基质金属蛋白酶-9与原发性肝癌侵袭转移关系的初步研究

目的　探讨原发性肝癌中MMP -9基因的表达 ,研究其与原发性肝癌侵袭转移的关系。方法　采用逆转录 -聚合酶链式反应技术 (RT -PCR ) ,对 41例原发性肝癌手术切除肿瘤组织、癌旁组织标本的MMP -9基因进行检测 ,并结合随访资料进行分析。结果　肝癌组织和癌旁 1cm、5cm组织中分别有 70 .7%、48.9%、41.5 %MMP -9表达阳性 ,癌组织MMP -9表达水平显著高于癌旁 5cm组织 (P <0 .0 1) ,癌组织与癌旁 1cm组织比较 ,MMP -9表达水平无显著性差异。癌组织MMP -9表达水平与肿瘤复发、有无包膜、门静脉癌栓显著相关 (P <0 .0 5 )。结论　MMP -9基因的过度表达与肝癌侵袭转移密切相关 ,并可作为判断其预后的指标之一     

小鼠肝癌细胞淋巴结转移的基质金属蛋白酶谱

背景与目的:肿瘤转移是恶性肿瘤致死的主要因素之一,淋巴结转移是癌早期最常见的转移方式,且淋巴结转移灶可以成为进一步血道转移的桥头堡,但癌淋巴道转移的分子机制尚不清楚。本实验旨在探讨小鼠肝癌细胞分泌基质金属蛋白酶(MMPs)及表达Fas-L与其淋巴结转移的关系。方法:采用酶谱法检测具有不同转移能力的小鼠肝癌细胞Hca-F(高转移)和Hca-P(低转移)分别在淋巴结匀浆、肝组织匀浆或脾组织匀浆存在的情况下分泌的基质金属蛋白酶谱;以流式细胞仪检测荷瘤鼠淋巴细胞的生长分数;以TUNEL检测淋巴结内淋巴细胞的凋亡情况;以免疫组化法检测Hca-F和Hca-P细胞表达Fas-L的情况。结果:在淋巴结匀浆存在下Hca-F和Hca-P细胞分泌MMP-9显著增多(P<0.01):RPMI-1640培养基中,Hca-F和Hca-P细胞分泌MMP-9分别为1256±157、2642±385;加入淋巴结匀浆后,则分别为12403±894、9086±686,同时分泌大量的MMP-2(Hca-F为7364±2001,Hca-P为2997±1990)和MMP-9的活性型(Hca-F为7297±1657,Hca-P为3914±1253),且Hca-F细胞分泌量多于Hca-P(P<0.05)。而在肝组织或脾组织匀浆存在下,Hca-F和Hca-P细胞均不分泌基质金属蛋白酶。Hca-F组荷瘤鼠引流淋巴结内的淋巴细胞的增殖高峰出现于荷瘤后第14日,并迅速下降,而Hca-P组荷瘤鼠引流淋巴结内     

利用酶谱法测定基质金属蛋白酶—2判断肝癌侵袭转移性的研究

通过酶谱法对肝细胞癌（ＨＣＣ）癌组织、癌旁肝组织及血浆中基质金属蛋白酶－２（ＭＭＰ－２）含量进行研究，并与反应ＨＣＣ侵袭转移性的病理指标间进行统计学分析。结果显示ＨＣＣ癌组织、癌旁肝组织及血浆中均存在ＭＭＰ－２；ＨＣＣ癌组织及血浆中ＭＭＰ－２含量升高与ＨＣＣ侵袭转移性有关；ＨＣＣ癌组织ＭＭＰ－２含量高于其癌旁肝组织含量可作为通过ＭＭＰ－２判断ＨＣＣ侵袭转移的指标。     

