沉默GPC3对肝癌Huh-7细胞增生、迁移和侵袭能力的影响

目的探讨Hippo通路靶向GPC3后对肝癌Huh-7细胞增生、迁移、侵袭能力的影响。方法采用Western blot检测法及RT-PCR分别测量肝癌Huh-7不同对数生长期的肝癌细胞株GPC3蛋白表达水平及GPC3 mRNA水平,并筛选出可有效沉默GPC3基因的siRNA。将肝癌Huh-7细胞株分为实验组、未转染组及对照组,实验组导入GPC3-siRNA-1633转染Huh-7,其余两组不作处理。EdU实验检查三组细胞增殖率,划痕实验测定各组细胞迁移率,Transwell实验测定各组细胞侵袭能力。结果实验组GPC3 mRNA表达量及GPC3蛋白水平显著低于未转染组及对照组,差异有统计学意义(P<0.05)。实验组Hippo通路中YAP mRNA表达量显著低于未转染组及对照组,差异有统计学意义(P<0.05)。转染48 h后,实验组细胞增生率、迁移率及侵袭率显著低于未转染组及对照组,差异有统计学意义(P<0.05)。结论 GPC3可促进肝癌Huh-7细胞增殖、侵袭及转移,从而促进肝癌细胞生长。CPG3可通过上调Hippo通路中YAP表达水平从而促进肝癌细胞增生。通过沉默GPC3 mRNA后可有效抑制肝癌Huh-7细胞生长。     

木瓜凝乳蛋白酶与5-Fu或MTX对人肝癌细胞7402的细胞毒试验

目的：测定木瓜凝乳蛋白酶及其与5-氟尿嘧啶(5-Fu)、氨甲喋呤(MTX)单独或联合作用对人肝癌细胞7402的细胞毒性。方法：采用MTT法，不同浓度的酶和药物在不同的培养时间下，通过其细胞抑制率和半数抑制率(IC50)测定木瓜凝乳蛋白酶及联合药物的细胞毒性。结果：木瓜凝乳蛋白酶对7402细胞的：IC50约为125μg／mL，随浓度增加对细胞的抑制率增加。培养36h时酶对细胞的毒性最大。酶与5-Fu、MTX联合使用比单独使用效果更好。结论：木瓜凝乳蛋白酶对人肝癌细胞7402具有细胞毒作用，酶在培养36h时细胞毒作用最大；联合化疗药物有一定的效果。     

姜黄素对人SPC-A549细胞生长抑制的影响

目的　研究姜黄素对人肺腺癌细胞SPC -A5 4 9的生长抑制作用。方法　采用细胞培养 ,倒置相差显微镜下观察细胞形态变化 ;MTT法检测姜黄素对肺腺癌细胞的抑制作用。结果　①姜黄素作用癌细胞后 ,光镜下可见细胞脱壁 ,胞体缩小 ,胞核固缩。② 5、10、2 0和 4 0mg/L姜黄素均能抑制人肺腺癌细胞SPC -A5 4 9的生长 ,4 0mg/L姜黄素作用 12h细胞存活率达2 8.5 %。结论　姜黄素能有效的抑制人肺腺癌细胞SPC -A5 4 9的生长 ,且对人SPC -A5 4 9细胞存活率具有浓度和时间依赖性     

Comparative effects of α, β and γ human interferons on a HBsAg-producing human hepatoma cell line

Summary The effects of all the three types of human interferons (α, β and γ) on a human hepatoma cell line with hepatitis B virus (HBV)-DNA sequences integrated into the host DNA and producing hepatitis B surface antigen (HBsAg) in culture medium were assayed. The aim of the present research was to test human interferon preparations in anin vitro system for hepatitis B virus, and to compare the observed effects. The results evidenced both the antireplicative activity principally showed by preparations of β and γ human interferons and the inhibition of HBsAg production by high concentrations of γ human interferon. This work was supported in part by a grant from theConsiglio Nazionale delle Ricerche (CNR), Roma, Italy (contract № 83.00631.52).     

