不同转移潜能肝癌细胞株血管内皮生长因子及其受体的表达和意义

目的 探讨血管内皮生长因子受体-1(VEGFR-1)在不同转移潜能肝癌细胞株中的表达及其意义.方法 应用半定量RT-PCR、酶联免疫吸附试验和(或)Western blot检测4株不同转移潜能的肝癌细胞株及一株正常肝细胞中VEGFR-1、VEGF-A、VEGF-B及VEGFR-2的mRNA和(或)蛋白质表达.结果 MHCC97-H、MHCC97-L和SMMC-7721细胞均有VEGFR-1 mRNA和蛋白质表达,且VEGFR-1 mRNA和蛋白质在MHCC97-H中的表达高于MHCC97-L、SMMC-7721的表达,两者比较,差异有统计学意义(P＜0.05),而其配体VEG-A和VEGF-B在检测的4种肝癌细胞株和正常肝细胞株L-02中均有表达.同时在检测的四种肝癌细胞株和正常肝细胞株L-02中均能检测到VEGFR-2 mRNA和蛋白质表达,但各组间表达差异无统计学意义(P＞0.05).结论 具有转移潜能的肝癌细胞株有VEGFR-1表达,而且其表达强弱与肝癌细胞株的转移潜能呈正相关,VEGFR-1的表达可能促进了肝细胞癌的侵袭转移.     

膜-细胞骨架联接分子ezrin对肝癌细胞系生长和侵袭能力的影响

目的探讨膜-细胞骨架联接蛋白ezrin在肝细胞肝癌生长和转移过程中的作用。方法分别应用免疫荧光、逆转录聚合酶链反应（RT—PCR）和Western blot检测ezrin和骨架蛋白在不同转移潜能肝癌细胞系中的表达。选取高转移潜能SF7721（SMMC-7721经转基因后稳定表达肝细胞生长因子，从而获得高转移潜能的细胞系）和MHCC97-H细胞系为研究对象。通过RNA干扰技术下调SF7721和MHCC97-H细胞系中ezrin蛋白的表达，观察其运动和侵袭能力的变化：通过四甲基偶氮唑盐检测细胞增殖能力变化；电子显微镜观察细胞伪足；Transwell检测细胞的运动侵袭能力。结果免疫荧光显示ezrin和骨架蛋白表达于细胞质，且双色荧光证实两者存在共表达，高转移潜能细胞系SF7721。MHCC—I、MHCC97-Hezrin和骨架蛋白的表达明显高于低转移潜能细胞系SMMC-7721、Hep3B、HepG2细胞（x^2=13．277，P=0．010；x^2=21．815，P〈0．01）。D-肌动蛋白在高低转移潜能细胞系的表达差异无统计学意义。通过RNA干扰技术抑制ezrin蛋白表达后。SF7721和MHHC97-H的细胞的增殖和侵袭能力均显著下降。结论ezrin和骨架蛋白的过表达与肝癌的转移潜能相关，通过下调ezrin的表达可明显抑制肝癌细胞系SMMC-7721和MHCC97-H细胞的增殖和运动侵袭能力。     

组织蛋白酶-S在肝癌细胞中的表达及意义

目的研究组织蛋白酶-S（Cat-S）在肝癌细胞和正常肝细胞中的表达情况,探讨其与肝细胞癌（HCC）发生、发展的关系。方法体外培养高转移人肝癌细胞株MHCC97-H、低转移人肝癌细胞MHCC97-L和正常肝细胞LO2,采用荧光定量PCR技术定量检测其Cat-S mRNA表达水平。结果 Cat-S mRNA在LO2细胞中呈弱表达,MH-CC97-H细胞及MHCC97-L细胞表达均明显高于LO2细胞（P〈0.05）;MHCC97-H细胞表达明显高于MHCC97-L细胞（P〈0.05）。结论 Cat-S在高转移、低转移肝癌细胞株和正常肝细胞株中的表达存在差异性,此可能与肝癌的发生发展有关;Cat-S可作为肝癌诊治的分子靶标。     

不同转移潜能人肝癌HCCLM3、MHCC97-L细胞系血管生成拟态基因的表达

目的采用细胞外基质与粘连分子Real-time PCR基因芯片筛选高、低转移潜能人肝癌HCCLM3、MHCC97-L细胞系差异表达的血管生成拟态基因,探讨血管生成拟态的机制。方法建立HCCLM3、MHCC97-L三维培养体系,倒置显微镜下观察其血管样结构形成情况。细胞培养24 h后,采用细胞外基质与粘连分子Real-timePCR基因芯片对HCCLM3、MHCC97-L的表达基因进行筛选。结果培养24 h,HCCLM3形成的血管样结构较MHCC97-L的长（P〈0.01）。筛选出20个差异基因,其中CD44、COL6A1、COL4A2、ECM1、ITGA1、ITGA3、MMP1、MMP2、SPP1、TNC、COL6A2、MMP7和MMP10共13个基因在HCCLM3中的表达较MHCC97-L明显上调,CTNND2、COL12A1、COL14A1、ITGAL、TIMP3、MMP16和VTN共7个基因明显下调。结论高、低转移潜能人肝癌HCCLM3、MHCC97-L细胞系体外形成血管生成拟态有差异,其原因可能与HCCLM3差异表达某些细胞外基质和粘连分子相关基因有关。     