MAG-1对人肺癌细胞转移能力的影响及其在24例肺癌组织中的表达

背景与目的 肺癌转移相关基因-1(Pulmonary cancer Metastasis Associated Gene-1,MAG-1)是利用抑制消减杂交技术获得的与肺癌转移相关的基因.此研究的目的在于探讨基因MAC-1对肺巨细胞癌细胞的转移相关生物学行为的影响及其在24例肺癌组织中的表达情况.方法 构建MAG-1的正反义表达载体,分别转染PLA801C和PLA801D,获得稳定表达相应序列的细胞株,观察稳定转染细胞的多种肿瘤转移相关能力的改变,并检测MAG-1对CD44、MMP-2等分子的影响,最后还检测TMAG-1在源于不同转移状况患者的肺癌标本中的表达情况.结果 MAG-1对细胞生长、细胞与胞外基质的粘附能力以及侵袭能力均有促进作用;并能促进细胞中CD44和MMP-2的表达;MAC-1 mRNA在有转移的肺癌中的阳性率(7/12)高于其在无转移的肺癌标本中的阳性率(2/12).结论 MAG-1具有增强肺癌细胞转移能力的功能,并且在转移性肺癌组织中其mRNA具有较高的阳性检出率,是可能的肺癌转移相关基因.     

FTY720对人类肝癌细胞HepG-2侵袭能力的影响

Objective To explore the effect of FTY720 on human hepatocellular carcinoma cell line HepG-2 of invasion ability. Methods HepG-2 cells were cultured,and divided into 4 groups,namely control and FTY720 (0.05 g/ml,0.10 g/ml,0.15 g/ml) groups.The cell invasion ability was observed by the cell invasion assay.mRNA expression of MMP-2,MMP-9 was measured by RT-PCR.MMP-2,MMP-9 protein expression was measured by ELISA method.Results The invasive ability of cancer cells in each group significantly weaken,with the FTY720 concentration increased.The number of control group was (110±3)(P<0.05),the number of O.05 g/ml FRY720 treatment group cells was (74±4),while the number of cells of O.10 g/ml,0.15 g/ml the drug treated group,Was (42±3),(25±5)(P<0.01).Compared with control group,the MMP-2,MMP-9 expression of different concentrations of FTY720 groups decreased significantly (P<0.05).As the expression of FTY720 concentration increased,the MMP-2,MMP-9 expression decreased (P<0.01).Compared with control group,the MMP-2,MMP-9 protein expression of different concentrations of FTY720 groups decreased significantly (P<0.05), but the protein expression and concentration of FTY720 is inversely proportional in a certain range. Conclusion The invasive ability of HepG-2 cells can be inhibited by FTY720,and with FTY720 concentration increased,the invasive ability decreased gradually.One of the mechanisms may be related to reduce the MMP-2,MMP-9 protein and gene expression.     

FTY720对人类肝癌细胞HepG-2侵袭能力的影响

Objective To explore the effect of FTY720 on human hepatocellular carcinoma cell line HepG-2 of invasion ability. Methods HepG-2 cells were cultured,and divided into 4 groups,namely control and FTY720 (0.05 g/ml,0.10 g/ml,0.15 g/ml) groups.The cell invasion ability was observed by the cell invasion assay.mRNA expression of MMP-2,MMP-9 was measured by RT-PCR.MMP-2,MMP-9 protein expression was measured by ELISA method.Results The invasive ability of cancer cells in each group significantly weaken,with the FTY720 concentration increased.The number of control group was (110±3)(P<0.05),the number of O.05 g/ml FRY720 treatment group cells was (74±4),while the number of cells of O.10 g/ml,0.15 g/ml the drug treated group,Was (42±3),(25±5)(P<0.01).Compared with control group,the MMP-2,MMP-9 expression of different concentrations of FTY720 groups decreased significantly (P<0.05).As the expression of FTY720 concentration increased,the MMP-2,MMP-9 expression decreased (P<0.01).Compared with control group,the MMP-2,MMP-9 protein expression of different concentrations of FTY720 groups decreased significantly (P<0.05), but the protein expression and concentration of FTY720 is inversely proportional in a certain range. Conclusion The invasive ability of HepG-2 cells can be inhibited by FTY720,and with FTY720 concentration increased,the invasive ability decreased gradually.One of the mechanisms may be related to reduce the MMP-2,MMP-9 protein and gene expression.     

