生存素、p27和PTEN在肝细胞癌中的表达及临床病理意义

目的 探讨生存素蛋白、生存素mRNA、p27蛋白、p27 mRNA和第10号染色体同源丢失性磷酸酶张力蛋白基因(PTEN)蛋白在肝细胞癌(HCC)中的表达及其临床病理意义.方法 自制组织芯片,采用免疫组织化学和原位杂交法检测生存素蛋白、p27蛋白、PTEN蛋白和生存素mRNA、p27 mRNA在141份肝细胞癌、128份癌旁肝组织、97份远癌肝组织及17份正常肝组织中的表达,探讨各指标的关系并建立预测肝癌发生的模型.结果 肝癌组中生存素蛋白(Ridit值的95%CI为0.689±0.048,P《0.01)、生存素mRNA(Ridit值的95% CI为0.690±0.049,P《0.01)和p27蛋白(Ridit值的95% CI为0.556±0.053,P《0.05)表达明显增高,PTEN(Ridit值的95% CI为0.282±0.048)表达明显下降(P《0.01);肝癌中生存素的表达与p27、PTEN表达均显著相关;生存素mRNA、p27蛋白和PTEN蛋白对判断肝癌发生与否有重要意义.结论 生存素mRNA、p27蛋白的表达上调和PTEN蛋白的表达下调可作为判断肝癌发生的有价值指标.     

p73基因在肝细胞癌中的表达及其临床意义

背景:p73基因是新发现的一种抑癌基因,其在肿瘤发生、发展中的作用尚不明确.目的:探讨p73基因在肝细胞癌中的表达、突变程度及其与临床预后的关系.方法:采用定量逆转录聚合酶链反应(RT-PCR)检测35例肝细胞癌和配对癌旁组织,以及10例转移淋巴结和10例正常淋巴结组织中p73基因的表达;采用内含子PCR产物StyⅠ酶切分析法调查标本的杂合性;采用PCR-单链构象多态性(SSCP)检测p73基因的突变情况.结果:35例肝细胞癌组织中有26例p73 mRNA呈高表达,癌旁组织则呈低表达,两者表达率有显著差异(P＜0.01);20例中高分化癌组织中有13例p73 mRNA呈中高表达,15例低分化癌组织中有13例p73 mRNA呈中高表达,两者阳性率之间有显著差异(P＜0.01);18例Ⅰ～Ⅱ期肝细胞癌标本中有9例p73 mRNA呈中高表达,17例Ⅲ～Ⅳ期肝细胞癌标本中p73 mRNA均呈中高表达,两者阳性率之间有显著差异(P＜0.05).10例转移淋巴结组织中p73 mRNA均呈高表达,而10例正常淋巴结组织中均呈低表达,两者之间有显著差异(P＜0.01).12例杂合性肝细胞癌组织标本中,有10例p73基因存在G/C:A/T双等位基因表达,在癌旁组织中则均为G/C表达,未见A/T表达.肝细胞癌和癌旁组织中未见p73基因突变.结论:p73基因的表达与肝细胞癌的分化程度、临床分期和预后有关,突变可能不是p73基因起作用的主要方式.     

甘露糖受体在肝细胞癌中的表达及意义

目的:观察甘露糖受体(mannose receptor,M R)在肝细胞癌组织/肝癌细胞系中的表达,探讨其与肝细胞癌发生、发展及恶性程度的关系.方法:应用免疫组织化学检测50例肝细胞癌组织及癌旁组织、10例正常肝组织中MR的表达,免疫荧光法、Western blot法检测肝癌细胞系、肝细胞系中MR的表达.结果:免疫组织化学染色:肝细胞癌组织中M R的表达水平明显高于癌旁组织及正常肝组织(86%vs 76%,30%,P0.05).免疫荧光:MR在肝癌细胞系BEL-7402、Hep G2和人肝细胞系HL-7702中均有表达.在肝癌细胞系BEL-7402、Hep G2上有很强的MR表达,在肝细胞系HL-7702上MR的表达分布明显较少.Western blot法:MR在肝癌细胞系Hep G2、BEL-7402和人肝细胞系HL-7702上均有表达,肝癌细胞BEL-7402中MR的表达水平明显高于肝癌细胞Hep G2(P0.01)、肝细胞HL-7702(P0.01).结论:MR在肝细胞癌组织/肝癌细胞系中高表达提示可能与肝细胞癌的发生、发展及恶性程度密切相关.     

