分析鉴定.结果 转HBsAg基因小鼠肝脏呈现肝炎表现,而正常对照C57小鼠肝脏未见异常.蔗糖密度梯度离心结合磁珠纯化法可以有效富集小鼠肝质膜组分,并减少线粒体的污染.小鼠肝脏质膜组分中,共获得30个≥2倍的差异蛋白质点,成功鉴定到11个可能与HBV感染相关的差异蛋白质,其中9个蛋白质在转基因鼠肝质膜中表达上调,2个蛋白质表达下调.这些差异蛋白质包括细胞支架蛋白、心脏钙离子释放通道蛋白、细胞色素B5和ATP合成酶α亚基等.结论 本研究鉴定了一批与HBsAg基因表达相关的小鼠肝脏质膜蛋白质,它们可能是乙型肝炎的药物作用靶标,本研究将为进一步探讨HBV感染的机制提供指导.     

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。     

锚蛋白重复序列22(ANKRD22)对人肝癌细胞的影响及其机制

目的 探讨锚蛋白重复序列22(ANKRD22)对人肝癌细胞增殖、侵袭和迁移的影响及其分子机制。方法 通过TCGA数据库分析正常肝组织及肝细胞癌组织中ANKRD22的表达水平及其与预后的关系。通过qRT-PCR和Western Blot检测人正常肝细胞（L-02）和人肝癌细胞系（Huh7、Hep G2、MHCC-97H、SK-HEP-1、SMMC-7721）中ANKRD22的表达情况。通过CCK-8、EdU、划痕实验及Transwell检测ANKRD22对肝癌细胞增殖、侵袭和迁移能力的影响。通过Western Blot检测ANKRD22与细胞周期蛋白、EMT相关蛋白之间的关系。通过KEGG、ssGSEA分析进一步探究ANKRD22在肝癌细胞中的作用机制,并进行实验验证。计量资料两组间比较采用成组t检验,多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。结果 TCGA数据库中ANKRD22在肝细胞癌组织中较正常肝组织高表达（t=5.083,P<0.05）,且ANKRD22高表达患者的总生存期及疾病相关生存期均显著低于ANKRD22低表达的患者（P值均<0.05）。...     

德氮吡格影响人肝癌细胞株QGY-7701亚细胞的蛋白质组学研究

目的 观察德氮吡格(TNBG)对人肝癌细胞QGY-7701亚细胞蛋白表达的影响,探讨其影响脂代谢的分子机制. 方法 分别提取TNBG处理前后人肝癌细胞QGY-7701的细胞质、细胞膜和细胞核蛋白进行双向电泳,PDQuest 7.4.0软件分析比对图像,采用基质辅助激光解吸电离飞行时间质谱技术(MALDI-TOF-MS)鉴定差异蛋白质点. 结果 TNBG作用于人肝癌细胞QGY-770172h后,细胞质的差异表达蛋白质点有56个,细胞膜的差异表达蛋白质点有65个,细胞核的差异表达蛋白质点有34个,共计155个.利用MALDI-TOF-MS技术鉴定出其中33个差异表达蛋白质点,其中包括与脂质合成有关的10个蛋白质点和与脂质降解、转运有关的7个蛋白质点,如3-羟基-3甲基戊二酸单酰辅酶A还原酶、角鲨烯合成酶、低密度脂蛋白受体、三磷酸腺柠檬酸裂解酶、甘油醛-3-磷酸脱氢酶、甘油-3-磷酸酰基转移酶、长链酯 辅酶A脱氢酶等,这些都是固醇调节元件结合蛋白调控的靶基因.结论 TNBG可能通过固醇调节元件结合蛋白途径增加胆固醇和甘油三酯合成,导致肿瘤细胞内脂滴大量聚积.     

肝细胞癌人源抗c-Met抗体Fab的筛选及鉴定

目的 从大容量人源Fab抗体库中筛选全人源抗c-Met抗体,并对抗体与肝癌细胞的结合活性进行初步鉴定.方法 利用Met-Fc融合蛋白对大容量人源Fab抗体库进行固相筛选,经过5轮固相筛选,随机挑选30个克隆经酶联免疫吸附法差减鉴定,阳性克隆进行可溶性表达,用c-Met表达阳性的人肝癌细胞株鉴定抗c-Met抗体Fab的结合活性.结果 Western blot、免疫荧光结果显示,c-Met分子表达于SMMC721、BEL7402人肝癌细胞膜上;从大容量人源Fab抗体库中筛选出1株抗c-Met抗体Fab(AM2-26),经免疫共沉淀、荧光激活细胞分类术、免疫荧光分析,结果显示AM2-26与人肝癌细胞表面c-Met分子有较好的结合活性.结论 从人源Fab抗体库中筛选的AM2-26能够与肝癌细胞表面c-Met分子特异性结合,为研制用于肝癌生物治疗的靶向药物,提供了候选分子.     