siRNA靶向干扰GPC3基因慢病毒载体的构建及其对肝癌Huh-7细胞凋亡的影响

目的构建GPC3基因短发夹干扰RNA(shRNA)慢病毒载体,观察GPC3基因对人肝癌细胞系Huh-7凋亡的影响,探讨GPC3基因影响肝癌细胞生长的机制,为肝癌的基因治疗提供理论基础。方法采用RNA干扰技术转染肝癌细胞系Huh-7,荧光定量PCR检测GPC3基因在多种肝癌细胞株中的表达情况。构建重组靶向GPC3基因的小干扰RNA序列PGC-shRNA-GPC3,以脂质体(lipofectamineTM2000)为载体转染人肝癌细胞系,筛选并建立稳定表达siRNA的细胞株。采用RT-PCR及Western blot检测GPC3 siRNA转染后mRNA表达情况,验证siRNA的干扰效率。流式细胞仪检测阴性对照组、未转染组和转染组细胞凋亡的情况。结果在肝癌细胞株Huh-7中,GPC3基因显示高表达。测序验证PGC-shRNA-GPC3重组质粒构建成功。将重组质粒稳定转染入肝癌细胞株Huh-7后能明显抑制GPC3 mRNA表达水平;同时,流式细胞仪检测发现,GPC3表达下调后,诱导Huh7细胞的凋亡。结论靶向GPC3的siRNA能有效抑制GPC3的表达,并能促进肝癌Huh7细胞凋亡。     

姜黄素体外抑制人结肠癌癌细胞Caco-2增殖

目的研究姜黄素在体外对人结肠癌细胞Caco-2生长增殖及其抗肿瘤的相关分子机制。方法体外培养人结肠癌细胞Caco-2,四甲基偶氮唑盐(MTT)法检测姜黄素在相同浓度下、不同时间对于结肠癌细胞增殖的影响,并计算半数抑制量(IC50)值;反转录聚合酶链式反应(RT-PCR)法检环氧合酶-2(COX-2)、基质金属蛋白酶-9(MMP-9)的mRNA的表达影响。结果 MTT:相同干预浓度姜黄素抑制人结肠癌细胞增殖,其24 h,48 h,72 h的IC50分别为:205.737±14.929μmol/L、102.023±6.809μmol/L、86.627±9.005μmol/L;RT-PCR:人结肠癌细胞Caco-2正常对照组及姜黄素干预组MMP-9 mRNA灰度值分别为:0.786±0.101、0.310±0.046,P<0.05;COX-2 mRNA灰度值分别为0.830±0.173、0.063±0.035,(P<0.05)。结论姜黄素可能通过抑制COX-2及MMP-9的mRNA的表达来抑制结肠癌细胞Caco-2增殖。     

姜黄素对荷瘤裸鼠皮下移植瘤的影响

目的观察姜黄素在体内对人肝癌细胞株SMMC-7721的抗肿瘤作用。方法用姜黄素在SMMC-7721肝癌细胞荷瘤裸鼠的瘤体内进行注射治疗,12 d后处死裸鼠,摘除瘤体,称瘤重,观察肿瘤生长变化,并通过免疫组化法检测Bcl-2、Bax、Caspase-3等与细胞凋亡相关因子的表达。结果姜黄素可抑制SMMC-7721细胞对裸鼠的致瘤能力,肿瘤体积较对照组显著减小（P〈0.01）,质量也明显小于对照组（P〈0.01）,肿瘤生长抑制率达47.7%,免疫组化结果显示阿的平能明显上调与细胞凋亡相关因子Bax和Caspase-3的表达和下调Bcl-2和Survivin的表达。结论姜黄素能抑制SMMC-7721细胞的体内致瘤能力。     

姜黄素对荷瘤裸鼠皮下移植瘤的影响

目的观察姜黄素在体内对人肝癌细胞株SMMC-7721的抗肿瘤作用。方法用姜黄素在SMMC-7721肝癌细胞荷瘤裸鼠的瘤体内进行注射治疗,12 d后处死裸鼠,摘除瘤体,称瘤重,观察肿瘤生长变化,并通过免疫组化法检测Bcl-2、Bax、Caspase-3等与细胞凋亡相关因子的表达。结果姜黄素可抑制SMMC-7721细胞对裸鼠的致瘤能力,肿瘤体积较对照组显著减小(P0.01),质量也明显小于对照组(P0.01),肿瘤生长抑制率达47.7%,免疫组化结果显示阿的平能明显上调与细胞凋亡相关因子Bax和Caspase-3的表达和下调Bcl-2和Survivin的表达。结论姜黄素能抑制SMMC-7721细胞的体内致瘤能力。     