基质金属蛋白酶及其抑制物与肿瘤侵袭转移的关系

基质金属蛋白酶（MMP）家族是降解细胞外基质的重要酶类。组织金属蛋白酶抑制物（TIMP）是MMP的天然抑制物。在细胞外基质中MMP和TIMP的失衡已证实与多种病理状态,尤其是与肿瘤的侵袭与转移密切相关。抑制MMP活性是抗肿瘤转移的一个新的靶点。     

乳腺癌组织中转化生长因子β1的表达及其介导乳腺癌侵袭和转移的机制

目的 探讨转化生长因子β1(TGF-β1)蛋白和mRNA在乳腺痛组织中的表达,及其与明胶酶和明胶酶抑制物的关系.方法 建它组织芯片平台,应用免疫组化SP法检测160例乳腺痛组织TGF-β1、基质金属蛋白酶(MMP)-2、MMP-9、组织金属蛋白酶抑制物(TIMP)-1和TIMP-2蛋白的表达;应用原位分子杂交方法检测乳腺癌组织中TGF-β1 mRNA的表达.结果 160例乳腺癌TGF-β1、MMP-2、MMP-9、TIMP-1和TIMP-2蛋白表达的阳性率分别为73.7%、96.9%、95.0%、87.5%和89.4%,TGF-β1 mRNA表达的阳性率为56.2%.TGF-β1的蛋白表达与腋窝淋巴结转移、TNM分期和c-erbB-2表达有关(P＜0.05,P＜0.05和P＜0.01),TGF-β1的mRNA表达与腋窝淋巴结转移有关(P＜0.05).TGF-β1蛋白表达阳性组中位总生存期(OS)为60个月,中位无复发生存期(RFS)为59个月;TGF-β1蛋白表达阴性组中位OS为61个月,中位RFS为61个月,两组生存率差异无统计学意义(P=0.090),无复发生存率有统计学意义(P=0.023).TGF-β1的蛋白表达与MMP-2和MMP-9的表达均呈正相关(r=0.170,P＜0.05;r=0.221,P＜0.01).结论 TGF-β1的表达与乳腺癌侵袭和转移密切相关,TGF-β1的蛋白产物通过调控MMP-2和MMP-9促进乳腺癌的侵袭和转移.     

肝癌组织中细胞间粘着分子—1的表达与肝癌侵袭转移关系的研究

目的研究肝癌组织中细胞间粘着分子－１（ＩＣＡＭ－１）的表达与肝癌侵袭转移的关系。方法采用斑点印迹方法测定ＩＣＡＭ－１在肝癌组织、癌旁组织及正常肝组织中的表达，分析其与肿瘤生长转移状态和肿瘤特性的关系。结果肝ＩＣＡＭ－１的含量在肝癌组织中明显高于癌旁组织（Ｐ＜０．０１）和正常组织（Ｐ＜０．０１），而与肿瘤大小和有无包膜无关（Ｐ＞０．０５）。有转移组肝癌组织中ＩＣＡＭ－１的表达明显高于无转移组，而癌旁组织中两组无差别。结论ＩＣＡＭ－１有可能作为预测肝癌转移、复发及预后的指标。     

肺成纤维细胞对裸鼠肺癌转移影响及其机制的探讨

目的:探讨成纤维细胞在裸鼠肺癌生长和转移过程中的作用。方法:将裸鼠40只随机分成实验组和对照组两组,将H460细胞、H460细胞+成纤维细胞分别接种裸鼠左侧胸腔,观察两组裸鼠共3周。统计其生存率,测量瘤质量,蛋白质印迹法测定动物肿瘤组织内基质金属蛋白酶1(MMP-1)的表达水平,明胶酶普法检测血清中基质金属蛋白酶-2(MMP-2)的表达。结果:裸鼠肺癌胸腔内接植成瘤率为100%,胸腔种植瘤模型逐渐出现远处转移。对照组于第18、19、20和21天共有4只裸鼠死亡,实验组分别于第12、15和19天共有3只死亡,第18、20天各有2只死亡,第21天有3只死亡,差异有统计学意义,P=0.004 8;实验组裸鼠死亡时间均较对照组早。实验组瘤质量(1.711±0.175)高于对照组(1.521±0.369),P=0.000。实验组动物血液中MMP-2的表达及瘤组织内MMP-1表达水平明显高于对照组,P<0.05。结论:成纤维细胞能够促进裸鼠肺癌的生长和转移,其作用机制可能是同肿瘤细胞相互协同,促进裸鼠体内MMP-1、MMP-2的表达有关。     

Characterization of somatic mutations and pathway alterations during hepatocellular carcinoma vascular invasion using next-generation sequencing