细胞外调节蛋白激酶信号通路在乙型肝炎病毒X基因调控c-met基因启动子中的作用

目的 明确在乙型肝炎病毒X基因(HBx)对原癌蛋白质(c-met)基因启动子区的调控中所涉及的信号传导通路及其在肝癌侵袭和转移中的作用.方法 用Western blot比较可能涉及的信号通路特异性阻断剂对HBx调控c-met表达的影响,分析在该调控过程中所涉及的信号通路.体外侵袭试验验证信号传导通路在HBx调控c-met表达中的作用.对数据进行析因设计的方差分析.结果 HBx引起c-met表达水平的增加,细胞外调节蛋白激酶信号通路抑制剂U0126能够降低该作用,而p38MAPK信号通路抑制剂SB203580、PI-3K信号通路抑制剂wortmanin则未显示出该作用.体外细胞侵袭试验也证实了U0126能够降低HBx引起的HepG2细胞的强侵袭性[(74.3±6.2)个与(34.6±5.2)个,F=113.45,P＜0.01].结论 HBx引起肝癌细胞的侵袭转移可能与其通过细胞外调节蛋白激酶信号通路对c-met基因启动子区(-183 bp～-100 bp)的调控有关.     

Protein profile of human hepatocarcinoma cell line SMMC-7721： Identification and functional analysis

AIM: To investigate the protein profile of human hepatocarcinoma cell line SMMC-7721, to analyze the specific functions of abundant expressed proteins in the processes of hepatocarcinoma genesis, growth and metastasis, to identify the hepatocarcinoma-specific biomarkers for the early prediction in diagnosis, and to explore the new drug targets for liver cancer therapy. METHODS: Total proteins from human hepatocarcinoma cell line SMMC-7721 were separated by two-dimensional electrophoresis (2DE). The silver-stained gel was analyzed by 2DE software Image Master 2D Elite. Interesting protein spots were identified by peptide mass fingerprinting based on matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and database searching. RESULTS: We obtained protein profile of human hepatocarcinoma cell line SMMC-7721. Among the twenty-one successfully identified proteins, mitofilin, endoplasmic reticulum protein ERp29, ubiquinol- cytochrome C reductase complex core proteinⅠ, peroxisomal enoyl CoA hydratase, peroxiredoxin-4 and probable 3-oxoacid CoA transferase 1 precursor were the six novel proteins identified in human hepatocarcinoma cells or tissues. Specific functions of the identified heat-shock proteins were analyzed in detail, and the results suggested that these proteins might promote tumorigenesis via inhibiting cell death induced by several cancer-related stresses or via inhibiting apoptosis at multiple points in the apoptotic signal pathway. Other identified chaperones and cancer-related proteins were also analyzed.CONCLUSION: Based on the protein profile of SMMC-7721 cells, functional analysis suggests that the identified chaperones and cancer-related proteins have their own pathways to contribute to the tumorigenesis, tumor growth and metastasis of liver cancer. Furthermore, proteomic analysis is indicated to be feasible in the cancer study.     

铜绿假单胞菌注射液对肺癌A549细胞自噬及PI3 K/Akt通路的影响

目的 探究铜绿假单胞菌注射液(PA-MSHA)对肺癌A549细胞自噬及磷脂酰肌醇-3激酶(PI3K)/蛋白激酶B(Akt)通路的影响.方法 体外培养人肺癌A549细胞,随机分为空白对照组、LY294002组(加入20μmol/L LY294002),PA-MSHA干预组(加入0.5×109/mL、1.0×109/mL、...     

SELDI-TOF-MS联合LCM技术筛选大肠癌肝转移早期诊断标志蛋白

目的:应用表面增强激光解吸电离飞行时间质谱蛋白质芯片(SELDI-TOF-MS)联合激光显微切割(LCM)技术筛选大肠癌及其肝转移标志蛋白.方法:采用LCM技术获取24例大肠癌肝转移患者正常大肠、原发灶及肝转移灶癌细胞;应用SELDI-TOF-MS技术对其行蛋白质谱分析;采用Biomarker Wizard软件分析差异蛋白;通过查询蛋白库对特定分子质量所对应的标志蛋白进行初步确定.结果:比较3组细胞间的SELDI质谱图,发现大肠癌原发灶与正常大肠两组间存在15个标志蛋白,12个表达上调,3个表达下调;大肠癌肝转移灶与原发灶两组间存在9个标志蛋白,5个表达上调,4个表达下调.其中质荷比4676.63Da,11740.87Da,21063.59Da和22783.36Da,蛋白峰差异性最明显(P<0.01).通过查询ExPasy蛋白库筛选出20个差异蛋白,包括整合膜蛋白2C、DNA修复蛋白RAD51同系物4、细胞周期检查点蛋白RAD1、人附睾蛋白4、着丝粒蛋白R、Pleckstrin同源结构域家族成员3等.其中细胞凋亡调节Bax蛋白γ亚型、蛋白质S100A11(Protein S100-A11)、Raf激酶抑制蛋白(RKIP)和热休克蛋白27(HSP-27)在正常大肠、原发灶及肝转移灶癌细胞中均呈差异性表达,并且差异性最明显(P<0.01).结论:SELDI蛋白质芯片联合LCM技术有可能筛选出敏感性高、特异性强的大肠癌标志蛋白,筛选出的差异蛋白可能是大肠癌及其肝转移特异性生物标志物.     

