肝癌细胞系Hep3B中CD133+细胞亚群的分离及其特性的研究

目的研究CD133在人肝癌细胞系Hep3B中的表达以及CD133+细胞的体外增殖、自我更新及体内成瘤能力,初步探讨肝癌中CD133+细胞亚群的干细胞特性。方法流式细胞仪检测未分选的Hep3B细胞中CD133+细胞表达情况;免疫磁珠分选技术纯化CD133+肿瘤细胞;MTT法检测CD133+细胞体外增殖能力;无血清培养纯化...     

RNA干扰CD133基因对结肠癌干细胞生物学行为的影响

目的探讨CD133基因表达、活化被阻断后对结肠癌干细胞生物学行为的影响。方法从EpcAMhighCD44＋结肠癌干细胞中流式分选获得CD133＋细胞,感染LV-CD133shRNA载体慢病毒后观察CD133＋结肠癌干细胞在生长方式、成球能力、克隆形成率、成瘤能力以及ABCC2mRNA的变化;Westernblot分析CD133-细胞中CD133蛋白表达情况。结果EpcAMhighCD44＋结肠癌干细胞中CD133＋细胞比例为89．2％。实验组经过LV-CD133shRNA载体病毒感染后,在干细胞养液中细胞改悬浮生长的方式为贴壁生长,不能形成细胞球。MTT法测定发现细胞增殖减慢,克隆形成率明显下降。将感染细胞移植在Balb／C裸鼠体内,在观察期间,感染LV—CD133shRNA载体病毒的CD133＋细胞无肿瘤形成。ABCG2mRNA表达水平明显降低（P〈0．01）。从EpcAMhighCD44＋结肠癌干细胞中流式分选获得CD133-细胞,其中也有CD133蛋白的表达。结论CD133维持结肠癌干细胞生物学特性。     

无血清分离人结直肠癌细胞的体外培养形态及标志物表达特征

目的探讨人结直肠肿瘤干细胞在体外分化过程中细胞形态和干细胞相关标志物CD133的表达变化,为进一步研究结直肠肿瘤干细胞分化走向提供实验依据。方法取来源于人结直肠癌的细胞系HCT116,无血清培养分离出CD133+细胞,加血清诱导分化,相差显微镜下观察其形态变化;在未分化状态下无血清培养第7天和14天与血清诱导分化后收集细胞,利用流式细胞仪检测干细胞标志物CD133的表达量,采用激光共聚焦检测CD133表面标记分子的表达。结果 1)细胞形态:无血清培养分离的CD133+细胞,在生长过程中聚集成规则的细胞球,血清诱导后即贴壁生长,贴壁形态与同来源细胞形态一致,且再次无血清悬浮培养后聚集成球稳定生长。2)标志物变化:结直肠肿瘤干细胞未分化时CD133表面标记分子高表达,流式细胞仪检测未分化细胞CD133第7天表达率为(20.4±0.52)%,第14天表达率为(78.5±2.80)%,分化后表达率为(0.50±0.17)%。结论细胞形态和标志物表达改变均表明高表达CD133+的HCT116结直肠癌肿瘤干细胞可定向分化为同源的结直肠癌细胞,CD133+细胞经血清诱导后表达下调而使细胞失去干细胞特性。     

si—DNMT1对肝门部胆管癌细胞抑癌基因甲基化的作用

目的研究RNA干扰对肝门部胆管癌细胞株QRC939抑癌基因甲基化的影响,初步探谤其在胆管癌治疗中的价值。方法构建靶向hDNMT1的发夹式siRNA表达载体;运用脂质体介导法将其转染人胆管癌细胞QBC939;RT-PCR法检测不同时间点hDNMT1、CDH1、p15的表达水平;MSP方法检测转染前后抑癌基因CDH1、p15的甲基纯状态;MTT检测各组细胞的增殖能力。结果1）hDNMT1的基因沉默恢复了抑癌基因CDH1、p15的表达水平;2）CDH1、p15的表达沉默是由启动子高甲基亿导致的;3）转染靶向hDNMT1的发夹式siRNA表达载体能有效地抑制QBC939的增殖能力。结论靶向hDNMT1的发夹式siRNA表达载体能有效、持续、稳定发挥对hDNMT1的基因沉默作用,恢复抑癌基因CDH1、p15的表达水平,从而抑制QBC939肿瘤细胞增殖。     