原发性肝细胞癌组织Smad4、p21蛋白的表达及其临床意义

目的 探讨原发性肝细胞癌组织Smad4和p21蛋白表达及其相关性.方法 采用免疫组化法检测60例原发性肝细胞癌组织及20例癌旁组织中Smad4、p21蛋白的表达,同时结合临床资料进行分析.结果 癌及癌旁组织中Smad4蛋白阳性表达率分别为36.7%、80.0%,p21蛋白阳性表达率分别为41.7%、70.0%,两种组织Smad4和p21蛋白表达差异均有统计学意义(P＜0.01,＜0.05).癌组织中Smad4蛋白表达与组织分级、肿瘤转移有关(P＜0.05).Smad4与p21蛋白的表达呈正相关(r=0.69,P＜0,01).结论 Smad4及p21蛋白在原发性肝细胞癌组织中表达均显著下降,并与肝癌生物学行为关系密切.     

肝癌组织中TGF-β1、TGF-β1RⅡ和NF-κB的表达

目的:探讨转化生长因子β1(TGF-β1)、转化生长因子β1Ⅱ型受体(TGF-β1RⅡ)、核转录因子-κB(NF-κB)在肝细胞癌(HCC)中表达.方法:在30例肝癌组织和癌旁肝组织中,用免疫组化技术,分别检测TGF-β1 TGF-β1RⅡ及NF-κB蛋白的表达;用原位杂交方法检测TGF-β1mRNA的表达,以CD34标记血管内皮细胞,观察TGF-β1及TGF-β1RⅡ蛋白与微血管密度(MVD)、TGF-β1蛋白与TGF-β1mRNA、NF-κB与TGF-β1的关系.结果:肝癌组织TGF-β1mRNA平均光密度明显高于癌旁组织(0.1 043±0.035 vs 0.0 620±0.0 225,P＜0.01)、TGF-β1蛋白平均光密度表达明显高于癌旁组织(0.0 72 5±0.0 102 vs 0.0 442±0.0 103,P＜0.01);肝癌组织TGF-β1RⅡ蛋白平均光密度表达明显低于癌旁组织(0.0 402±0.0 113 vs 0.0 669±0.0 157,P＜0.01);肝癌组织NF-KB蛋白表达均明显高于癌旁组织(0.0 723±0.0 210 vs 0.0 305±0.0116,P＜0.01),肝癌组织MVD明显高于癌旁组织(31.23±9.25 vs 4.24±2.10,P＜0.01),肝癌组织TGF-β1蛋白阳性表达与MVD呈正相关(t=3.25,P＜0.01);TGF-β1 mRNA与TGF-β1蛋白呈正相关(χ2=8.21,P＜0.01);NF-κB蛋白阳性表达与TGF-β1蛋白阳性表达呈正相关(χ2=9.075,P＜0.01);而TGF-βRⅡ蛋白与MVD呈负相关.结论:肝癌组织中TGF-β1基因的变化发生在多个水平,肝癌细胞中TGF-β1RⅡ、NF-KB蛋白表达异常,并与MVD相关,提示他们在肝细胞癌血管形成中发挥重要作用,NF-κB可能在介导TGF-β1的活化或产生中发挥一定的作用.     

