足叶苦素抑制胰岛素样生长因子Ⅰ受体表达对肝癌细胞增殖与运动的影响

目的 观察足叶苦素(PPP)抑制胰岛素样生长因子Ⅰ受体(IGF-IR)后对肝癌细胞增殖和运动的影响. 方法 体外培养肝细胞L02和肝癌细胞(Bel-7404、Bel-7402、HepG2和Huh-7),以Western blot分析IGF-IR表达,以磺酰罗丹明B法分析细胞活性;在PPP抑制IGF-IR后,以流式细胞术分析细胞周期,以AnnexinV-FITC法分析细胞凋亡;在z-VAD-FMK抑制caspases后,以均质发光法检测caspase-3/7活性;划痕试验分析细胞运动能力.对数据进行析因设计方差分析,采用t检验或one-way ANOVA法进行组间比较.结果 肝细胞L02中几乎检测不到IGF-IR,各肝癌细胞中IGF-IR呈不同程度表达.PPP抑制肝癌细胞增殖呈时间和剂量依赖性,经1.0μmol/L PPP处理HepG2细胞24h,G1、S期和G2/M期细胞比例分别为2.1％±0.4％、11.0％±0.7％和87.1％±0.6％,且划痕不愈合;caspase-3/7活性显著增加(t=11.83,P＜0.01);HepG2细胞凋亡率为16.4％±0.4％,明显高于对照组的5.8％±0.2％ (t=14.05,P＜ 0.01);z-VAD-FMK抑制caspases表达,HepG2细胞凋亡率为11.3％±0.7％,明显高于对照组的5.8％±0.2％ (t=11.83,P＜0.01). 结论 IGF-IR表达与肝癌细胞增殖、运动和凋亡相关,可能是肝癌分子靶向治疗的有效靶点.     

TRAIL-induced apoptosis of hepatocellular carcinoma cells is augmented by targeted therapies

AIM: To analyze the effect of chemotherapeutic drugs and specific kinase inhibitors, in combination with the death receptor ligand tumor necrosis factor-related apoptosis inducing ligand (TRAIL), on overcoming TRAIL resistance in hepatocellular carcinoma (HCC) and to study the efficacy of agonistic TRAIL antibodies, as well as the commitment of antiapoptotic BCL-2 proteins, in TRAIL-induced apoptosis.METHODS: Surface expression of TRAIL receptors (TRAIL-R1-4) and expression levels of the antiapoptotic BCL-2 proteins MCL-1 and BCL-xL were analyzed by flow cytometry and Western blotting, respectively.Knock-down of MCL-1 and BCL-xL was performed by transfecting specific small interfering RNAs. HCC cells were treated with kinase inhibitors and chemotherapeutic drugs. Apoptosis induction and cell viability were analyzed via flow cytometry and 3-(4,5-Dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide assay.RESULTS: TRAIL-R1 and -R2 were profoundly expressed on the HCC cell lines Huh7 and Hep-G2.However, treatment of Huh7 and Hep-G2 with TRAIL and agonistic antibodies only induced minor apoptosis rates. Apoptosis resistance towards TRAIL could be considerably reduced by adding the chemotherapeutic drugs 5-fluorouracil and doxorubicin as well as the kinase inhibitors LY294002 [inhibition of phosphoinositol-3-kinase (PI3K)], AG1478 (epidermal growth factor receptor kinase), PD98059 (MEK1), rapamycin (mammalian target of rapamycin) and the multi-kinase inhibitor Sorafenib. Furthermore, the antiapoptotic BCL-2 proteins MCL-1 and BCL-xL play a major role in TRAIL resistance: knock-down by RNA interference increased TRAIL-induced apoptosis of H CC cells. Additionally,knock-down of MCL-1 and BCL-xL led to a significant sensitization of HCC cells towards inhibition of both c-Jun N-terminal kinase and PI3K.CONCLUSION: Our data identify the blockage of survival kinases, combination with chemotherapeutic drugs and targeting of antiapoptotic BCL-2 proteins as promising ways to overcome TRAIL resistance in HCC.     