Functional alpha 1 protease inhibitor produced by a human hepatoma cell line

Alpha 1 protease inhibitor antigen was identified in the culture medium of the human ascites hepatoma cell line SK-HEP-1. Trypsin inhibitory activity and alpha 1 Pl antigen accumulated in serum-free medium concomitantly over a period of several days. Radioactive alpha 1 Pl antigen was detected in conditioned medium from cultures supplemented with 35S-L-methionine, indicating a synthesis and release of the protein. Alpha 1 Pl antigen in conditioned medium appeared to be antigenically identical to that in human plasma, and the newly synthesized (radiolabeled) antigen co-migrated with plasma, alpha 1 Pl after immunoelectrophoresis or SDS-polyacrylamide gel electrophoresis. Moreover, evidence is presented that the synthesized inhibitor exhibits functional activity, since the 35S-labeled alpha 1 Pl in conditioned medium complexes with trypsin. We conclude that SK-HEP-1 cells in culture produce functionally active alpha 1 Pl which may be identical to that in plasma.     

凝血酶敏感蛋白1对人肝癌细胞株HCCLM3生长和黏附的作用

目的:观察凝血酶敏感蛋白1对人肝癌细胞株HCCLM3的生长抑制和黏附的作用。方法:实验于2006-05/2007-01在河南省高等学校省级开放实验室完成。①采用浓度为2×107L-1的HCCLM3对数生长期单细胞悬液接种培养后,采用MTT法检测凝血酶敏感蛋白1对HCCLM3的生长抑制作用。②对数生长期的HCCLM3细胞株接种于纤黏连蛋白覆盖的96孔板后,按照处理因素分组:空白对照组、凝血酶敏感蛋白1组(加入凝血酶敏感蛋白1蛋白40mg/L)、CD36阻断组和CD47阻断组[先加入对应的单抗(浓度5mg/L),在体积分数为0.05CO2,37℃温箱孵育30min使单抗与细胞充分结合后再加入凝血酶敏感蛋白1]。培养72h后MTT方法检测凝血酶敏感蛋白1对细胞黏附力的作用,染色后直接计数黏附细胞。结果:①凝血酶敏感蛋白1对HCCLM3细胞的生长抑制率随着浓度的提高而增加,40mg/L与50mg/L相比抑制作用差异无显著性。抑制率随着时间的延长而增加,在72h抑制作用最明显。②黏附力的检测显示:凝血酶敏感蛋白1组,CD36阻断组,CD47阻断组细胞黏附力均显著低于对照组(P<0.05),但凝血酶敏感蛋白1组,CD36阻断组,CD47阻断组细胞黏附力差异无显著性(P>0.05)。结论:凝血酶敏感蛋白1对HCCLM3的生长有抑制作用,在一定范围内呈剂量依赖性和时间依赖性。凝血酶敏感蛋白1可抑制人肝癌细胞株HCCLM3与细胞外基质的黏附能力,作用途径可能与受体CD36,CD47无关。     

HSP90抑制剂GA对人肝癌BEL7404细胞株凋亡作用的研究

目的：研究热休克蛋白90 (heat shock protein 90, hsp90）抑制剂格尔德霉素（geldanamycin,GA）对人肝癌细胞株BEL7404的凋亡作用,并探讨相关机制及意义。方法：体外培养BEL7404细胞株,以HSP90抑制剂GA作用处理;噻唑兰比色（MTT）法检测GA对BEL7404细胞株的增殖抑制作用;吖啶橙荧光染色法及Annexin V-EGFP/PI双染法、透射电镜等方法研究GA对BEL7404细胞株的凋亡作用;流式细胞仪检测BEL7404细胞株的细胞周期变化。结果：HSP90抑制剂GA对BEL7404细胞株具有增殖抑制作用,呈时间剂量依赖关系;GA各剂量组（5 umol/L、10 umol/L、15 umol/L）作用24h凋亡率（%）分别为10.2±1.35、21.3±1.30、38.7±1.43,明显高于阴性对照组(6.31±0.82);流式细胞仪检测各剂量组GA均可使BEL7404细胞株细胞周期阻滞于G0/G1期。结论：通过抑制HSP90的功能,GA能明显抑制肝癌细胞增殖,促进肝癌细胞凋亡,使肝癌细胞周期阻滞于G0/G1期,其抗癌活性可能与其抑制HSP90的分子伴侣功能有关。     