携SiRNA的聚乳酸羟基乙酸微球对Bel-7402/5-Fu细胞MDR1基因沉默作用的研究

目的 探讨携SiRNA的聚乳酸羟基乙酸(PLGA)微球通过沉默肝癌耐药细胞多药耐药基因(MDR1)基因,实现肝癌耐药的逆转.方法 合成针对MDR1的RNA干扰小片段(SiRNA),通过超声复乳法制备携药微球,转染肝癌耐药细胞Bel-7402/5 -Fu.运用Real-time PCR与Western blot方法,通过检测其mRNA及P糖蛋白表达水平,评价RNA干扰对MDR1表达的影响.MTT法检测RNA干扰逆转Bel-7402/5 Fu细胞对药性的效果,通过实验组细胞和对照组细胞之间的半数抑制剂量(IC50),计算RNA干扰组细胞的耐药逆转倍数.结果 在肝癌耐药细胞Bel-7402/5-Fu中,RNA干扰明显抑制了MDR1 mRNA和蛋白产物P糖蛋白的表达水平,且出现了P糖蛋白的持续低表达.MTT法显示,相同药物浓度下,实验组细胞生长明显受到抑制,表明其耐药性明显下降,其对药逆转倍数为11.56.结论 携SiRNA的PLGA微球能够有效减少肝癌耐药细胞Bel-7402/5-Fu的MDR1mRNA及P糖蛋白的表达水平,降低其耐药性.     

RNA干扰靶向沉默COX 2基因对MG 63骨肉瘤细胞生物学特性的研究

目的 通过构建小干扰RNA(small interfering RNA,siRNA)降低MG 63骨肉瘤细胞环氧合酶 2(COX 2)基因的表达,并进一步研究其对MG 63骨肉瘤细胞增值、侵袭、迁移能力的影响及分子机制。方法 设计靶向干扰COX 2基因的siRNA,通过脂质体转染MG 63骨肉瘤细胞,使其抑制MG 63骨肉瘤细胞COX 2基因的表达,后采用噻唑蓝(MTT)比色法、Transwell小室实验研究其对MG 63骨肉瘤细胞增殖、侵袭、迁移能力的影响,采用RFQ PCR和Western blot分别从基因和蛋白的水平检测MG 63骨肉瘤细胞侵袭性相关因子基质金属酶(MMP 9)的表达及血管内皮生长因子(VEGF)的表达。结果 转染MG 63骨肉瘤细胞后,实验组与阴性对照组和空白对照组比较,通过RFQ PCR和Western blot检测COX 2基因表达降低约90%(P0.05),MTT检测MG 63骨肉瘤细胞增值能力明显受到抑制(P0.05),Transwell实验检测MG 63骨肉瘤细胞侵袭、迁移能力明显下降(P0.05),经RFQ PCR、Western blot检测侵袭性相关因子MMP 9和血管内皮生长因子VEGF的mRNA及蛋白表达降低(P0.05)。空白对照组和阴性对照组比较无明显变化,差异无统计学意义(P0.05)。结论 人MG 63骨肉瘤细胞COX 2基因被抑制后,MG 63骨肉瘤细胞增值、侵袭、迁移能力明显下降。     