Natural and activated cytotoxic lymphocytes reactivity to human hepatocellular carcinoma cell lines in hepatocellular carcinoma patients

The status of cellular cytotoxic activity in Hepatocellular Carcinoma (HCC) patients was compared to that in normal individuals by testing the cytotoxicity against K562 and five established HCC cell line targets. Natural killer (NK) activity of fresh peripheral blood mononuclear (PBM) cells in HCC patients to K562 cell line target was lower than that in normal donors. NK activity of unstimulated PBM cells from either source was minute against all five HCC cell line targets. Three different activation systems were employed to examine the cellular cytotoxicity of activated PBM cells: (1) conventional mixed lymphocyte culture (MLC), (2) allogeneic mixed lymphocyte tumor culture (MLTC), and (3) lymphokine-activated killer (LAK) cell culture. The cytotoxic effects of PBM cells in all three activation conditions were significantly lower in HCC patients than in normal donors (P less than 0.05 to P less than 0.01). These results suggest that, in addition to naturally present NK cells, the degree of in vitro activation of PBM cells may also have decreased in HCC patients.     

Effect of human noxa on irinotecan-induced apoptosis in human gastric carcinoma cell lines

BACKGROUND/AIMS: A protein BH3-only member bcl-2 family, Noxa is a proapoptotic mediator for p53-induced apoptosis. We analyzed the effect of Noxa on p53-induced apoptotic gastric carcinoma cell lines. METHODOLOGY: The expressions of human Noxa (hNoxa) mRNA on human gastric carcinoma cell lines were assessed with RT-PCR. Further, hNoxa antisense and sense S-oligodeoxynucleotide (ODN) were used to analyze the effect of hNoxa on p53-induced apoptotic gastric carcinoma cell lines. RESULTS: Various levels of hNoxa mRNA expression were detected in all gastric cell lines. MKN45 that has wild-type p53 showed severe inhibition by irinotecan compared with MKN28, which has mutated p53. Cell growth under hNoxa antisense S-ODN treatment did not differ from that under sense S-ODN treatment in MKN28. On the other hand, the suppression of cell growth in MKN45 decreased with hNoxa antisense S-ODN treatment as compared to hNoxa sense S-ODN treatment. MKN45 cells exhibited DNA fragmentation clearly after 24 hr of 3 mM hNoxa sense S-ODN treatment. The DNA fragmentation in MKN45 was inhibited by hNoxa antisense S-ODN treatment. CONCLUSIONS: It seems that hNoxa plays an important role in induction of apoptosis on p53 wild type gastric carcinoma cell lines.     

人类肝细胞肝癌的细胞凋亡和癌组织内血管形成的调控

目的研究肝细胞肝癌（ＨＣＣ）中细胞凋亡和血管形成的相互关系．方法采用原位末端标记技术和抗第Ⅷ因子的抗体分别检测ＨＣＣ凋亡指数和瘤内微血管的密度，同时用免疫组化法检测血管内皮细胞生长因子（ＶＥＧＦ）及其受体ｆｌｔ１和肝（癌）细胞凋亡启动基因ｆａｓ的表达．结果ＶＥＧＦ的表达主要见于微血管形成处的血管内皮细胞及其周围的瘤细胞和胆管上皮细胞，阳性率为８２７％，在ＨＣＣ组织中的表达具有普遍性．平均微血管计数为２８～４１６个／２００倍视野．在瘤组织中，微血管的密度越高的区域，ＶＥＧＦ的表达就越丰富．ＶＥＧＦ的受体ｆｌｔ１的表达见于血管内皮细胞和部分窦内皮细胞，特别是新生的微血管内皮细胞．ＶＥＧＦ阳性区域很少或不表达Ｆａｓ抗原．凋亡细胞的分布和凋亡指数与肿瘤的微血管密度呈明显的负相关（ｒ＝－０９１７，Ｐ＜００１）．结论ＨＣＣ中血管形成主要是由ＶＥＧＦ／ｆｌｔ１系统介导的．血管形成丰富的区域，细胞凋亡的敏感性和发生率降低     

Radiation-induced apoptosis and cell cycle alterations in human carcinoma cell lines with different radiosensitivities

Radiosensitivity of examined human neoplastic cell lines was assessed with the aid of MTT assay. Differences between radiosensitive and radioresistant human neoplastic cell lines were as follow: a) radiation-induced apoptosis detected by flow cytometry was apparent in the most radiosensitive (i.e. CH-1 ovarian carcinoma cell line), but not in the radioresistant (i.e. SKOV-3 ovarian carcinoma) cell lines, b) radiation-induced G2/M arrest appeared early after irradiation (6 hours) in both the radioresistant SKOV-3 cells and in the radiosensitive CH-1 human ovarian carcinoma cell line, but a different pattern was observed 24 hours after irradiation with 2 Gy dose with G2/M arrest only in radiosensitive cell line. The radiosensitivity and resistance to radiation-induced apoptosis in the radioresistant human breast carcinoma MDA-MB-231 cell line were similar to those observed in SKOV-3 cells. These data suggest that radiation-induced apoptosis and cell cycle alterations can predict radiosensitivity at least in some examined human malignant cells in vitro.     

