体外培养的小鼠抗原负载树突状细胞的形态学特征

目的:研究体外培养的小鼠抗原负载树突状细胞(dentritic cells,DCs)的形态学特征,为肿瘤的生物学治疗提供形态学基础.方法:分离和培养DC,制备B16黑色素瘤细胞抗原,进行共培养,即为抗原负栽的DC.建立B16黑色素瘸小鼠模型,于瘤周围皮下注射抗原负载的DC.应用光镜、免疫组化方法和透射电镜观察抗原负载DC的形态学特征.结果:培养的抗原负载DC与DC比较,体积较大,表面突起较粗大且弯曲.免疫组化染色显示抗原负载树突状细胞主要分布于肿瘤周围皮肤的乳头层、网织层和肿瘤周围,于局域淋巴结的被膜下窦和副皮质区有散在分布.电镜下抗原负载DC细胞体积较大,核有切迹,细胞表面的突起,与肿瘤细胞和淋巴细胞接触密切.结论:抗原负载DC表现出比一般树突状细胞功能更加活跃的形态特征,冻融法全细胞来源的肿瘤抗原负载DC可以获得理想的DC疫苗.     

软骨多糖诱导MCF-7乳腺癌细胞凋亡的实验研究

研究软骨多糖诱导MCF-7乳腺癌细胞凋亡及其作用机理。方法:选用MCF-7人类乳腺癌细胞系体外培养,应用MTT法检测细胞生长抑制率,TUNEL法检测细胞凋亡率,HE染色法观察细胞形态学改变,流式细胞仪检测细胞周期的变化,免疫荧光方法检测BCL-2BAD及波形蛋白Vimentin的表达率。结果:软骨多糖对MCF-7细胞体外生长具有明显的抑制作用,且呈时间和浓度依赖性;软骨多糖可诱导MCF-7细胞发生凋亡并伴随有凋亡小体出现等形态学变化;软骨多糖促进BCL-2蛋白的表达水平下降,BAD表达水平上升,及Vimentin的降解。结论:软骨多糖能够在体外诱导MCF-7细胞凋亡,是一种新型的抗乳腺癌活性物质。     

67kD层粘连蛋白受体的克隆、表达及其对肝癌细胞侵袭能力的影响

探讨细胞膜表面 6 7kD层粘连蛋白受体 (6 7kDlamininreceptor ,6 7LR)在肝癌细胞侵袭转移中的作用 ,从肝癌细胞中提取RNA ,通过RT PCR扩增 6 7LR的前体——— 37kD层粘连蛋白受体前体(37kDlamininreceptorprecursor,37LRP)基因并定向克隆到真核表达载体pcDNA3.1 myc His(- )A ,采用脂质体将重组质粒转染到HepG2肝癌细胞中 ,通过G4 1 8筛选和RT PCR、流式细胞术鉴定 ,获得了细胞膜表面 6 7LR高表达 (阳性率为 6 9.2 % )和低表达 (阳性率为 1 1 .7% )的细胞克隆 ,采用体外细胞侵袭实验测定不同细胞的侵袭能力 ,发现膜表面 6 7LR高表达的细胞侵袭能力明显高于低表达及不表达细胞 ,说明 6 7LR在肝癌细胞侵袭转移过程中可能具有重要意义     

用重组痘苗病毒感染动物细胞表达的Epstein-Barr病毒膜抗原检查IgA/MA抗体

用EB病毒膜抗原基因重组的痘苗病毒感染动物细胞,其细胞表面可表达EB病毒膜抗原,以此膜抗原作为诊断抗原检测血清中IgA/MA抗体,明显优于常用的B95-8细胞表面膜抗原,从而为研究人群血清抗体反应与鼻咽癌的关系,为鼻咽癌的诊断和普查开辟了新的途径。     