Givinostat inhibition of hepatic stellate cell proliferation and protein acetylation

AIM: To explore the effect of the histone deacetylase inhibitor givinostat on proteins related to regulation of hepatic stellate cell proliferation.METHODS: The cell counting kit-8 assay and flow cytometry were used to observe changes in proliferation, apoptosis, and cell cycle in hepatic stellate cells treated with givinostat. Western blot was used to observe expression changes in p21, p57, CDK4, CDK6, cyclin D1, caspase-3, and caspase-9 in hepatic stellate cells exposed to givinostat. The scratch assay was used to analyze the effect of givinostat on cell migration. Effects of givinostat on the reactive oxygen species profile, mitochondrial membrane potential, and mitochondrial permeability transition pore opening in JS-1 cells were observed by laser confocal microscopy.RESULTS: Givinostat significantly inhibited JS-1 cell proliferation and promoted cell apoptosis, leading to cell cycle arrest in G0/G1 phases. Treatment with givinostat downregulated protein expression of CDK4, CDK6, and cyclin D1, whereas expression of p21 and p57 was significantly increased. The givinostat-induced apoptosis of hepatic stellate cells was mainly mediatedthrough p38 and extracellular signal-regulated kinase 1/2. Givinostat treatment increased intracellular reactive oxygen species production, decreased mitochondrial membrane potential, and promoted mitochondrial permeability transition pore opening. Acetylation of superoxide dismutase(acetyl K68) and nuclear factor-κB p65(acetyl K310) was upregulated, while there was no change in protein expression. Moreover, the notable beneficial effect of givinostat on liver fibrosis was also confirmed in the mouse models.CONCLUSION: Givinostat has antifibrotic activities via regulating the acetylation of nuclear factor-κB and superoxide dismutase 2, thus inhibiting hepatic stellate cell proliferation and inducing apoptosis.     

Expression of gap junction genes connexin 32, connexin 43 and their proteins in hepatocellular carcinoma and normal liver tissues

AIM To investigate the significance and mechanism of cx32 mRNA, cx43 mRNA and their proteins in hepatocarcinogenesis.METHODS Sixty-one cases of HCC and 14 cases of normal liver tissues were detected by immunohistochemical and in situ hybridization (ISH) methods.RESULTS In HCC grades Ⅰ,Ⅱ,Ⅲ and normal liver tissues, the positive rates of Cx32 protein were 55.6%, 42.1%, 18.2% and 92.9%,respectively. The detection rates of Cx43 protein were 44.4%, 26.3%, 12.1% and 78.6%,respectively. There was significant difference in Cx32 and Cx43 protein between HCC and normal liver tissues (P＜0.01). ISH the positive rates of cx32 mRNA shown by ISH in HCC grades Ⅰ,Ⅱ,Ⅲ and normal liver tissues were 88.9%, 84.2%,87.9% and 92.9%, respectively. Those of cx43 mRNA were 77.8%, 78.6%, 78.8% and 85.7%,respectively. There was no statistical difference in the positive rates of cx32 mRNA and cx43 mRNA between HCC and normal liver tissue (P＞0.05).CONCLUSION The aberrant location of Cx32 and Cx43 proteins could be responsible for progression of hepatocarcinogenesis, and the defect of cx genes in post-translational processing might be the possible mechanism.     