乙型肝炎病毒蛋白及环氧合酶在乙肝相关性肝细胞癌发展与转移中的作用

目的探讨乙型肝炎病毒蛋白及环氧合酶(COX)-2在乙肝相关性肝细胞癌发展与转移中的作用及机制。方法选取53例手术切除的人肝细胞癌组织,采用免疫组化法检测乙型肝炎病毒X蛋白、COX-2、CD34的表达水平,Werdner法计算微血管密度;分析上述因子与乙肝相关性肝细胞癌组织微血管生成的相关性。RT-PCR和Western印迹检测人肝癌细胞系Hep G2和稳定转染乙型肝炎病毒X蛋白的Hep G2-X细胞中COX-2 mRNA和蛋白表达情况;酶联免疫吸附(ELISA)法检测细胞上清液中PGE2表达水平和不同浓度COX-2抑制剂西乐葆作用后PGE2水平。结果乙型肝炎病毒X蛋白阳性表达组织中COX-2阳性率明显高于乙型肝炎病毒X蛋白阴性表达组织和非乙型肝炎相关性肝癌组织(P0.01)。乙型肝炎病毒X蛋白阳性表达组织中早期肝癌组织中微血管密度明显低于进展期肝癌组织,乙型肝炎病毒X蛋白阴性表达组织中微血管密度明显低于阳性表达组织(P0.01);COX-2阳性表达组织中微血管密度明显高于COX-2阴性表达组织(P0.01);非乙型肝炎相关性肝癌组织中微血管密度明显低于乙型肝炎病毒X蛋白、COX-2阳性表达组织(P0.01),乙型肝炎病毒X蛋白阴性表达组织和COX-2阴性表达组织差异无统计学意义(P0.05);乙型肝炎病毒X蛋白、COX-2在乙型肝炎相关性人肝细胞癌组织微血管生成呈正相关。Hep G2-X细胞中COX-2 mRNA和蛋白表达水平明显高于空载体对照Hep G2细胞,并且细胞培养上清液中PGE2水平明显增加;与Hep G2细胞相比,西乐葆对Hep G2-X细胞分泌PGE2具有更强的抑制作用。结论乙型肝炎病毒X蛋白、COX-2在乙肝相关性肝细胞癌组织中高表达,促进癌组织微血管生成;乙型肝炎病毒X蛋白可通过COX-2/PEG2信号通路促进肝癌的发生和发展。     

Plk1与p21WAF1/CIP1在肝细胞癌中的表达

目的 细胞周期抑制因子p21<'WAF1/CIP1>(p21)可在转录水平抑制Polo-like kinase1(Plk1)基因的表达,本文旨在探讨Plk1和p21蛋白在肝细胞癌中的表达及相关性.方法 应用Westem Blot方法检测10对原发性肝细胞癌和癌旁组织中Plk1和p21蛋白的表达情况.通过将pFlex-21表达质粒瞬时转染人肝癌细胞系HepG2细胞,进一步检测p21过表达对Plk1mRNA和蛋白表达的影响.结果 Plk1在10例肝癌组织中的表达均高于配对癌旁组织,p21蛋白在7对肝癌组织中的表达高于配对癌旁组织.HepG2细胞瞬时表达p21质粒后,Plk1的蛋白表达和mRNA水平均无变化(P>0.05).结论 Plk1和p21在肝细胞癌组织中的表达具有一定的肿瘤特异性.肝癌组织中p21蛋白过表达可能不具有抑制Plk1表达的作用,具体机制还有待于进一步研究.     