积雪草甙对乳腺癌MCF-7细胞凋亡及VEGF、bFGF蛋白表达水平的影响

目的:探讨积雪草甙对乳腺癌MCF-7细胞凋亡及VEGF、bFGF蛋白表达水平的影响。方法:选取人乳腺癌细胞MCF-7细胞系进行体外培养后,根据是否进行积雪草甙干预而分为两组,应用积雪草苷进行干预后,HE染色后用光学显微镜法观察细胞形态学变化,干预后的24h、48h以及72h时,应用TUNEL技术对对细胞凋亡情况进行检测,同时应用免疫荧光法检测血管内皮生长因子(VEGF)和碱性成纤维细胞生长因子(bFGF)的表达。结果:(1)与对照组相比较,积雪草甙干预的乳腺癌MCF-7细胞出现空泡、胞质外溢以及胞核皱缩等细胞凋亡现象,大量癌MCF-7细胞发生破碎死亡;(2)TUNEL技术法检测结果证实积雪草甙能够提高人乳腺癌MCF-7细胞凋亡率,与对照组比较差异具有统计学意义(P0.05),且呈时间依赖性;(3)积雪草甙干预的MCF-7细胞VEGF阳性表达和bFGF阳性表达显著低于对照组,差异具有统计学意义(P0.05),积雪草甙的抑制作用且呈时间依赖性。结论:积雪草甙不仅能够促进乳腺癌MCF-7细胞凋亡,而且能够降低VEGF和bFGF表达。     

硫酸化茯苓多糖对人乳腺癌细胞株MCF-7凋亡的影响

为了探讨硫酸化茯苓多糖(SP)对人乳腺癌细胞株MCF-7凋亡的影响,采用MTT法检测不同浓度、作用时间SP对乳腺癌MCF-7细胞的抑制作用,倒置显微镜观察MCF-7细胞的形态学变化,RT-PCR检测SP处理MCF-7细胞凋亡相关基因(Bcl-2,Bax)的表达;Western blotting技术检测SP对乳腺癌细胞凋亡蛋白Bcl-2、Bax表达变化。结果表明,SP对MCF-7细胞增殖有抑制作用,且在一定范围内呈剂量效应;细胞贴壁能力减弱,细胞间隙增大,胞膜褶皱;Bcl-2基因表达水平和蛋白表达水平明显降低(p0.05),Bax基因表达水平和蛋白表达水平明显升高(p0.05)。基于以上研究,SP通过促凋亡基因Bax的表达,抑制抗凋亡基因Bcl-2的表达来下调Bcl-2/Bax比值,激活凋亡途径,诱导MCF-7细胞的凋亡。     

细胞因子基因转导对人乳腺癌细胞MHC抗原及细胞膜糖蛋白表达的影响

为探讨细胞因子基因(人IL-2、IL-6)转导对于肿瘤细胞膜MHC抗原及细胞膜糖蛋白表达调控的影响,本文利用脂质体介导的方法,将含人IL-6、IL-2基因的逆转录病毒载体分别导入人乳腺癌细胞系MCF-7细胞中,采用间接免疫荧光染色流式细胞仪测定法,对基因转导的瘤细胞细胞膜糖蛋白及MHC抗原表达进行测定。结果表明经两种基因修饰的MCF-7细胞MHCⅠ型抗原表达均获得增强,此外,基因转导细胞可程度不同地表现出细胞膜多种糖蛋白表达的变化。提示肿瘤细胞膜抗原及糖蛋白表达的改变可能是细胞因子基因转导影响肿瘤细胞免疫原性的重要结构基础。     

2-甲氧基雌二醇对乳腺癌MCF-7细胞HIF-1α、CXCR4、VEGF表达的影响

目的:探讨2-甲氧基雌二醇(2-Methoxyestradiol,2ME2)对乳腺癌MCF-7细胞生长及缺氧诱导因子-1α(Hypoxia-inducible factor1 α,HIF-1α)、趋化因子受体-4(CXC chemokine receptor-4,CXCR4)、血管内皮生长因子(Vascular endothelial growth factor,VEGF)表达的影响.方法:采用MTT法检测不同浓度2ME2对MCF-7细胞的增殖抑制作用;Hoechest 33258染色观察细胞凋亡形态学改变;RT-PCR、Western blot分别检测不同浓度2ME2对乳腺癌MCF-7细胞中HIF-1α、CXCR4、VEGF mRNA及蛋白表达水平的影响.结果:2ME2可较强的抑制MCF-7细胞增殖,并呈时间和剂量依赖性;经2ME2作用48h后MCF-7细胞表现出典型的凋亡形态特征;不同浓度2ME2作用于MCF-7细胞48小时后,随着药物浓度的增加细胞中HIF-1α、CXCR4、VEGF在mRNA及蛋白表达水平逐渐降低,与对照组相比有统计学意义(P＜0.05).结论:2ME2可通过降低HIF-1α、CXCR4、VEGFmRNA及蛋白表达,抑制乳腺癌MCF-7细胞的增殖及肿瘤血管生成和侵袭转移相关因子的表达.     