沉默Maspin基因对人乳腺癌细胞MCF-7侵袭迁移能力的影响

目的 探讨靶向沉默maspin基因对乳腺癌MCF-7细胞侵袭和迁移能力的影响.方法 构建针对maspin基因的具有荧光蛋白表达的shRNA真核表达载体,稳定转染入乳腺癌细胞MCF-7中.通过划痕实验和Transwell小室侵袭实验分别观察转染前后细胞迁移及侵袭能力的影响.分别用RT-PCR及Western Blot检测转染重组质粒前后细胞maspin mRNA和蛋白表达情况.结果 成功构建表达质粒pGenesil-HK、pGenesil-maspin-1和pGenesil-maspin-2,并成功稳定转染MCF-7细胞.与未转染组和pGenesil-HK组相比,转染pGenesil-maspin-1和pGenesil-maspin-2后的MCF-7细胞划痕伤口愈合速度加快(P＜0.05),细胞侵袭至小室下层的细胞数明显高于对照组(P＜0.05).RT-PCR及Western Blot结果表明转染pGenesil-maspin-1和pGenesil-maspin-2后的细胞maspin的mRNA和蛋白表达明显下降.结论 靶向maspin基因的RNA干扰能够下调maspin基因在乳腺癌MCF-7细胞中的表达,并能增强肿瘤细胞的侵袭和迁移能力.     

pEGFPN1-dnEGFR对胃癌细胞的化疗增敏作用

目的研究人 EGFR显性负性突变体真核表达载体(pEGFPN1 dnEGFR)对人胃癌细胞株 SGC 7901和 NCI N87化疗敏感性的影响,并探讨其可能机制.方法 MTT法测定奥沙利铂对稳定转染 pEGFPN1 dnEGFR和 pEGFP N1载体的两种胃癌细胞的量效反应.奥沙利铂作用各组细胞24h后,RT PCR检测各组细胞中 Caspase 3和 CyclinD1的 mRNA表达情况;Westernblot检测各组细胞中 Caspase 3和 CyclinD1蛋白表达情况.结果转染 pEGFPN1 dnEGFR后,两种胃癌细胞对奥沙利铂的敏感性增加,奥沙利铂对 pEGFPN1 dnEGFR转染组细胞的增殖抑制率(VI)与对照组相比有显著提高(P<0.05).RT PCR显示 pEGFPN1 dnEGFR转染组细胞 CyclinD1mRNA表达较对照组下降,而 Caspase 3mRNA表达较对照组升高(P<0.05);Westernblot显示 pEGFPN1 dnEG FR转染组细胞 CyclinD1蛋白表达较对照组下降,而 Caspase 3蛋白表达较对照组升高(P<0.05).结论 EGFR显性负性突变体能提高胃癌细胞对化疗药物奥沙利铂的敏感性,其机制可能与 Caspase 3和 CyclinD1有关     

Survivin过表达对MS1细胞Survivin基因及蛋白表达与凋亡的作用

目的 探讨Survivin过量表达对MS1细胞Survivin基因及蛋白表达与凋亡的影响.方法 构建Survivin真核表达载体,以脂质体转染MS1细胞,采用流式细胞仪的方法检测细胞凋亡;Real-time PCR和Western blot检测Survivin的mRNA及蛋白水平.结果 转染Survivin实验组与空载体对照组比较,Survivin mRNA和蛋白表达明显增高,实验组细胞凋亡率较对照组下降.结论 Survivin过表达可使MS1细胞的Survivin蛋白及mRNA高表达并能使细胞凋亡凋亡率下降,为后续的动物实验打下了理论依据.     

siRNA靶向沉默TAK1基因对前列腺癌细胞DU145耐药性和增殖能力的影响

目的研究siRNA沉默转化生长因子β激活激酶(TAK1)基因对前列腺癌细胞DU145增殖及药物敏感性的影响,对其相关作用机制进行初步初探。方法用Lipofectamine 2000将TAK1 siRNA及阴性对照siRNA序列转入细胞DU145中,用MTT法检测两组细胞增殖能力的变化,同时检测其对多西紫杉醇(艾素)、奥沙利铂(L-OHP)、5-氟尿嘧啶(5-FU)的敏感性的变化,应用western blot检测细胞TAK1、COX-2、bcl-2及JNK的表达。结果 siRNA-TAK1组细胞增殖能力显著低于siRNA-control组(P0.05)。siRNA-TAK1组,艾素、L-OHP和5-FU的24 h、48、72H IC50分别为(mg·L~(-1)):0.52±0.02、0.35±0.03、0.17±0.01;1.79±0.10、0.99±0.05、0.57±0.04和53.41±2.15、25.91±1.55、10.89±0.81;siRNA-control组分别为(mg·L~(-1)):0.62±0.04、0.32±0.02;2.10±0.05、0.63±0.04和28.31 4-1.14、13.73±0.82、5.77±0.43。siRNA-TAK1组的IC50均明显低于siRNA-control组(P0.05),转染48 h后siRNA-TAK1组TAK1、COX-2、bcl-2蛋白的表达显著低于siRNA-control组(P0.05)。结论干扰TAK1蛋白表达抑制TGF-β和COX-2信号通路可能是降低细胞增殖能力和增强细胞DU145药物敏感性的一个机制。     