MIF与JNK1在原发性肝细胞癌中的表达及相关性研究

目的研究巨噬细胞移动抑制因子(MIF)和c-Jun氨基末端蛋白激酶1(JNK1)蛋白在肝细胞癌(HCC)组织和HepG2肝癌细胞中的表达,并探讨二者的相互关系。方法选择有乙肝肝硬化基础的HCC患者52例,取手术切除的癌组织及相应的癌旁组织为实验组,另选取20例肝血管瘤、肝破裂手术切除的肝组织标本作正常对照,应用免疫组织化学法检测MIF、JNK1和pJNK1的表达;原位杂交法检测MIF mRNA和JNK1 mRNA表达。将不同浓度的rMIF分别与HepG2肝癌细胞和L-O2人正常肝细胞共培养,通过Western blotting法检测JNK1和pJNK1蛋白的表达,通过RT-PCR检测相应蛋白在核酸水平的表达。结果 MIF、JNK1、pJNK1和MIF mRNA、JNK1 mRNA在HCC癌组织和癌旁组织中的阳性表达率均明显高于正常肝组织(P均0.05);经不同浓度的rMIF刺激后,HepG2肝癌细胞中pJNK1和JNK1mRNA的相对表达量均呈浓度依赖性升高,尤以rMIF浓度为200 ng/ml升高最明显,与L-O2细胞比较,差异有统计学意义(P0.05)。结论 MIF、MIF mRNA、JNK1、JNK1 mRNA、pJNK1在HCC组织和HepG2肝癌细胞中均呈高表达,MIF参与HCC进展,可能是通过激活JNK1信号通路来实现的。     

非小细胞肺癌组织中凋亡抑制蛋白Livin的表达及意义

目的 研究肺癌相关Livin基因在非小细胞肺癌(NSCLC)组织中的表达以及与p53基因的关系.方法 采用RT-PCR方法检测48例NSCLC组织Livinα mRNA的表达,用免疫组化SABC法检测livin和p53蛋白在48例NSCLC患者肺癌组织切片中的表达.结果 48例NSCLC中15例检测到Livinα mRNA表达,其阳性表达率为31.3%,而正常肺组织和癌旁及良性疾病肺组织均未检测到Livinα mRNA表达.Livin蛋白在腺癌、低分化癌、有淋巴结转移者中的表达阳性率明显高于鳞癌和大细胞癌、高中分化癌和无淋巴结转移者(P＜0.01),而癌旁组织和良性疾病肺组织未检出阳性表达.Livinα mRNA表达与p53呈正相关(r=0.457,P＜0.01);livin蛋白表达与p53呈正相关(r=0.563,P＜0.01).结论 Livin在NSCLC组织中高表达,可能成为肺癌诱导凋亡治疗的新靶点.     

肝癌组织巨噬细胞移动抑制因子的表达及对肿瘤细胞增殖和血管形成的影响

目的:研究巨噬细胞移动抑制因子(MIF)在原发性肝癌中的表达及对肝癌细胞增殖和血管形成的影响.方法:免疫组化法检测40例肝癌、癌旁组织和10例正常肝组织中MVD及MIF,IL-8和VEGF蛋白的表达情况,RQ-PCR检测MIF,IL-8和VEGF的mRNA表达水平.MTT法检测人重组MIF(rMIF)对Bel-7402肝癌细胞株增殖的影响,酶联免疫吸附试验(ELISA)和实时定量聚合酶链反应(RQ-PCR)检测VEGF和IL-8的分泌和mRNA的表达.结果:MIF,VEGF和IL-8在肝癌组织中均高表达(72.5%,67.5%,62.5%),经χ2检验,与正常组织相比(30.0%,20.0%,30.0%),差异具有显著性(P＜0.01).MIF和IL-8的表达与VEGF的表达呈正相关(r=0.271,P＜0.05),并且与MVD显著相关(r=0.284,P＜0.05).rMIF和Bel-7402共培养24 h后,上清中VEGF含量为900±265 ng/L,对照组为180±50 ng/L(P＜0.01),而IL-8含量改变无明显的变化(P＞0.05);实验组肝癌细胞株VEGF mRNA表达量是对照组的6.86倍(P＜0.05);而IL-8 mRNA是对照组的1.23倍(P＞0.05).结论:MIF是一种血管生成因子,MIF和IL-8的参与肿瘤血管形成有可能是通过VEGF发挥作用.     