Chemical sensitization and regulation of TRAIL-induced apoptosis in a panel of B-lymphocytic leukaemia cell lines

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) effectively kills tumour cells but not normal cells. We investigated TRAIL sensitivity and the TRAIL-induced apoptosis signalling pathway in a panel of B-lymphocytic leukaemia cell lines. Depending upon TRAIL sensitivity, leukaemia cells could be divided into three groups: highly sensitive, moderately sensitive and resistant. TRAIL receptor-2 (DR5) plays an important role in transducing apoptosis signals. DR5 was internalized into the cytoplasm where it recruited FAS-associated death domain protein (FADD) under TRAIL stimulation in both sensitive and resistant cells. However, the active form of caspase-8 was recruited to FADD and only sensitive cells showed increased caspase-8 activity upon TRAIL stimulation. The caspase-8 specific inhibitor, Z-IETD, impaired caspase-8 activation and completely abrogated TRAIL-induced apoptosis. These results suggest that TRAIL resistance in B-lymphocytic leukaemia cells is due to negative regulation at the level of caspase-8 activation and that caspase-8 activation is an indispensable process in TRAIL-induced apoptosis. However, FADD-like interleukin-1 beta-converting enzyme inhibitory protein (c-FLIPL) was similarly expressed and down-regulated after TRAIL stimulation in both sensitive and resistant cells. Interestingly, in some cell lines, TRAIL sensitivity and caspase-8 activity was enhanced or restored with the treatment of cycloheximide (CHX). In addition, X-linked inhibitor of apoptosis (XIAP) levels decreased significantly and rapidly following treatment with CHX. Down-regulation of XIAP may be responsible for enhancement or restoration of TRAIL sensitivity after CHX treatment in B-lymphocytic leukaemia cells.     

香芹酚对肝细胞癌HepG2细胞凋亡的诱导作用及其分子机制

目的:探讨香芹酚(carvacrol,CV)对人肝癌细胞(HepG2)的抗癌作用及其分子机制.方法:予以不同浓度的香芹酚(0.00、0.05、0.10、0.20、0.40 mmol/L)处理肝癌细胞HepG2后,采用四甲基偶氮唑蓝(MTT)比色法检测细胞活力;Hoechst33258染色法及流式细胞仪(FCM)技术检测...     

Augmentation of tumor necrosis factor family-induced apoptosis by E3330 in human hepatocellular carcinoma cell lines via inhibition of NFκB

AIM: To investigate the reduction of cell viability in human hepatocellular carcinoma (HCC) cell lines induced by inhibition of nuclear factor κB (NFκB).METHODS: HLE, SKHep1, and HepG2 were incubated and E3330 was used to compare the stimulation of some chemotherapeutic drugs with that of TNF family, Fas ligand, TNFα and TNF-related apoptosis-inducing ligand (TRAIL) at the point of the reduction of cell viability by inhibiting NFκB.RESULTS: E3330 decreased NFκB levels in HLE cells stimulated by TNF and TRAIL. The cytotoxicity of the combination of TRAIL, TNFα, Fas ligand, and E3330increased synergistically in a dose-dependent manner compared to either E3330 alone in all HCC cell lines by MTT assay. However, the combination of some chemotherapeutic drugs and E3330 did not decrease the cell viability.CONCLUSION: Inhibition of NFκB sensitizes human HCC cell lines to TNF-mediated apoptosis including TRAIL, and TRAIL-based tumor therapy might be a powerful potential therapeutic tool in the treatment of human HCC.     

Augmentation of tumor necrosis factor family-induced apoptosis by E3330 in human hepatocellular carcinoma cell lines via inhibition of NFκB

AIM: To investigate the reduction of cell viability in human hepatocellular carcinoma (HCC) cell lines induced by inhibition of nuclear factor κB (NFκB).METHODS: HLE, SKHep1, and HepG2 were incubated and E3330 was used to compare the stimulation of some chemotherapeutic drugs with that of TNF family, Fas ligand, TNFα and TNF-related apoptosis-inducing ligand (TRAIL) at the point of the reduction of cell viability by inhibiting NFκB.RESULTS: E3330 decreased NFκB levels in HLE cells stimulated by TNF and TRAIL. The cytotoxicity of the combination of TRAIL, TNFα, Fas ligand, and E3330increased synergistically in a dose-dependent manner compared to either E3330 alone in all HCC cell lines by MTT assay. However, the combination of some chemotherapeutic drugs and E3330 did not decrease the cell viability.CONCLUSION: Inhibition of NFκB sensitizes human HCC cell lines to TNF-mediated apoptosis including TRAIL, and TRAIL-based tumor therapy might be a powerful potential therapeutic tool in the treatment of human HCC.     