HAb18G/CD147-mediated calcium mobilization and hepatoma metastasis require both C-terminal and N-terminal domains

HAb18G/CD147 is a heavily glycosylated protein containing two immunoglobulin superfamily domains. Our previous studies have indicated that overexpression of HAb18G/CD147 enhances metastatic potentials in human hepatoma cells by disrupting the regulation of store-operated Ca²⁺ entry by nitric oxide (NO)/cGMP. In the present study, we investigated the structure-function of HAb18G/CD147 by transfecting truncated HAb18G/CD147 fragments into human 7721 hepatoma cells. The inhibitory effect of HAb18G/CD147 on 8-bromo-cGMP-regulated thapsigargin-induced Ca²⁺ entry was reversed by the expression of either C or N terminus truncated HAb18G/CD147 in T7721C and T7721N cells, respectively. The potential effect of HAb18G/CD147 on metastatic potentials, both adhesion and invasion capacities, of hepatoma cells was abolished in T7721C cells, but not affected in T7721N cells. Release and activation of matrix metalloproteinases (MMPs), MMP-2 and MMP-9, were found to be enhanced by the expression of HAb18G/CD147, and this effect was abolished by both truncations. Thapsigargin significantly enhanced release and activation of MMPs (MMP-2 and MMP-9) in non-transfected 7721 cells, and this effect was negatively regulated by SNAP. However, no effects of thapsigargin or SNAP were observed in T7721 cells, and expression of HAb18G/CD147 enhanced secretion and activation of MMPs at a stable and high level. Taken together, these results suggest that both ectodomain and intracellular domains of HAb18G/CD147 are required to mediate the effect of HAb18G/CD147 on the secretion and activation of MMPs and metastasis-related processes in human hepatoma cells by disrupting the regulation of NO/cGMP-sensitive intracellular Ca²⁺ mobilization although each domain may play different roles.Received 1 April 2004; received after revision 15 June 2004; accepted 22 June 2004     

Overexpression of HAb18G/CD147 promotes invasion and metastasis via α3β1 integrin mediated FAK-paxillin and FAK-PI3K-Ca2+ pathways

Mechanism of HAb18G/CD147 underlying the metastasis process of human hepatoma cells has not been determined. In the present study, we found that integrin α3β1 colocalizes with HAb18G/CD147 in human 7721 hepatoma cells. The enhancing effect of HAb18G/CD147 on adhesion, invasion capacities and matrix metalloproteinases (MMPs) secretion was decreased by integrin α3β1 antibodies (p<0.01). The expressions of integrin downstream molecules including focal adhesion kinase (FAK), phospho-FAK (p-FAK), paxillin, and phospho-paxillin (p-paxillin) were increased in human hepatoma cells overexpressing HAb18G/CD147. Deletion of HAb18G/CD147 reduces the quantity of focal adhesions and rearranges cytoskeleton. Wortmannin and LY294002, specific phosphatidylinositol kinase (PI3K) inhibitors, reversed the effect of HAb18G/CD147 on the regulation of intracellular Ca²⁺ mobilization, significantly reducing cell adhesion, invasion and MMPs secretion potential (p<0.01). Together, these results suggest that HAb18G/CD147 enhances the invasion and metastatic potentials of human hepatoma cells via integrin α3β1-mediated FAK-paxillin and FAKPI3K-Ca²⁺ signal pathways. Received 5 June 2008; received after revision 16 July 2008; accepted 23 July 2008     