肝癌细胞中Wnt/β-连环素信号通路与caspase-3、X连锁凋亡抑制蛋白和葡萄糖调节蛋白78的表达

目的 研究肝细胞癌中Wnt/β-连环素信号传导通路与caspase-3、X连锁凋亡抑制蛋白(XIAP)、葡萄糖调节蛋白78(Grp-78)、热休克蛋白(HSP)27的关系及其意义.方法 用RNAi技术将针对β-连环素的siRNA转染入肝癌HepG2细胞中沉默β-连环素基因表达,于72、96h提取蛋白质,用Western blot法检测β-连环素、caspase-3、XIAP、Grp-78、HSP27蛋白质的表达.用方差分析的方法进行统计学分析.结果 对照组、转染72h组、转染96h组β-连环素蛋白质表达的灰度值分别为18.9、1.5和3.2 ; β-肌动蛋白表达的灰度值分别为41.1、45.6和47.8 ;caspase-3蛋白质表达的灰度值分别为49.8、5.2和45.1 ; p-caspase-3蛋白质表达的灰度值分别为16.0、67.7和16.3 ; XIAP蛋白质表达的灰度值分别为28.2、21.9和49.9 ; Grp-78蛋白质表达的灰度值分别为23.3、49.9和26.8 ; HSP27蛋白质表达的灰度值分别为29.6、34.8和35.6 ; β-肌动蛋白表达的灰度值分别为32.1、33.6和34.2.针对β-连环素的siRNA转染肝癌HepG2细胞72h和96h均可抑制β-连环素蛋白质的表达(F=160.72,P＜0.01),而96h比72h的表达略有增加,差异有统计学意义.caspase-3的蛋白质表达于72 h被抑制,96h升高恢复至原有水平(F=136.10,P＜0.01);而p-caspase-3的蛋白质表达于72h增加,96h减少至原有水平(F=98.65,P＜0.01).XIAP的蛋白质表达于72 h被抑制,96h表达升高(F=37.29,P＜0.01).Grp-78的蛋白质表达于72 h增加,96h减少至原有水平(F=58.72,P＜0.01).HSP27的蛋白质表达转染前后一致,差异无统计学意义.结论 HepG2细胞中,Wnt/β-连环素信号传导通路与caspase-3、XIAP、Grp-78蛋白质的表达有关,而与HSP27的蛋白质表达无关.Wnt/β-连环素信号传导通路正是通过调节这些因子参与HepG2细胞凋亡及增殖、分化等调节.     

Expression and clinical significance of TAp73α, p53, PCNA and apoptosis in hepatocellular carcinoma

AIM: To study the prognostic role of TAp73α, p53, proliferating cell nuclear antigen (PCNA) and apoptosis inpatients with hepatocellular carcinoma (HCC) atter surgical tumor ablation.METHODS: Forty-seven human resected HCC tissues and 42 adjacent non-cancerous tissues were studied with 10 normal liver tissues as control group. TAp73α, p53, and PCNA were detected with Elivision immunohistochemistry.Terminal deoxynucleotidyl transferase (TdT)-mediatedd-UTP-biotin nick-end labeling (TUNEL) method wasused to detect the apoptosis cells. All clinical and pathological materials were analyzed by SPSS10.0statistical package.RESULTS: TAp73α overexpressed in HCC tissues (36.2%) when compared with adjacent non-cancerous tissues (2.38%, P＜0.005) and normal liver tissues (0, P＜0.01).Mutant type p53 (mt-p53) overexpressed in HCC tissues (38.3%) when contracted with adjacent non-cancerous tissues (16.7%, P＜0.05) and normal liver tissues (0, P＜0.01). Proliferation index (PI) level in HCC tissues was significantly higher than that in adjacent non-cancerous tissues (30.34%±4.46% vs 27.88%±5.89%, t, P= 0.028). Apoptosis index (AI) level in HCC tissues was higher than that in adjacent non-cancerous tissues (8.62%±2.28% vs 7.38%±2.61%, t, P = 0.019). Expression of TAp73α was associated with lymph node metastasis and mt-p53, with r = 0.407 and 0.265, respectively. Expression of mtp53 was associated with Edmondson's stage and AFP, with r = 0.295 and -0.357, respectively. In Kaplan-Meier univariant analysis, TAp73α, AFP, TNM stage, portal vein invasion, liver membrane invasion and HBsAg correlated with prognosis (log rank, P = 0.039, 0.012, 0.002, 0.000,0.014, 0.007, respectively). Multivariant Cox regression analysis showed that TAp73α, AFP, TNM stage, portal vein invasion, liver membrane invasion and age were independent factors of prognosis. CONCLUSION: These results suggest that TAp73α can be used as a prognostic indicator of patients with HCC undergoing surgical tumor ablation. AFP, TNM, portal vein invasion, liver membrane invasion and age also have a potency of predicting the prognosis of HCC.     