Op18：一个潜在的肝癌诊断标志物

目的探讨人原癌基因蛋白18（Op18）在肝癌细胞和组织中的表达及临床意义。方法应用RT-PCR、Western blot和免疫组化染色法检测常见肝癌细胞和88对配对肝癌组织及癌旁组织中Op18的表达。结果 Hep3B、SMMC-7721、MHCC97L、MHCC97H、HCCLM3和HCCLM6等常见肝癌细胞株中均见Op18表达;配对肝癌及癌旁组织中,RT-PCR发现肝癌组织中Op18阳性率达68.75%（11/16）,Western blot发现Op18的阳性率为56.25%（9/16）,免疫组化检测发现肝癌组织中Op18的阳性率为65.28%（47/72）,P〈0.05;Op18的表达与肿瘤大小数目、临床分化、门脉侵犯和TMN分期相关（P〈0.05）。结论 Op18可能是一个潜在的肝癌诊断标志物。     

人肝癌细胞系Slit/Robo启动子甲基化状态及基因表达的研究

目的 研究Slit/Robo信号通路相关基因Slit1、Slit2、Slit3和Robo1、Robo3在多种人肝癌细胞系中的表达及甲基化状态,探讨与肝癌发生和发展的关系. 方法提取9种人肝细胞癌细胞株(Hep3B、HepG2、PLC/PRF/5/PRF/5、SMMC-7721、BEL-7402、MHCC97-H,MHCC97-L、LM3、LM6)及对照细胞株L02的基因组DNA和总RNA,采用半定量逆转录聚合酶链反应技术和甲基化特异性聚合酶链反应技术检测Slit1、Slit2,Slit3和Robo1,Robo3基因的基因表达水平与启动子甲基化状态.实验数据应用Paired t检验. 结果 Slit1、Slit2、Slit3基因除个别细胞株外,在不同转移潜能细胞株中均发生了DNA甲基化,同时Slit1和Slit3在mRNA水平几乎均不表达,Slit2基因表达程度在不同转移潜能的细胞株之间存在差异,随着转移潜能的增加表达大致呈下降趋势.作为Slit2受体的Robo1基因在10株肝癌细胞株中均发生甲基化修饰,但除在SMMC-7721、BEL-7402、L02不表达外,其余7种细胞株均有表达.Robo3基因相关CpG岛在9种肝癌细胞株中均未发生甲基化,同时其在mRNA水平均无表达. 结论 Slit/Robo可能在肝癌发生和发展中发挥作用.而Robo3则在肝癌中不发生表达而且其表达沉默可能不受甲基化方式调控.     

DLC-1基因表达与肝细胞癌复发转移的关系

目的已报道人类染色体8p缺失可能与肝癌转移有关,本文应用实时定量聚合酶链反应(RQ- PCR)研究位于8p的DLC-1基因mRNA表达与肝细胞癌侵袭转移的关系。方法收集51例复旦大学中山医院外科手术切除的肝细胞癌(HCC)及癌旁正常组织标本,根据临床病理学指标,分为高低侵袭性两组,用RQ-PCR对不同侵袭性HCC之间的DLC-1基因表达进行分析。对不同侵袭转移潜能MHCC97-L、MHCC97-H、HCCLM3、Hep3B及HepG2肝癌细胞系用同样方法分析DLC-1基因的表达差异。结果非转移细胞系Hep3B和HepG2与转移细胞系MHCC97-L、MHCC97-H和HCCLM3之间DLC—1基因表达差异有统计学意义(P相似文献   

不同转移潜能肝癌细胞株的差异蛋白质组的二维液相色谱分析

目的:对不同转移潜能肝癌细胞株MHCC97- H(高转移)和MHCC97-L(低转移)差异表达的蛋白质进行二维液相色谱分离和MALDI-TOF质谱鉴定.方法:将肝癌细胞株MHCC97-H和MHCC97- L细胞裂解样品按蛋白质PI进行一维的色谱聚焦分离,然后每个PI组分再按疏水性经二维反相无孔硅胶HPLC分离,利用ProteoVue软件将UV光吸收图谱转换成PI对疏水性的胶图,再利用DeltaVue软件比较升高或降低的差异蛋白.收集差异蛋白峰进行胰酶酶解,然后进行MALDI-TOF质谱鉴定.结果:2D图谱显示共有72个差异蛋白条带,共鉴定出了9个差异蛋白.分别为M2型丙酮酸激酶、ATP合成酶α亚单位、热休克蛋白60、Toll样受体9、含黄素单加氧酶、钙网硬蛋白前体、锰超氧化物岐化酶、nm23-H1、G-蛋白偶连受体激酶5;其中4个蛋白在高转移细胞株MHCC97-H中表达升高,5个蛋白在低转移细胞株MHCC97-L表达升高.结论:这些差异蛋白可能在肝癌的转移中起关键作用.     