Recombinant expression of perchloric acid-soluble protein reduces cell proliferation

Perchloric acid-soluble protein (PSP) may play an important role in the regulation of cellular physiological functions because it has been highly conserved throughout evolution; however, this role has not been well elucidated. In previous reports, we suggested that PSP regulates cell proliferation. In this study, we examined the effect of PSP expression on proliferation of the normal rat kidney cell line NRK-52E, the rat hepatocyte cell line RLN-10, and the rat hepatoma cell line dRLh-84. Cells transfected with pcDNA-sense-PSP (pcDNA-S-PSP) over-expressed PSP mRNA and protein, and cell proliferation of the transfected cells was suppressed compared with that of cells transfected with pcDNA-empty (pcDNA-E). Cell viability of pcDNA-S-PSP-transfected cells was similar to that of pcDNA-E-transfected cells. Thus, over-expression of PSP suppresses cell proliferation without any influence on cell viability. These findings are the first to report an inhibitory activity of PSP on cell proliferation. Received 27 April 2001; received after revision 8 June 2001; accepted 8 June 2001     

Thyroid hormone receptor-mediated regulation of the methionine adenosyltransferase 1 gene is associated with cell invasion in hepatoma cell lines

The thyroid hormone T₃ regulates differentiation, growth, and development. We demonstrated that methionine adenosyltransferase 1A (MAT1A) was positively regulated by T₃ identified by cDNA microarray previously. The expression of the MAT1A was upregulated by T₃ in hepatoma cell lines overexpressing thyroid hormone receptors (TRs). Additionally, these findings indicate that MAT1A may be regulated by CCAAT/enhancer binding protein (C/EBP). The critical role of the C/EBP binding sites was confirmed by the reporter or chromatin immuno-precipitation (ChIP) assay. In addition, C/EBP was upregulated in hepatoma cells after T₃ treatment and ectopic expression of MAT1A inhibited cell migration and invasion in J7 hepatoma cells. Conversely, knockdown of MAT1A expression increased cell migration. Together, these findings suggest that the expression of the MAT1A gene is mediated by C/EBP and is indirectly upregulated by T₃. Finally, TR was downregulated in a small subset of hepatocellular carcinoma cells concomitantly reduced the expression of C/EBPα and MAT1A.     

Lumican基因对人肺腺癌细胞株A549体外增殖和侵袭的影响

目的 观察基膜聚糖(Lumican)基因过度表达对人肺腺癌细胞株A549体外增殖和侵袭的影响.方法 以携带人Lumican基因的重组慢病毒感染A549细胞,用嘌呤霉素(puromycin)筛选法建立稳定细胞株.分别用Real-time PCR和Western blotting方法检测A549细胞组、空载体组及Lumican感染组目的基因mRNA和蛋白的表达.运用MTT法研究Lumican基因对A549细胞增殖的影响.运用Transwell法检测Lumican基因对A549细胞侵袭的影响.结果 ①各组Lumican mRNA表达差异显著(x2=21.60,P＜0.01),Lumican感染组明显高于其它两组(F=102.86,P＜0.000 1),Lumican感染组的蛋白表达水平高于其它两组.②Lumican感染组在不同时间(1、2、3、4、5 d)OD(吸光度)值与A549细胞组和空载体组差异不显著(P＞0.05).③各组穿膜细胞数差异显著(x2=23.49,P＜0.01),Lumican感染组明显高于其它两组(F=73.92,P＜0.001).结论 成功构建了Lumican高表达的人肺腺癌A549稳定细胞株,Lumican基因对人肺腺癌细胞的体外增殖能力无影响,但能增强人肺腺癌细胞的体外侵袭能力.     