柴胡皂甙-d对HepG2细胞恶性表型的逆转作用

目的 探讨柴胡皂甙-d (SSd)对人肝癌细胞株HepG2细胞恶性表型的逆转作用及其机制. 方法 常规培养HepG2细胞至对数生长期,四甲基偶氮唑盐(MTT)比色法观察SSd不同浓度、作用不同时间对HepG2细胞增殖情况的影响.实验组选择10 mg/L的SSd作用HepG2细胞48h,Giemsa染色法观察细胞形态学改变;化学发光法测定细胞上清液中甲胎蛋白(AFP)浓度;放射免疫法检测细胞上清液中白蛋白浓度;transwell小室实验观察细胞迁移率;RT-PCR检测p27基因mRNA表达.并与空白对照组做比较.数据在多组间比较采用随机分组单因素方差分析,在两组间比较采用t检验. 结果 10 mg/L的SSd作用48 h,对HepG2细胞的生长抑制最明显(F=265.06,P＜0.01).实验组HepG2细胞形态倾向于正常分化,形态变小、变圆,核变小、变圆或卵圆形,核/质比例缩小.与对照组比较,实验组穿膜细胞数明显减少[(50.60+4.04)个与(41.00+4.64)个,t= -7.32,P＜0.01];细胞上清液中白蛋白含量明显增多[(24.7+2.8)×10-3g/L与(31.1 +4.9)×10-3g/L,t=7.83,P＜ 0.05];AFP含量明显减少[(118.2±15.6)×10μ g/L与(70.3+5.7)×103μg/L,t=-10.72,P＜0.0l].实验组p27基因mRNA的相对表达量明显多于对照组(t=22.00,P＜0.05). 结论 SSd对人肝癌细胞株HepG2细胞恶性表型具有明显的逆转作用,其机制可能与SSd上调HepG2细胞p27基因mRNA表达使其进入分化过程有关.     

Rab23 is a potential biological target for treating hepatocellula carcinoma

AIM:To elucidate the role of Rab23 in hepatocellular carcinoma(HCC)by assessing the expression of Rab23 in HCC tissue and in HCC cell lines.METHODS:Primary tumors(n = 100)were stained with Rab23 antibodies using immunohistochemistry and in situ hybridization in tissue microarrays.Relationships between gene expression and pathology parameters were analysed.The biological significance of Rab23 in Hep-3B cells was examined by knocking down Rab23 gene expression.We designed a pair of doublestranded RNAs against human rab23 and transfected siRNA into Hep-3B cells.Rab23 expression in these cells was examined using RT-PCR and Western blots.We investigated cell growth by MTT assays and fluorescenceactivated cell sorting.RESULTS:High cytoplasmic and nuclear expression of Rab23 was found in 38 of 71(53.5%)and in 49 of 68 HCC patients(72%)respectively,which correlated with tumor size.HCC cell lines expressed Rab23.In Hep3B cells,siRNA for Rab23 decreased Rab23 mRNA by 4.5-fold and protein expression by 2-fold.Survival rates at 24 and 48 h for Hep-3B cells transfected with siRNA were lower and about 30% Hep-3B cells were apoptotic.Knocking down rab23 suppressed Hep3B cell growth,suggesting that rab23 could play an important role in Hep3B cell growth.CONCLUSION:Rab23 is overexpressed and/or activated in HCC.Rab23 may be both a HCC predictor and a target for treating HCC.     