肝癌组织DNA-PKcs过量表达及其靶向siRNA分子的抗增殖作用

目的:研究肝癌组织中DNA依赖蛋白激酶催化亚基DNA-PKcs的表达差异及其生物学意义.方法:用组织芯片和免疫组化法检测肝胆癌组织86例,肝胆管结石切除肝组织17例标本和正常肝组织70例中DNA-PKcs蛋白表达情况,Western blot检测培养细胞中DNA-PKcs表达,Lipofectamine 2000介导DNA-PKcs的siRNA质粒DNA转染,细胞生长曲线分析细胞增殖,克隆形成法分析细胞辐射敏感性.结果:组织芯片检测显示,60例肝癌组织细胞中DNA-PKcs阳性表达率<25%(极低表达),25%-50%(低表达),51%-75%(中等表达)和>75%(高表达)分别占15%,20%,23.3%和41.7%,而64例正常肝组织中各DNA-PKcs阳性表达率分别为68.7%,10.9%,12.6%和7.8%,表明癌组织DNA-PKcs的表达水平显著高于正常组织(P=0.0008).26例肝癌组织病理切片的免疫组化结果也显示,其DNA-PKcs表达水平显著高于23例非肿瘤性肝组织(P= 0.001).Western blot检测结果显示,体外培养的肝癌细胞HepG2,7721和7402中DNA-PKcs表达水平,同样显著高于正常肝细胞LO2.siRNA分子靶向抑制DNA-PKcs表达,不但显著提高HepG2细胞对电离辐射的敏感性,同时还降低肝癌细胞的增殖速度.随着DNA-PKcs表达的抑制,癌基因c-Myc蛋白表达水平也显著降低.结论:肝癌组织细胞中DNA-PKcs表达水平显著高于正常肝组织和非肿瘤性肝病理组织,siRNA抑制DNA-PKcs表达,具有抗癌细胞增殖和放射增敏作用,c-Myc蛋白表达抑制可能是其抗增殖作用的相关机制之一.     

Melatonin modulation of intracellular signaling pathways in hepatocarcinoma HepG2 cell line: role of the MT1 receptor

Melatonin reduces proliferation in many different cancer cell lines. However, studies on the oncostatic effects of melatonin in hepatocarcinoma are limited. We have previously demonstrated that melatonin administration induces cycle arrest, apoptosis, and changes in the expression of its specific receptors in HepG2 human hepatocarcinoma cells. In this study, we used the receptor antagonist luzindole to assess the contribution of MT1 melatonin membrane receptor to melatonin effects on cell viability, mitogen-activated protein kinase (MAPKs) activation, and cAMP levels. Additionally, effects of MT1 inhibition on mRNA levels of cytosolic quinone reductase type-2 (NQO2) receptor and nuclear retinoic acid-related orphan receptor alpha (RORα) were tested. Melatonin, at 1000 and 2500 μm, significantly reduced cell viability. Pre-incubation with luzindole partially inhibited the effects of melatonin on cell viability. Melatonin at 2500 μm significantly reduced cAMP levels, and this effect was partially blocked by luzindole. Both melatonin concentrations increased the expression of phosphorylated p38, ERK, and JNK. ERK activation was completely abolished in the presence of luzindole. NQO2 but not RORα mRNA level significantly increased in luzindole-treated cells. Results obtained provide evidence that the melatonin effects on cell viability and proliferation in HepG2 cells are partially mediated through the MT1 membrane receptor, which seems to be related also with melatonin modulation of cAMP and ERK activation. This study also highlights a possible interplay between MT1 and NQO2 melatonin receptors in liver cancer cells.     

Induction of apoptosis and cell cycle arrest in human HCC MHCC97H cells with Chrysanthemum indicum extract

AIM： To investigate the effects of Chrysanthemum indicum extract （CIE） on inhibition of proliferation and on apoptosis, and the underlying mechanisms, in a human hepatocellular carcinoma （HCC） MHCC97H cell line. METHODS： Viable rat hepatocytes and human endothelial ECV304 cells were examined by trypan blue exclusion and MTT assay, respectively, as normal controls. The proliferation of MHCC97H cells was determined by MTT assay. The cellular morphology of MHCC97H cells was observed by phase contrast microscopy. Flow cytometry was performed to analyze cell apoptosis with annexin V/propidium iodide （PI）, mitochondrial membrane potential with rhodamine 123 and cell cycle with PI in MHCC97H cells. Apoptotic proteins such as cytochrome C, caspase-9, caspase-3 and cell cycle proteins, including P21 and CDK4, were measured by Western blotting. RESULTS： CIE inhibited proliferation of MHCC97H cells in a timeand dose-dependent manner without cytotoxicity in rat hepatocytes and human endothelial ceils. CIE induced apoptosis of MHCC97H cells in a concentration-dependent manner, as determined by flow cytometry. The apoptosis was accompanied by a decrease in mitochondrial membrane potential, release of cytochrome C and activation of caspase-9 and caspase-3. CIE arrested the cell cycle in the S phase by increasing P21 and decreasing CDK4 protein expression. CONCLUSION： CIE exerted a significant apoptotic effect through a mitochondrial pathway and arrested the cell cycle by regulation of cell cycle-related proteins in MHCC97H cells without an effect on normal cells. The cancer-specific selectivity shown in this study suggests that the plant extract could be a promising novel treatment for human cancer.