5-氟尿嘧啶联合热疗对人肝癌细胞株的增殖抑制作用

目的 探讨5-氟尿嘧啶(5-FU)联合热疗诱导人肝癌细胞株(SSMC7721细胞)的增殖抑制率,细胞凋亡率及其对细胞周期进程的影响,旨在为肝细胞癌的综合治疗提供理论依据.方法 应用MTT比色法检测不同条件下对SSMC7721细胞的增殖抑制率,流式细胞仪检测细胞周期进程的变化及凋亡率,电子显微镜检测亚细胞结构改变.结果 单纯热疗组、5-FU组及联合组对SSMC7721细胞抑制率分别为18.4%、28.3%和52.7%,与对照组相比差异均有统计学意义(P均＜0.01).流式细胞仪结果显示:G1期细胞增多,S期细胞减少、G2/M期细胞相对增多,细胞凋亡率升高,组间比较差异均有统计学意义(P＜0.01或P＜0.05),电子显微镜结果显示细胞核染色质趋边凝集、线粒体肿胀.结论 5-FU联合热疗能显著提高SSMC7721细胞的增殖抑制率,抑制SSMC7721细胞G1期向S期转化进程,诱导SSMC7721细胞凋亡及亚细胞结构改变.     

着丝粒蛋白CENP-H对乳腺癌细胞株MCF7增殖能力的影响

目的 研究CENP-H对乳腺癌细胞增殖能力的影响,初步探讨CENP-H与乳腺癌发生、发展的关系.方法 将反转录病毒质粒pMSCV和pMSCV-CENP-H经脂质体转染至293FT细胞制备病毒,并感染MCF7细胞,用嘌呤霉素筛选及Western blot鉴定,建立CENP-H基因稳定表达的MCF7细胞株;应用噻唑盐(MTT)法、平板集落形成实验、5-溴-2-脱氧尿苷(BrdU)掺入法检测CENP-H对MCF7细胞增殖的影响.结果 成功建立稳定表达CENP-H的MCF7细胞株,并发现CENP-H过表达可上调细胞增殖相关分子cyclin D1的表达;MTT、平板克隆实验及Brdu掺入实验结果显示CENP-H过表达后,MCF7的增殖能力模型增强.结论 CENP-H可上调cyclin D1的表达,增强MCF7的增殖能力,提示CENP-H可能在乳腺癌发生、发展中起重要作用.     

小干扰RNA对人肝癌细胞株HepG2 Survivin基因表达的影响

目的构建Survivin基因特异性小干扰RNA(siRNA),检测siRNA-Survivin对Survivin基因表达的抑制,在人肝癌细胞株HepG2中研究survivin和survivin siRNA对细胞凋亡的影响。方法设计survivin siRNA序列,构建靶向Survivin siRNA真核载体。利用脂质体转染人肝癌HepG2细胞,通过相差显微镜下观察、四甲基偶氮唑盐微量酶反应比色法(MTT法)及反转录-聚合酶链反应(RT-PCR)观察转染后survivin的表达,以及HepG2细胞的凋亡。结果成功构建了survivin siRNA表达载体,并且si-survivin对survivin有明显抑制效应,可显著抑制HepG2细胞的发生凋亡。转染Survivin-siRNA细胞活性受到显著抑制(P<0.05),光镜下出现明显的凋亡形态,DNA电泳出现典型的凋亡"梯状"带。RT-PCR结果显示细胞转染24 h、48 h和72 h后HepG2 Survivin mRNA分别减少了56%、78%和50%,而siR-NA阴性对照与未转染细胞相比差异不显著。结论转染的Survivin-siRNA可特异性抑制肝癌细胞株HepG2凋亡抑制基因的表达,从而为肿瘤的基因治疗提供新的实验基础。     

Transforming potential of DNA of the human PLC/PRF/5 hepatoma cell line

DNA of the human PLC/PRF/5 hepatoma cell line can induce the appearance of colonies on soft agar after transfection of BK-BK cells (BALB/c mouse kidney cells partially transformed by BK virus). Blot hybridization analyses indicate that the transformants contained the hepatitis B virus DNA sequence. This sequence disappeared during serial passage of transformants. The amplification and rearrangement of the integrated BK virus genome appears to be specifically associated with transformation.     