BackgroundVascular invasion is an independent risk factors for recurrence and poor prognosis in patients with hepatocellular carcinoma (HCC). However, the molecular mechanisms of HCC vascular invasion are largely unknown. Deciphering the molecular changes associated with the vascular invasion process will aid in the identification of therapeutic targets and treatment for patients with HCC.MethodsDNA was extracted from tumor specimens and blood samples collected from 50 patients with HCC. Next-generation sequencing (NGS) was performed to detect HCC gene variants. Bioinformatics methods were used to comprehensively analyze the three sets of sequencing data grouped by vascular invasion, including differences in tumor mutation burden (TMB), mutation characteristics, and alterations in signaling pathways.ResultsBioinformatics analysis detected a total of 762 single nucleotide variants (SNVs). The TMB was not significantly different between patients with macrovascular invasion, microvascular invasion (MVI), or avascular invasion. Ten genes related to prognosis or recurrence, and one oncogene related to vascular invasion were screened. Compared with the avascular invasion cluster, the variant genes in the macrovascular and MVI clusters were mainly enriched in the thyroid hormone signaling pathway. In addition, macrovascular invasion variant genes were also enriched in the insulin signaling pathway and the Fanconi anemia pathway.ConclusionsSomatic mutations and pathway changes associated with vascular invasion in HCC were identified. The discovery of the molecular drivers of vascular invasion in HCC provides novel insights that can help guide further patient diagnosis and personalized therapy.     

奥沙利铂治疗肝细胞癌耐药的相关机制研究进展

奥沙利铂在我国已被批准用于晚期肝细胞癌（HCC）的治疗,以奥沙利铂为基础的FOLFOX 4方案已成为晚期HCC的标准治疗之一,显著延长了患者的生存期,展现出安全有效、耐受性良好等特点。尽管如此,奥沙利铂治疗HCC的临床疗效仍十分有限,其治疗失败与HCC产生耐药密切相关。本文就奥沙利铂治疗HCC的耐药机制研究进展作一综述,希望能帮助临床和科研工作者了解最新动态,并为他们的工作拓宽思路。     

A novel classification in predicting prognosis and guiding postoperative management after R0 liver resection for patients with hepatocellular carcinoma and microvascular invasion

BackgroundMicrovascular invasion (MVI) is a significant risk factor affecting survival outcomes of patients after R0 liver resection (LR) for hepatocellular carcinoma (HCC). The current classification of MVI is not refined enough to prognosticate long-term survival of these patients, and a new MVI classification is needed.MethodsPatients with HCC who underwent R0 LR at the Eastern Hepatobiliary Surgery Hospital from January 2013 to December 2013 and with resected specimens showing MVI were included in this study with an aim to establish a novel MVI classification. The classification which was developed using multivariate cox regression analysis was externally validated.ResultsThere were 180 patients in the derivation cohort and 131 patients in the external validation cohort. The following factors were used for scoring: α-fetoprotein level (AFP), liver cirrhosis, tumor number, tumor diameter, MVI number, and distance between MVI and HCC. Three classes of patients could be distinguished by using the total score: class A, ≤3 points; class B, 3.5–5 points and class C, >5 points with distinct long-term survival outcomes (median recurrence free survival (mRFS), 22.6, 10.2, and 1.9 months, P < 0.001). The predictive accuracy of this classification was more accurate than the other commonly used classifications for HCC patients with MVI. In addition, the mRFS of class C patients was significantly prolonged (1.9 months vs. 6.2 months, P < 0.001) after adjuvant transcatheter arterial chemoembolization (TACE).ConclusionsA novel MVI classification was established in predicting prognosis of HCC patients with MVI after R0 LR. Adjuvant TACE was useful for class C patients.     