肝癌转移相关的骨桥蛋白表达及其糖基化的改变

目的 检测骨桥蛋白(OPN)在不同转移潜能肝癌细胞株及肝癌组织中的蛋白表达水平及其糖基化水平,探讨OPN糖基化改变与肝癌转移的相关性及其意义. 方法 用免疫组织化学和Western blot法检测人肝癌组织(非转移组6例、转移组7例)及不同转移潜能人肝癌细胞株(L02、Hep3B、MHCC97L、MHCC97H、HCCLM3、HCCLM6)中OPN蛋白水平;采用免疫沉淀技术纯化肝癌组织中的OPN蛋白,并采用多重凝集素印迹技术检测转移与非转移肝癌组织中OPN糖基化水平差异.数据统计采用t检验和方差分析.结果 OPN在不同转移潜能肝癌细胞株的表达差异具有统计学意义(F=5.04,P＜0.01).在肝癌组织中,转移组OPN蛋白表达水平明显高于非转移组(t=2.447,P＜0.05),相对吸光度值分别为0.69±0.21和0.45±0.14.免疫沉淀技术成功纯化肝癌组织中的OPN蛋白,后续的凝集素印迹结果显示:与非转移组相比,转移组OPN蛋白对凝集素朝鲜槐、红腰果E型、蔓陀罗、刀豆素A的亲和力较低(P值均＜0.05).结论 OPN蛋白表达水平与肝癌转移潜能增强呈正相关;OPN蛋白的α2,3-唾液酸、平分型GlcNAc、多天线、偏二天线的糖链、高甘露糖型N-糖等糖基化水平改变可能与肝癌转移潜能增高有关.     

不同分化肝癌细胞株中上皮细胞黏附分子、CD90及CD24的表达分析

目的 验证不同分化肝癌细胞株MHCC97L、MHCC97H和HCCLM3的恶性表型,并探索其中肿瘤干细胞表面标志分子CD90、上皮细胞黏附分子(EpCAM)及CD24的表达情况.方法 Transwell侵袭小室检测三种不同分化细胞株MHCC97L、MHCC97H和HCCLM3的侵袭能力,细胞计数试剂盒8检测三种不同分化细胞株对奥沙利铂的敏感性,流式细胞仪检测肿瘤干细胞表面标志分子CD90、EpCAM、CD24在三种细胞株中的表达情况.多个样本均数比较采用方差分析. 结果 MHCC97L、MHCC97H和HCCLM3三种细胞穿膜细胞数存在明显差异,HCCLM3(30.57±8.95)个＞MHCC97H (21.33±4.17)个＞HCC97L (9.33±3.85)个,F=38.484,P＜ 0.01.对奥沙利铂的半数抑制浓度IC50 HCCLM3为(36.57±6.95)μmol/L,MHCC97H (26.35±3.88) μmol/L,MHCC97L (17.68±3.25) μmol/L,F=40.181,P＜0.01. CD90在MHCC97L、MHCC97H和HCCLM3细胞中的表达比例分别为0.92％±0.21％、1.98％±0.23％和2.55％±0.34％,差异有统计学意义(F=60.481,P＜0.01);EpCAM在三种细胞中的表达比例分别为2.11％±0.32％、3.23％±0.18％和4.38％±0.49％,表达差异也有统计学意义(F=84.800,P＜0.01);CD24在三种细胞中的表达比例分别为0.68％±0.37％、1.22％±0.26％和1.36％±0.24％,表达差异有统计学意义(F=15.677,P＜0.01).结论 分化越低的肝癌细胞株侵袭能力越强,对化疗敏感性越差,其中的肝癌干细胞比例则越高.     