Synergistic inhibitory effects of curcumin and 5-fluorouracil on the growth of the human colon cancer cell line HT-29

The synergistic effect of combination treatment with COX-2 inhibitors and chemotherapy may be another promising therapy regimen in the future treatment of colorectal cancer. Curcumin, a major yellow pigment in turmeric which is used widely all over the world, inhibits the growth of human colon cancer cell line HT-29 significantly and specifically inhibits the expression of COX-2 protein. However, the worldwide exposure of populations to curcumin raised the question of whether this agent would enhance or inhibit the effects of chemotherapy. In this report, we evaluated the growth-inhibitory effect of curcumin and a traditional chemotherapy agent, 5-FU, against the proliferation of a human colon cancer cell line (HT-29). The combination effect was quantitatively determined using the method of median-effect principle and the combination index. The inhibition of COX-2 expression after treatment with the curcumin-5-FU combination was also evaluated by Western blot analysis. The IC(50) value in the HT-29 cells for curcumin was 15.9 +/- 1.96 microM and for 5-FU it was 17.3 +/- 1.85 microM. When curcumin and 5-FU were used concurrently, synergistic inhibition of growth was quantitatively demonstrated. The level of COX-2 protein expression was reduced almost 6-fold after the combination treatment. Our results demonstrate synergism between curcumin and 5-FU at higher doses against the human colon cancer cell line HT-29. This synergism was associated with the decreased expression of COX-2 protein.     

MCF7细胞中肿瘤干细胞的增殖特征

背景:肿瘤干细胞学说认为,肿瘤干细胞是肿瘤不断增殖的根源,并与肿瘤的浸润转移、耐药现象密切相关。目的:在单细胞水平研究体外传代培养的MCF7细胞中肿瘤干细胞含量变化规律,认识肿瘤干细胞在体外培养环境下的增殖特点。方法:利用单细胞分离种植-成瘤性克隆方法对传代培养后不同时间点MCF7细胞中肿瘤干细胞的比例连续检测,流式细胞仪同步检测相应细胞样本中CD44+CD24-/low亚群的含量变化。结果与结论:传代后培养的MCF7细胞在不同时间点其肿瘤干细胞比例及CD44+/CD24-/low亚群比例均呈现有规律的变化。但CD44+/CD24-/low亚群变化更为显著(36.84%到81.95%),而肿瘤干细胞含量仅在小范围波动(38.54%～47.39%)。结果提示传代培养的MCF7细胞中,肿瘤干细胞具有通过调节自身增殖来保持其比例相对稳定的特点;CD44+/CD24-/low亚群并不代表或者富集MCF7细胞株中的肿瘤干细胞亚群。     

MCF7细胞中肿瘤干细胞的增殖特征

背景：肿瘤干细胞学说认为,肿瘤干细胞是肿瘤不断增殖的根源,并与肿瘤的浸润转移、耐药现象密切相关。目的：在单细胞水平研究体外传代培养的MCF7细胞中肿瘤干细胞含量变化规律,认识肿瘤干细胞在体外培养环境下的增殖特点。方法：利用单细胞分离种植-成瘤性克隆方法对传代培养后不同时间点MCF7细胞中肿瘤干细胞的比例连续检测,流式细胞仪同步检测相应细胞样本中CD44＋CD24-/low亚群的含量变化。结果与结论：传代后培养的MCF7细胞在不同时间点其肿瘤干细胞比例及CD44＋/CD24-/low亚群比例均呈现有规律的变化。但CD44＋/CD24-/low亚群变化更为显著（36.84%到81.95%）,而肿瘤干细胞含量仅在小范围波动（38.54%～47.39%）。结果提示传代培养的MCF7细胞中,肿瘤干细胞具有通过调节自身增殖来保持其比例相对稳定的特点;CD44＋/CD24-/low亚群并不代表或者富集MCF7细胞株中的肿瘤干细胞亚群。     

Inhibition of plasminogen activators by conditioned medium of human hepatocytes and hepatoma cell line Hep G2

Primary cultures of human hepatocytes and the human hepatocellular cell line Hep G2 are shown to produce fast-acting inhibitors of tissue-type plasminogen activator (tPA) and urokinase. The tPA inhibitory activities in conditioned medium of these liver cell types are very similar to those present in human endothelial cell conditioned medium. They are stable at pH 2.5, have similar dissociation constants with tPA (1.5 to 5 pmol/L), and are similar in thermostability. Addition of tPA to conditioned medium of Hep G2 and endothelial cells that has been depleted of tPA and urokinase reveals a 100 kilodalton tPA-inhibitor complex. The fast-acting tPA inhibitory activity in human plasma has comparable properties, and may originate from the liver or the vascular endothelium or both. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis of conditioned medium from hepatocytes, Hep G2, and endothelial cells, additional fibrinolytic inhibition at 52 kilodaltons was visualized. This was not found with human plasma.