Rab23 is a potential biological target for treating hepatocellular carcinoma

AIMTo elucidate the role of Rab23 in hepatocellular carcinoma(HCC)by assessing the expression of Rab23 in HCC tissue and in HCC cell lines.METHODSPrimary tumors(n = 100)were stained with Rab23 antibodies using immunohistochemistry and in situ hybridization in tissue microarrays.Relationships between gene expression and pathology parameters were analysed.The biological significance of Rab23 in Hep-3B cells was examined by knocking down Rab23 gene expression.We designed a pair of doublestranded RNAs against human rab23 and transfected siRNA into Hep-3B cells.Rab23 expression in these cells was examined using RT-PCR and Western blots.We investigated cell growth by MTT assays and fluorescenceactivated cell sorting.RESULTSHigh cytoplasmic and nuclear expression of Rab23 was found in 38 of 71(53.5%)and in 49 of 68 HCC patients(72%)respectively,which correlated with tumor size.HCC cell lines expressed Rab23.In Hep3B cells,siRNA for Rab23 decreased Rab23 mRNA by 4.5-fold and protein expression by 2-fold.Survival rates at 24 and 48 h for Hep-3B cells transfected with siRNA were lower and about 30% Hep-3B cells were apoptotic.Knocking down rab23 suppressed Hep3B cell growth,suggesting that rab23 could play an important role in Hep3B cell growth.CONCLUSIONRab23 is overexpressed and/or activated in HCC.Rab23 may be both a HCC predictor and a target for treating HCC.     

肝硬化及肝癌组织中整合素β1表达的差异

目的 探讨整合素β1在肝癌发生、发展中的作用.方法 应用逆转录聚合酶链反应和激光扫描共聚焦显微镜免疫荧光染色方法分别检测23份正常肝脏组织、152份肝硬化肝组织和105份肝癌组织中整合素β1 mRNA及蛋白的表达水平和分布情况,分析整合素β 1基因的表达与肝细胞肝癌临床、病理参数的关系.采用SPSS12.0统计分析软件对整合素β1表达结果及其临床病例参数的关系进行pearson x2检验,直线相关分析,pearson列联相关分析,单因素方差分析.结果 (1)整合素β1 mRNA及蛋白的相对表达量在肝癌组织分别为1.30±0.24和90.50±33.50、肝硬化组织分别为1.58±0.31和123.10±38.90、正常肝组织分别为0.37±0.08和11.90±6.00,肝癌组织、肝硬化组织中整合素β 1 mRNA及蛋白的表达均明显高于正常肝组织,F值分别为9.584和7.633,P＜0.01,差异有统计学意义.(2)肝组织整合素β 1 mRNA及蛋白表达量与肝纤维化半定量评分呈正相关(mRNA:r=0.546,P＜0.05;蛋白:r=0.692,P＜0.01),而与肝功能无明显相关性.整合素β1的表达与肝癌Edmondson病理分级、有无包膜侵犯、有无转移、HBsAg感染呈正相关,而与肿瘤大小、结节数目、甲胎蛋白水平无明显相关性. 结论整合素素β1可能参与了肝纤维化--肝硬化--肝癌这一动态的病理过程,在肝细胞恶性转化中发挥重要作用.     