Augmentation of tumor necrosis factor family-induced apoptosis by E3330 in human hepatocellular carcinoma cell lines via inhibition of NF kappa B

AIM: To investigate the reduction of cell viability in human hepatocellular carcinoma (HCC) cell lines induced by inhibition of nuclear factor kappa B (NF kappa B). METHODS: HLE, SKHep1, and HepG2 were incubated and E3330 was used to compare the stimulation of some chemotherapeutic drugs with that of TNF family, Fas ligand, TNF alpha and TNF-related apoptosis-inducing ligand (TRAIL) at the point of the reduction of cell viability by inhibiting NF kappa B. RESULTS: E3330 decreased NF kappa B levels in HLE cells stimulated by TNF and TRAIL. The cytotoxicity of the combination of TRAIL, TNF alpha, Fas ligand, and E3330 increased synergistically in a dose-dependent manner compared to either E3330 alone in all HCC cell lines by MTT assay. However, the combination of some chemotherapeutic drugs and E3330 did not decrease the cell viability. CONCLUSION: Inhibition of NF kappa B sensitizes human HCC cell lines to TNF-mediated apoptosis including TRAIL, and TRAIL-based tumor therapy might be a powerful potential therapeutic tool in the treatment of human HCC.     

Role of cell adhesion signal molecules in hepatocellular carcinoma cell apoptosis

AIM: Cell adhesion molecules and their signal molecules play a very important role in carcinogenesis. The aim of this study is to elucidate the role of these molecules and the signal molecules of integrins and E-cadherins, such as (focal adhesion kinase) FAK, (integrin linked kinase)ILK, and β-catenin in hepatocellular carcinoma cell apoptosis.METHODS: We first synthesized the small molecular compound, S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and identified it, by element analysis and 1H NMR. To establish the apoptosis model of the SMMC-7721 hepatocellular carcinoma cell, we treated cells with DCVC in EBSS for different concentrations or for various length times in the presence of 20 μmol/L N,N-diphenyl-p-phenylenediamine,which blocks necrotic cell death and identified this model by flow cytometry and DNA ladder. Then we studied the changes of FAK, ILK, β-catenin, and PKB in this apoptotic model by Western blot.RESULTS: We found that the loss or decrease of cell adhesion signal molecules is an important reason in apoptosis of SMMC-7721 hepatocellular carcinoma cell and the apoptosis of SMMC-7721 cell was preceded by the loss or decrease of FAK, ILK, PKB, and β-catenin or the damage of cell-matrix and cell-cell adhesion.CONCLUSION: Our results suggested that the decrease of adhesion signal molecules, FAK, ILK, PKB, and β-catenin,could induce hepatocellular carcinoma cell apoptosis.     

不同转移潜能人肝癌细胞系酪氨酸磷酸化蛋白质差异分析

目的研究不同转移潜能人肝癌细胞系中酪氨酸磷酸化蛋白质表达的差异并筛查与肝癌转移相关的酪氨酸磷酸化蛋白质分子。方法应用双电泳(2-D E)、免疫印迹法及基质辅助激光解析电离飞行时间质谱分析,对三种不同转移潜能人肝癌细胞系Hep 3B、MHCC97L和MHCC97H进行酪氨酸磷酸化蛋白质组分析。结果对照2-DE胶和免疫印迹发光胶片,Hep3B检测到10个点,MHCC 9 7L检测到19个点, MHCC97H检测到17个点。经质谱鉴定,得到膜连蛋白Ⅰ等19个差异点。结论细胞内酪氨酸磷酸化蛋白的表达差异与肝癌的侵袭转移有关。     

Novel recurrent genetic imbalances in human hepatocellular carcinoma cell lines identified by comparative genomic hybridization