Lentiviral-mediated microRNA-26b up-regulation inhibits proliferation and migration of hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is a frequently occurred malignancy worldwide with a high mortality. The treatment for HCC is still controversial. Emerging evidences have demonstrated that microRNAs (miRs) play a role in HCC. This study aims to investigate the effects of lentiviral-mediated miRNA-26b (miR-26b) on the proliferation and metastasis of HCC cells. The normal hepatic cell line HL-7702 and HCC cell lines HepG2 (without metastatic potential), SMMC-7721 (with low metastatic potential) and MHCC97H (with high metastatic potential) were purchased for our experiment. The lentiviral-mediated miR-26b overexpression (miR-26b-LV) and low expression (sh-miR-26b) were constructed to transfect the cells. The miR-26b expression and expressions of Karyopherin α-2 (KPNA2), matrix metalloproteinase 1 (MMP-1), MMP-7 and MMP-14 were determined by RT-qPCR and western blot analysis. The proliferation and metastasis of transfected HCC cells were detected by MTT and Transwell assay respectively. The miR-26b expressions were decreased significantly in MHCC97H cells. With lentiviral-mediated miR-26b overexpression, the proliferation and migration of HepG2, MHCC97H and SMMC-7721 cells were decreased significantly. The RT-qPCR and western blot analysis results revealed that the mRNA and protein expressions of KPNA2, MMP-1, MMP-7 and MMP-14 were decreased by lentiviral-mediated miR-26b overexpression. All the above indexes in the HepG2, MHCC97H and SMMC-7721 cells treated by sh-miR-26b exhibited opposite trends. These results show that overexpressed miR-26b could inhibit the proliferation and metastasis of HCC cells significantly, which provides a novel target and theoretical foundation for the treatment of HCC.     

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.     

Identification of side population cells in human hepatocellular carcinoma cell lines with stepwise metastatic potentials

Purpose  To identify the side population (SP) cells from four hepatocellular carcinoma (HCC) cell lines with stepwise metastatic potentials. Methods  SP cells were sorted from HCCLM3, MHCC97-H, MHCC97-L and Hep3B by flow cytometry, and then analyzed by differentiation study, clonogenic assay, chemoresistance study and tumorigenicity assay in vivo. The expression of ABCG₂ in SP cells was detected by immunocytochemistry, western blotting and real-time quantitative PCR, respectively. Results  There was significant difference in SP proportion among HCCLM3, MHCC97-H, MHCC97-L and Hep3B (28.7 ± 1.6%, 14.5 ± 0.6%, 4.2 ± 0.4%, 0.9 ± 0.1%, respectively, P < 0.01). All the SP cells showed similar characteristics of self-renewal, high clonogenicity, remarkable chemo-resistance and high expression of ABCG₂. As low as 2,000 SP cells could initiate tumors in non-obese diabetic/severe combined immunodeficiency mice successfully. Conclusions  SP cells purified from HCC cell lines harbors cancer stem cell-like properties, and may be related to the metastatic potentials and therapeutic-resistance of HCC. Guo-Ming Shi and Yang Xu contributed equally to this work. This is an original work by all the authors and no previous presentations, reports, or publications contain any material that appears in the article.     

Stepwise metastatic human hepatocellular carcinoma cell model system with multiple metastatic potentials established through consecutive in vivo selection and studies on metastatic characteristics

PURPOSE: To establish a "stepwise metastatic human hepatocellular carcinoma (HCC) cell model system" for in-depth study of the underlying mechanisms of HCC metastasis. METHODS: Using MHCC97- a metastatic human hepatocellular carcinoma (HCC) cell line reported in 1999-as the parent cells, we subsequently established three cell lines (MHCC97-L, HMCC97-H, and HCCLM3) with increasing spontaneous metastatic potential. Now, the fourth cell line with unique multiple metastatic characteristics has been established by six rounds of in vivo selection. RESULTS: This cell line, designated as HCCLM6, is a polygonal epithelial cell with hypotriploid karyotype, the modal chromosomes are 55-58, and marker chromosomal abnormalities include i(1) (q10), i(8)(q10), der (4) t(4;8)(q31;q22), i(X)(q10). The cell population doubling time was 32 h. Fluorescent PCR showed HBV DNA integration in the cellular genome. Thirty-five days after HCCLM6 was injected subcutaneously into BALB/c nude mice, prominent lung metastases occurred in 100% of the recipient animals. When tumor tissue was orthotopically implanted into the liver of nude mouse, widespread loco-regional and pulmonary metastases occurred. Inoculation of this cell into the footpad of nude mice also produced 75% regional lymph node metastasis. Compared with MHCC97-L which was not metastastatic via subcutaneous or footpad inoculation and 40% metastatic via orthotopic inoculation, HCCLM6 had increased expression of matrix metalloproteinase (MMP-2 and MMP-9) and cytokeratin 19 (CK19), and decreased expression of Rb2/p130. The establishment of this new cell line has completed our stepwise metastatic HCC cell mode system, which was characterized by a similar genetic background but with significant differences in spontaneous metastasis behavior. CONCLUSIONS: The study supports the theory that cancer metastasis is a highly selective dynamic process and the cell model system could be a useful platform for the study of HCC metastasis.