MKP-4在肝细胞癌中的表达及临床意义

目的 探讨MKP-4在肝细胞癌（HCC）中的表达及其临床意义。方法 收集2005年1月至2010年12月在南通大学附属医院行肝癌根治术的100例HCC切除标本及对应癌旁组织,采用免疫组化SP法检测上述组织中MKP-4的表达,并分析其与临床病理特征及预后的关系。另收集8例新鲜HCC及对应癌旁组织,采用Western blotting法半定量检测MKP-4表达。结果 MKP-4蛋白在8例新鲜HCC组织中的表达量为0.611±0.136,显著低于癌旁组织的0.931±0.107（P＜0.05）。HCC组织中MKP-4的高表达率为20％,低于癌旁组织的34％,差异有统计学意义（P＜0.05）。MKP-4表达与组织学分级（P=0.047）、静脉侵袭（P=0.026）和TNM分期（P=0.036）有关。Kaplan-Meier法显示,MKP-4低表达者的中位总生存时间（OS）为52个月,MKP-4高表达者的中位OS未达到,后者OS明显长于前者（P＜0.05）。结论 MKP-4在HCC的发生、发展中起抑癌基因的作用,可能是HCC潜在的治疗靶点和判断预后的分子标志物。     

Quality of life and hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a common and rapidly fatal cancer ranking third among the leading causes of cancer-related deaths. Potentially curative therapies like surgery, transplant and ablation are not an option for most patients as they are often diagnosed when the disease is advanced. Liver directed therapy and oral targeted therapies are used in these patients to prolong life and palliate symptoms of the cancer and associated liver failure. Overall survival remains poor and hence health-related quality of life (HRQoL) is of paramount importance in these patients. As novel therapies are developed to improve outcomes, a comprehensive knowledge of available tools to assess impact on QoL is needed. Hence we reviewed all the studies in HCC patients published within the last 13 years from 2001-2013 which assessed HRQoL as a primary or secondary endpoint. A total of 45 studies and 4 meta-analysis were identified. Commonly used tools were European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 (EORTC QLQ-C30) (15 studies) and the Functional Assessment of Cancer Therapy-Hepatobiliary Questionnaire (FACT-Hep) (14 studies). Of the 45 publications which incorporated HRQoL as end-point only 24 were clinical trials, 17/24 (71%) assessed systemic therapies while 7/24 (29%) assessed liver-directed therapies. Majority of the publications (trials + retrospective reviews) that had HRQoL as an endpoint in HCC patients were studies evaluating liver-directed therapies (23/45 or >50%). We discuss the measures included in the tools, their interpretation, and summarize existing QoL data that will help design future HCC trials.     

Relationship of ethnicity and overall survival in patients treated with sorafenib for advanced hepatocellular carcinoma

Background

Although both the SHARP and the Asian-Pacific trials showed improved overall survival (OS) for sorafenib, the magnitude of benefit was substantially less for Asians, who have a higher prevalence of hepatitis B viral (HBV) infection. Whether the worse prognosis is related to ethnicity or to the etiology of hepatocellular carcinoma (HCC) remains unclear. The aim of this study was to identify prognostic factors among patients with HCC who received sorafenib in British Columbia (BC), which has a sizeable Asian population.

Methods

A total of 255 consecutive patients with advanced HCC who initiated sorafenib from January 2008 to February 2013 were identified using our pharmacy database. Clinicopathological variables and outcomes were retrospectively collected. Prognostic factors were assessed by univariate and multivariate analyses.

Results

Median age was 63 years, 80.2% were men, and 38% were Asian. Among them, 34.5% had HBV and 29.8% had hepatitis C viral (HCV) infection. In addition, 68.6% had cirrhosis and 45.9% had liver-limited disease. Median progression-free and OS were 3.7 [95% confidence interval (CI): 3.3-4.2] and 7.5 months (95% CI: 5.7-9.2), respectively. On multivariate analysis, Eastern Cooperative Oncology Group performance status (ECOG PS) and HCV positivity correlated with better OS (P<0.001 and 0.04, respectively), but ethnicity did not (P=0.622).

Conclusions

When treated with sorafenib at the same institution, Asians and Caucasians with advanced HCC had similar OS. ECOG PS and HCV were the only significant prognostic factors. A higher proportion of HCV positivity might explain why the SHARP trial achieved better OS when compared to the Asian-Pacific trial.     