改变细胞膜的脂肪酸组成可促进乳腺癌细胞凋亡

目的: 研究n-6脂肪酸脱氢酶 fat-1基因在人乳腺癌细胞内的表达,改变细胞膜脂肪酸组成,对乳腺癌细胞的凋亡作用。方法: 构建含有fat-1 基因的重组腺病毒载体 (Ad.GFP.fat-1),通过包装细胞系（293）产生的腺病毒,感染人乳腺癌细胞MCF-7。提取细胞的总RNA,以fat-1的反义mRNA 作探针,用Northern Blot检测fat-1 基因在MCF-7细胞内的表达。MTT法分析fat-1 基因对MCF-7细胞增殖的影响,凋亡染色试剂盒检测细胞的凋亡。气相色谱仪分析对MCF-7细胞的n-6 PUFAs/n-3 PUFAs含量影响。结果: 通过基因重组技术,得到预期的重组病毒;fat-1 基因在人乳腺癌细胞MCF-7 中能有效异源表达,2天后,可检测到fat-1 mRNA的条带。与对照细胞相比,fat-1基因有效地抑制了MCF-7细胞的增殖（23%,p<0.05）,促进了凋亡（增加35%）;同时降低了人乳腺癌细胞MCF-7细胞膜n-6 PUFAs/n-3 PUFAs的比率。结论: 腺病毒介导的fat-1 基因能在人乳腺癌细胞MCF-7内有效异源表达,且抑制了MCF-7细胞的增殖。机理为降低了细胞膜的n-6 PUFAs/n-3 PUFAs的比率。     

细胞膜脂肪酸组成改变促进乳腺癌细胞凋亡的研究

目的:研究n-6脂肪酸脱氢酶fat-1基因在人乳腺癌细胞内的表达,改变细胞膜脂肪酸组成,对乳腺癌细胞的凋亡作用.方法:构建含有fat-1基因的重组腺病毒载体(Ad.GFP.fat-1),通过包装细胞系(293)产生的腺病毒,感染人乳腺癌细胞MCF-7.提取细胞的总RNA,以fat-1的反义mRNA作探针,用Northern blot检测fat-1基因在MCF-7细胞内的表达.MTT法分析fat-1基因对MCF-7细胞增殖的影响,凋亡染色试剂盒检测细胞的凋亡.气相色谱仪分析对MCF-7细胞的n-6 PUFAs/n-3 PUFAs含量影响.结果:通过基因重组技术,得到预期的重组病毒;fat-1基因在人乳腺癌细胞MCF-7中能有效异源表达,2d后,可检测到fat-1 mRNA的条带.与对照细胞相比,fat-1基因有效地抑制了MCF-7细胞的增殖(23%,p＜0.05),促进了凋亡(增加35%);同时降低了人乳腺癌细胞MCF-7细胞膜n-6 PUFAs/n-3 PUFAs的比率.结论:腺病毒介导的fat-1基因能在人乳腺癌细胞MCF-7内有效异源表达,且抑制了MCF-7细胞的增殖.机理为降低了细胞膜的n-6 PUFAs/n-3 PUFAs的比率.     