To search for recurrent and specific genomic alterations in human hepatocellular carcinoma (HCC), we examined 18 cell lines by comparative genomic hybridization (CGH), a molecular cytogenetic approach that allows positional identification of gains and losses of DNA sequences of the entire tumor genome. We report here a distinct pattern of multiple recurrent DNA copy-number gains and losses that include alterations frequently seen in other neoplasias as well as changes potentially specific for HCC. The most frequent gains were localized on 1p34.3-35, 1p33-34.1, 1q21-23, 1q31-32, 6p11-12, 7p21, 7q11.2, 8q24.1-24.2, 11q11-13, 12q11-13, 12q23, 17q11. 2-21, 17q23-24, and 20p11.1-q13.2. Recurrent losses were mapped on 3p12-14, 3q25, 4p12-14, 4q13-34, 5q21, 6q25-26, 8p11.2-23, 9p12-24, 11q23-24, 13q12-33, 14q12-13, 15q25-26, 18q11.2-22.2, and 21q21-22. Seventeen genomic imbalances are novel in HCC, thus extending significantly the map of genetic changes and providing a starting point for the isolation of new genes relevant in pathogenesis of liver neoplasia, as well as providing molecular probes for both diagnosis and monitoring treatment of the disease.     

不同转移潜能人肝癌细胞株趋化因子受体谱差异表达

目的对比观察不同转移潜能人肝癌细胞株趋化因子受体谱差异性表达。方法Pre- mier软件设计18对趋化因子受体引物,RT-PCR分析SMMC-7721、MHCC97-L、MHCC97-H和HCCLM6细胞侵袭转移潜能逐渐增强的人肝癌细胞株趋化因子受体谱。结果4组不同转移潜能细胞株趋化因子受体表达谱存在明显差异（P＜0．01）,其中CCR10、CXCR4、CXCR6表达随转移潜能增加逐渐降低。HCCLM6表达谱中CCR3、CCR4、CCR10、CCR12及XCR1比SMMC-7721表达明显降低甚至缺失（P＜0．01）,而CXCR1（P=0．006）、CXCR5（P=0．003）表达高于低转移潜能组SMMC-7721。MHCC97-H和MHCC97-L比较,除CXCR2、CXCR6、XCR1外差异均有统计学意义,其中CCR1（P=0．002）、CCR2（P=0．004）、CCR5（P=0．046）表达高于MHCC97- L。CXCR4在模板减量时只能在SMMC-7721组检测到。结论高低转移潜能肝癌细胞株趋化因子受体表达在mRNA水平存在差异性表达,与肝癌细胞株差异性转移潜能相关。     

Role of cell adhesion signal molecules in hepatocellular carcinoma cell apoptosis

AIM: Cell adhesion molecules and their signal molecules play a very important role in carcinogenesis. The aim of this study is to elucidate the role of these molecules and the signal molecules of integrins and E-cadherins, such as (focal adhesion kinase) FAK, (integrin linked kinase) ILK, and p-catenin in hepatocellular carcinoma cell apoptosis. METHODS: We first synthesized the small molecular compound, S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and identified it, by element analysis and 1H NMR. To establish the apoptosis model of the SMMC-7721 hepatocellular carcinoma cell, we treated cells with DCVC in EBSS for different concentrations or for various length times in the presence of 20 μmol/L N,N-diphenyl-p-phenylenediamine, which blocks necrotic cell death and identified this model by flow cytometry and DNA ladder. Then we studied the changes of FAK, ILK, β-catenin, and PKB in this apoptotic model by Western blot. RESULTS: We found that the loss or decrease of cell adhesion signal molecules is an important reason in apoptosis of SMMC-7721 hepatocellular carcinoma cell and the apoptosis of SMMC-7721 cell was preceded by the loss or decrease of FAK, ILK, PKB, and β-catenin or the damage of cell-matrix and cell-cell adhesion. CONCLUSION: Our results suggested that the decrease of adhesion signal molecules, FAK, ILK, PKB, and β-catenin, could induce hepatocellular carcinoma cell apoptosis.     

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.     