Cortactin overexpression correlates with poor prognosis in hepatocellular carcinoma

Objective: To investigate cortactin expression in hepatocellular carcinoma (HCC) and explore its significance in the prognosis of HCC patients.Methods: Immunohistochemistry was performed for paraffin samples of 119 pairs of HCC tissues (HCCs) and paratumorous liver tissues (PTLTs) to evaluate cortactin expression. The cortactin expression difference in HCCs and PTLTs were analyzed by the McNemar's test. The relationship of cortactin expressions in HCCs and clinicopathologic factors was analyzed with Mann-Whitney U test. The Kaplan-Meier method and log-rank test were employed to compare the overall survival between Cortactin negative expression group, weak expression group and strong expression group. Expression of cortactin was further determined in 19 pairs of fresh HCCs and PTLTs specimens with Western blotting.Results: Cortactin expression rate was significantly higher in HCCs (53/119, 44.5%) than that in PTLTs (2/119, 1.7%) (P<0.001). The upregulated cortactin expression in HCCs was significantly correlated to absence of capsule formation (P=0.012), vascular invasion (P=0.037) and high Edmondson-Steiner grade (P=0.020), and predicted shorter overall survival. Western blotting demonstrated that cortactin expression was upregulated in 9 out of 19 HCCs (47.4%) compared to corresponding PTLTs.Conclusion: Cortactin expression is upregulated in HCC and is related to shorter overall survival of patients, suggesting that cortactin might play roles in the metastasis of HCC and predict a poor prognosis of HCC patients.     

BMP4 通过诱导EMT 促进肝癌迁移侵袭*

目的:检测BMP 4 在肝癌中的表达并探讨BMP 4 在诱导肝癌EMT 中的作用,进而研究其对肝癌细胞迁移侵袭能力的影响。方法:采用免疫组织化学方法检测肝癌组织中BMP 4 的表达,分析其与肝癌临床病理资料之间的关系。将BMP 4 表达质粒转染至肝癌细胞系HepG2 中,诱导BMP 4 外源性过表达。观察BMP 4 转染前、后HepG2 的细胞形态学改变;Westernblot检测转染前、后HepG2 中BMP 4、EMT 相关蛋白（E-cadherin、Vimentin）表达变化情况;划痕和侵袭实验检测BMP 4 对细胞迁移侵袭能力的影响。结果:BMP 4 与患者的年龄、病理分级、临床分期、不良预后密切相关。BMP 4 过表达后HepG2 呈现典型的EMT 形态学改变,E-cadherin 表达下调、Vimentin 表达上调、细胞的迁移侵袭能力显著增强。结论:BMP 4 与肝癌临床病理资料密切相关,并可能通过诱导EMT 促进肝癌细胞的迁移侵袭能力。      

Experimental study on enhancement of the metastatic potential of portal vein tumor thrombus-originated hepatocellular carcinoma cells using portal vein serum

Objective： Portal vein metastasis of hepatocellular carcinoma（HCC） results in a poor prognosis and seriously affects the survival rate of patients. The mechanism underlying the formation of portal vein tumor thrombus（PVTT） is complex and is not yet fully understood. This study was conducted to investigate the impact of portal vein blood on the proliferation, metastasis, invasion and apoptosis of PVTT cells and to explore its possible mechanisms, which was expected to lay a foundation for ascertaining the mechanism underlying the portal vein metastasis of HCC.
Methods： Peripheral blood and portal vein blood were collected from patients with HCC, and the sera from these two sources were used to culture the PVTT-originated HCC cell line CSQT-2. The cells were collected after 24 h, and flow cytometry was performed to detect cell proliferation, cell cycle stages and apoptosis. Transwell migration and invasion assays were applied to detect the metastasis and invasion of the cells in each group. The changes in the expression of MMP-2 and MMP-9 in cells were detected via Western blotting. The contents of IL-12, IFN-γ, IL-1β, IL-2 and TNF-α in the two groups of sera were quantified using corresponding kits.
Results： Compared with the group of cells cultured with peripheral serum, the cells cultured with portal vein serum showed significantly lower apoptosis（P〈0.01）, significantly enhanced cell metastasis and invasion（P〈0.01）, whereas cell proliferation and the stages of the cell cycle did not differ significantly（P〉0.05）. A significantly increased expression level of MMP-2 has been observed in tumor cells treated portal vein serum. In addition, compared with peripheral serum, the content of IL-12 was significantly decreased in portal vein serum（P〈0.05）, while the contents of IFN-γ, IL-1β, IL-2, and TNF-α did not differ significantly（P〈0.05）.
Conclusions： Portal vein serum from HCC patients could inhibit the apoptosis of PVTT-originated HCC cells and promote cell met