MCF—7细胞在亚多层中细胞膜极性的变化

Sub-multilayer of MCF-7 cell, an established human breast carcinoma cell line, was achieved by culturing the cells on millipore filters for a long time. The superficial layer cells maintained their membrane polarity features as MCF-7 cells in monolayers did. MAM-6, a human milk fat globule membrane antigen, was polarized distributed in apical domain of 97.5% superficial layer cells revealed by immunoperoxidase cytochemistry. Whereas, among the low layer cells, which had no free surface (apical domain) toward the culture medium and did not show morphological polarity features, only 12.9% expressed surface MAM-6 with weak immunoperoxidase staining and random distribution. But the immunostaining for detecting cytoplasmic MAM-6 in low layer cells was stronger than that in superficial layer cells, indicating that the vectorial delivery and insert of MAM-6 carrying glycoprotein to the plasma membrane seemed to be stopped or declined and became undirectional in the later situation. The study demonstrates that an asymmetric spatial environment, which is composed of a liquid phase space and a solid phase space, is crucial for the establishment of epithelial membrane polarity of MCF-7 cells.     

Polarized expression of an apical membrane glycoprotein is established before functional tight junctions have developed in MCF-7 cells

A milk-fat globule membrane antigen, designated MAM-6 and detected immunocytochemically by the monoclonal antibody 115D8, is expressed apically in confluent MCF-7 monolayer cultures. Immediately after preparation of a single-cell suspension, MAM-6 appears on the entire cell surface. However, polarized apical expression of MAM-6 is restored as early as 2-6 hr after plating of unpolarized cells, before functional tight junctions are established, as judged by freeze-fracture and ruthenium red permeability. Quantitative immunogold cytochemistry reveals that the apical:basal ratio of MAM-6 expression was about 17:1 after 6 hr. Tight junctions developed as late as 12-24 hr after plating. At this time the apical:basal MAM-6 ratio was about 30:1 (as compared to about 50:1 in control monolayers).     

Epithelial membrane polarity: a stable, differentiated feature of an established human breast carcinoma cell line MCF-7

Carcinoma cells typically show little or no polarity as compared to normal, differentiated epithelial cells. We have studied polarity in two established human breast carcinoma cell lines, T47D and MCF-7, by various techniques (electron microscopic enzyme- and immunocytochemistry, freeze-fracture) and show that one of them (MCF-7) is characterized by a high degree of polarity. Thus, in contrast to T47D cells, MCF-7 cells in monolayer culture form apical tight junctions, do not allow a ricin-horseradish peroxidase conjugate, which binds to terminal galactose residues on the apical surface, to stain the basolateral membrane domain, and express a surface antigen (MFGM-A) only in the apical surface membrane domain, as do normal mammary epithelial cells in vivo. This polarization is independent of a basement membrane, since it is maintained when MCF-7 cells, which do not deposit type IV collagen themselves, are grown directly on plastic. Moreover, even though MCF-7 cells express estrogen receptors rather homogeneously, estrogen has no effect on this polarity, neither in vitro nor after transplantation to nude mice. We conclude that polarity is a stable, differentiated feature of MCF-7 cells.     

Internalized ricin and the plasma membrane glycoprotein MAM-6 colocalize in the trans-Golgi network of T47D human breast carcinoma cells

We have analyzed the intracellular transport of endocytosed ricin in the human breast carcinoma cell line T47D. Cells were incubated with ricin (10 micrograms/ml) for 1 h at 37 degrees C. Marked reduction in the protein synthesis did not take place until the end of this period. To detect ricin immunocytochemically, a rabbit anti-ricin serum was used. Gel electrophoresis followed by immunoblotting revealed that the antiserum reacted specifically with ricin and detected both the ricin A-chain and the ricin B-chain. Immunofluorescence experiments showed endocytosed ricin in endosomal and lysosomal vacuoles throughout the cytoplasm, as well as in a typical perinuclear position corresponding to the Golgi region. Using the monoclonal mouse antibody 115D8 directed toward the high-molecular-weight membrane glycoprotein MAM-6 of human breast epithelial cells, we similarly obtained a swarms of small vesicles throughout the cytoplasm. To further analyze the apparent colocalization of ricin and MAM-6 in the perinuclear Golgi region, immunogold cytochemistry on ultracryosections was performed. MAM-6 was detected mainly in Golgi stacks and associated trans-Golgi network (TGN) profiles, in 0.1 to 0.2-micron secretory vesicles, and on the cell surface. Ricin was detected on the cell surface, in endosomes and lysosomes, and also in the TGN. Furthermore, by using immunogold double labeling, internalized ricin was found to colocalize with MAM-6 in the TGN.     