不同转移潜能人肝癌细胞系转录因子活性差异分析

目的分析不同转移潜能人肝癌细胞系细胞核内转录因子活性的差异，筛选与肝癌转移相关的转录因子。方法应用转录因子活性芯片技术，在功能水平分析三种不同转移潜能人肝癌细胞系（Hep3B、MHCC97L和MHCC97H）细胞核内转录因子活性的差异，并用电泳迁移率变动分析和蛋白免疫印迹实验验证芯片结果。结果在345个候选的转录因子中，筛选出7个活性差异转录因子。随人肝癌细胞转移潜能的增高（Hep3B〈MHCC97L〈MHCC97H），活性上调的转录因子有5个，包括p53、缺氧诱导因子-1α（HIF-1α）、核因子κb、信号传导及转录活化因子3（Stat3）和Sp1；活性下调的转录因子有2个，包括Rb和Smad3。结论转录因子活性异常与肝癌转移密切相关，本实验筛选出的转录因子可能有助于揭示肝癌转移的分子机制，并寻找新的预测指标及干预治疗的靶点。     

肝癌转移相关的微小RNAs在不同转移潜能肝癌细胞系的定量研究

目的 研究与转移密切相关的微小RNAs(miRNAs)在不同转移潜能肝癌细胞系的表达水平,探讨其在肿瘤转移过程中的生物学功能.方法 提取细胞系MHCC97H、MHCC97L、HepG2、L02的总RNA,通过反转录获得特异miRNA(miR-122a、miR-124a、miR-148a、miR-148b、miR-15a、miR-219、miR-30c、miR-338、miR-34a、Let-7g、miR-9)的cDNA,并应用TaqMan MGB探针法对其进行定量检测.采用AB17500系统软件V1.3.1采集Ct值,并使用miRNA内参基因RNU6B校正,相对定量计算公式RQ=2-△Ct,A Ct=CtmiRNAs-CtRNu6B.数据均经SPSS13.0统计软件包处理,采用t检验或非参数检验. 结果 转移相关的miRNAs(miR-124a除外)在MHCC97H与MHCC97L中表达,差异均有统计学意义.HepG2中miR-30c、miR-338、miR-34a和Let-g的表达水平明显高于L02,分别为miR-30c(8.41±0.40比6.82±0.29),miR-338(3.14±0.29比-2.36±0.32),miR-34a(0.71±0.40比-2.95±0.26),Let-7g(-4.07±0.55比-6.98±0.56),t值依次为2.948,12.656,7.484,3.684,P值均＜0.05,差异有统计学意义.而miR-148b,miR-9的表达则显著低于正常肝细胞,分别为miR-148b(1.96±0.51比3.76±0.28),miR-9(-4.38±0.86比-1.10±0.53),t值依次为-3.073,-3.324,P值均＜0.05,差异有统计学意义.miR-148家族中miR-48b在所测细胞系中的表达(5.46±1.21)均显著强于miR-148a的表达(1.29±0.35),Z=-5.097,P=3×10-7,差异有统计学意义.结论 可以利用肝癌细胞系列细胞平台进一步研究肝癌转移相关miRNAs在肿瘤转移过程中的生物学功能.     

Survivin在原发性肝细胞癌中的表达及意义

目的　Survivin是凋亡抑制蛋白中的一种 ,选择性地表达于恶性肿瘤组织。该文研究Sur vivin基因在原发性肝细胞癌中的表达及生物学意义。方法　收集 2株肝细胞癌细胞株 ,4 0例原发性肝癌组织标本及相应的癌旁组织 ,以Westernblotting法检测Survivin蛋白表达 ;半定量RT PCR法检测SurvivinmRNA表达 ;肝癌细胞凋亡指数采用原位末端标记法检测。结果　 2株肝癌细胞株和 85 % (34例 )的肝癌组织表达Survivin蛋白和mRNA ,而癌旁组织内无一例阳性表达。Survivin蛋白表达的阳性率在肝内转移组为 93.5 % ,显著高于肝内无转移组 (5 5 .6 % ,P <0 .0 5 ) ;在门静脉癌栓浸润组为 92 .8% ,显著高于无门静脉癌栓浸润组 (6 6 .7% ,P <0 .0 5 )。RT PCR显示 ,2株肝癌细胞株和 85 .0 % (34例 )的肝癌组织表达SurvivinmRNA ,与Westernblotting的结果一致 ,SurvivinmRNA的表达水平在肝内转移组 (1.10 5± 0 .396 )和门静脉癌栓浸润组 (1.137± 0 .4 0 4 )中 ,显著高于肝内无转移组 (0 .5 72± 0 .0 82 )和无门静脉癌栓浸润组 (0 .6 2 7± 0 .12 2 ,P <0 .0 5 )。所有肝癌组织标本中均可检测到凋亡细胞 ,但Survivin表达阳性组的凋亡指数 (1.15 2 %± 0 .32 6 % )显著低于Survivin表达阴性组 (4.5 0 2 %± 0 .830 % ,P <0 .0 5