肿瘤拒绝抗原1在肝硬化和肝癌组织中的表达及其与肝癌临床病理学特征的关系

目的 探讨肿瘤拒绝抗原1(TRA1)在肝细胞癌(HCC)和肝硬化组织中的表达水平及其与HCC临床病理学特征的关系.方法 采用RT-PCR法、Western blot和免疫组织化学法分别检测了HCC和肝硬化组织中TRA1 mRNA和蛋白的表达水平,并与HCC临床病理学特征进行相关分析.RT-PCR和Western blot结果采用单因素方差分析;免疫组织化学结果分析采用Fisher's确切概率法;相关性分析采用Spearman等级相关.结果 经对RT-PCR结果分析,HCC和肝硬化组织中TRA1 mRNA表达水平高于正常肝组织水平(F值分别为20.821和12.311,P值均＜0.05).Western blot检测结果显示,HCC和肝硬化组织中TRA1蛋白表达水平也高于正常肝组织(F值分别为21.231和20.125,P值均＜0.05).经免疫组织化学分析,TRA1蛋白在正常对照组、肝硬化组和HCC组的表达逐渐增强,阳性表达率分别为57.14%、78.95%和93.75%.其表达水平与HCC分化程度呈负相关(r=-0.4655,P＜0.01);与HCC患者TNM分期呈正相关(r=0.5157,P＜0.01).结论 TRA1在肝硬化和HCC中的过表达可能与肝癌的发生和发展有关,并有可能成为一个潜在的HCC早期诊断标志物;TRA1的检测有助于判断HCC的分化程度,与HBV的感染有一定关系,同时可作为判断预后的指标.     

Wnt/b-catenin signaling pathway affects the protein expressions of caspase-3, XIAP and Grp-78 in hepatocellular carcinoma

To investigate the relationship and significance of Wnt/b-catenin signaling pathway with caspase-3, XIAP, HSP27and Grp-78. The HCC cell line HepG2 was transfected with small interfering RNA (siRNA) directed against b-catenin. After 72 and 96 h, protein was extracted and the protein expressions of b-catenin, caspase-3, XIAP, Grp-78 and HSP27 were detected by Western blot. b-catenin protein expression was inhibited at both time points and the expression at 96 h was higher than that at 72 h (F = 160.72, P is less than to 0.01). Interestingly, Caspase-3 protein expression was decreased at 72 h and increased to normal at 96 h (F = 136.10, P is less than to 0.01), while p-caspase-3 protein expression increased at 72 h and decreased to normal at 96 h (F = 98.65, P is less than to 0.01). XIAP protein expression decreased at 72 h (F = 37.29, P is less than to 0.01) and increased at 96 h. Grp-78 protein expression increased at 72 h and decreased to normal at 96 h ( F = 58.72, P is less than to 0.01). HSP27 protein expression showed no change following transfection ( F = 1.91, P is more than to 0.05). Wnt/b-catenin signaling pathway is related to the protein expressions of caspase-3, XIAP and Grp-78, but not related to HSP27 protein expression in HCC. Wnt/b-catenin signaling pathway may participate in the regulation of HCC apoptosis, proliferation and differentiation through affecting these factors.     

Inhibition of autophagy significantly enhances combination therapy with sorafenib and HDAC inhibitors for human hepatoma cells

AIM:To clarify whether histone deacetylase inhibitors histone deacetylase inhibitors(HDACIs)can sensitize hepatocellular carcinoma(HCC)cells to sorafenib treatment.METHODS:Bax,Bcl-2,ATG5-ATG12,p21,and p27protein levels in Hep3B,HepG2,and PLC/PRF/5 cells were examined by Western blot.CCK8 and a fluorometric caspase-3 assay were used to examine cellular viability and apoptosis levels.The effect of Beclin-1 on sensitization of HCC cells to sorafenib was examined by transfecting Beclin-1 siRNA into Hep3B,HepG2,and PLC/PRF/5 cells.RESULTS:Autophagy inhibition enhances the inhibitory effects of vorinostat and sorafenib alone or in combination on HCC cell growth.Vorinostat and sorafenib synergistically induced apoptosis and cell cycle alterations.Western blot data indicated that HDACIs and Beclin-1 knockdown increased the p53 acetylation level.The knockdown of Beclin-1 enhanced the synergistic effect of the combination of vorinostat with sorafenib.CONCLUSION:HDACIs can sensitize HCC cells to sorafenib treatment by regulating the acetylation level of Beclin-1.