Dexamethasone enhances CTLA-4 expression during T cell activation

T cell activation is enhanced by the costimulatory interaction of B7 on antigen-presenting cells and CD28 on T cells, resulting in long-term T cell proliferation, differentiation and production of large amounts of cytokines, such as interleukin (IL)-2. CTLA-4 is a co-stimulation receptor that shares 31% homology with CD28 and binds B7 family members with higher affinity. CTLA-4 is transiently expressed intracellularly and on the cell surface following activation of T cells. We have studied the kinetics of CTLA-4 expression and the effects of dexamethasone on CTLA-4 expression during T cell activation in cultures of mouse spleen cells stimulated by a mixture of immobilized anti-CD3 and anti-CD28 monoclonal antibodies (anti-CD3/CD28 mAb) or concanavalin A (ConA). CTLA-4 expression peaked on day 2 and returned to background levels after 7 days. Dexamethasone was found to potentiate CTLA-4 expression in a dose-dependent manner with an EC₅₀ effective concentration 50%) of about 10⁻⁸ M. In contrast, other immunosuppressive agents, such as rapamycin or cyclosporin A had no or an inhibitory effect on CTLA-4 expression, respectively. Dexamethasone also stimulated CD28 expression, but inhibited IL-2R expression during anti-CD3/CD28 mAb-induced mouse splenic T cell activation. Western blot analyses of lysates of activated mouse T cells showed that dexamethasone increased CTLA-4 protein levels twofold during anti-CD3/CD28 mAb-induced activation. Dexamethasone also enhanced CTLA-4 messenger RNA twofold as quantified by ribonuclease protection assay. The effects of dexamethasone on CTLA-4 expression were glucocorticoid-specific and completely inhibited by the glucocorticoid receptor antagonist mifepristone (RU486), indicating that the effect of dexamethasone on CTLA-4 expression is mediated through the glucocorticoid receptor. In conclusion, the immunosuppressive agent dexamethasone actually stimulates CTLA-4 expression, which is involved in downregulation of T cell activation. Received 19 May 1999; received after revision 13 July 1999; accepted 13 July 1999     

枸杞多糖对人肺腺癌 A549细胞增殖和凋亡的影响

目的探讨枸杞多糖(LBP)对体外培养的人肺腺癌细胞 A549的增殖抑制作用及其可能的作用机制.方法用不同浓度的LBP处理 A549细胞,MTT法检测24、48、72h时间点 LBP对 A549细胞的生长抑制率,实验设为对照组和实验组(1/2IC50作用48小时),MTT法绘制生长曲线、细胞计数计算倍增时间、流式细胞仪检测凋亡率及其细胞周期、RT PCR检测 SurvivinmRNA的变化、Westernblot检测 CyclinB1蛋白的变化,transwell体外侵袭实验观察药物对细胞体外侵袭的影响.结果 MTT显示不同浓度的LBP均能明显抑制 A549细胞的增殖且成剂量 效应关系,实验组细胞的倍增时间、凋亡率与对照组相比,均有统计学意义(P<0.05);LBP使细胞阻滞在 G2期,SurvivinmRNA表达和 CyclinB1蛋白的表达均降低,与对照组相比差异有显著性(P<0.05).结论 LBP可抑制 A549细胞的增殖,其机理可能与 LBP使 SurvivinmRNA表达下降引起细胞凋亡及 CyclinB1蛋白的表达降低造成细胞周期阻滞及抑制细胞的侵袭能力有关     

Role of Chk1 in the differentiation program of hematopoietic stem cells

Hematopoietic stem cells (HSC) isolated from umbilical cord blood (UCB) were treated with ionizing radiation (IR) and sensitivity and IR induced checkpoints activation were investigated. No difference in the sensitivity and in the activation of DNA damage pathways was observed between CD133+ HSC and cells derived from them after ex vivo expansion. Chk1 protein was very low in freshly isolated CD133+ cells, and undetectable in ex vivo expanded UCB CD133+ cells. Chk1 was expressed only on day 3 of the ex vivo expansion. This pattern of Chk1 expression was corroborated in CD133+ cells isolated from peripheral blood apheresis collected from an healthy donor. Treatment with a specific Chk1 inhibitor resulted in a strong reduction in the percentage of myeloid precursors (CD33+) and an increase in the percentage of lymphoid precursors (CD38+) compared to untreated cells, suggesting a possible role for Chk1 in the differentiation program of UCB CD133+ HSC.