Internalized ricin and the plasma membrane glycoprotein MAM-6 colocalize in the trans-Golgi network of T47D human breast carcinoma cells

We have analyzed the intracellular transport of endocytosed ricin in the human breast carcinoma cell line T47D. Cells were incubated with ricin (10 μg/ml) for 1 h at 37 °C. Marked reduction in the protein synthesis did not take place until the end of this period. To detect ricin immunocytochemically, a rabbit anti-ricin serum was used. Gel electrophoresis followed by immunoblotting revealed that the antiserum reacted specifically with ricin and detected both the ricin A-chain and the ricin B-chain. Immunofluorescence experiments showed endocytosed ricin in endosomal and lysosomal vacuoles throughout the cytoplasm, as well as in a typical perinuclear position corresponding to the Golgi region. Using the monoclonal mouse antibody 115D8 directed toward the high-molecular-weight membrane glycoprotein MAM-6 of human breast epithelial cells, we similarly obtained a marked perinuclear fluorescence. In addition, MAM-6 fluorescence was observed in swarms of small vesicles throughout the cytoplasm. To further analyze the apparent colocalization of ricin and MAM-6 in the perinuclear Golgi region, immunogold cytochemistry on ultracryosections was performed. MAM-6 was detected mainly in Golgi stacks and associated trans-Golgi network (TGN) profiles, in 0.1 to 0.2-μm secretory vesicles, and on the cell surface. Ricin was detected on the cell surface, in endosomes and lysosomes, and also in the TGN. Furthermore, by using immunogold double labeling, internalized ricin was found to colocalize with MAM-6 in the TGN.     

甲胎蛋白在体外对人肝癌细胞生长的影响

本文用MTT比色法观察了甲胎蛋白(AFP)在体外对人肝癌细胞生长的影响。结果表明,AFP能促进SMMC-7721人肝癌细胞的生长。当AFP与AFP抗体合用时,AFP抗体能减弱AFP对SMMC-7721细胞生长的促进作用;AFP抗体单用对此种细胞的生长亦有抑制作用。另一方面,在相同的实验条件下,AFP和AFP抗体对HL-60人白血病细胞的生长无明显影响;提示AFP的促生长作用具有一定的肿瘤细胞特异性,并非一种蛋白质对培养细胞的非特异性营养作用。此外,AFP亦能促进MCF-7人乳腺癌细胞的生长,AFP抗体对此种细胞的生长有抑制作用。由于MCF-7细胞存在功能性AFP受体,也能合成和分泌AFP。这就提示,人肝癌细胞中的AFP很可能与其受体特异性结合,产生促生长效应。确切机制尚待进一步阐明。     

Membrane raft microdomains mediate front-rear polarity in migrating cells

The acquisition of spatial and functional asymmetry between the rear and the front of the cell is a necessary step for cell chemotaxis. Insulin-like growth factor-I (IGF-I) stimulation of the human adenocarcinoma MCF-7 induces a polarized phenotype characterized by asymmetrical CCR5 chemokine receptor redistribution to the leading cell edge. CCR5 associates with membrane raft microdomains, and its polarization parallels redistribution of raft molecules, including the raft-associated ganglioside GM1, glycosylphosphatidylinositol-anchored green fluorescent protein and ephrinB1, to the leading edge. The non-raft proteins transferrin receptor and a mutant ephrinB1 are distributed homogeneously in migrating MCF-7 cells, supporting the raft localization requirement for polarization. IGF-I stimulation of cholesterol-depleted cells induces projection of multiple pseudopodia over the entire cell periphery, indicating that raft disruption specifically affects the acquisition of cell polarity, but not IGF-I-induced protrusion activity. Cholesterol depletion inhibits MCF-7 chemotaxis, which is restored by replenishing cholesterol. Our results indicate that initial segregation between raft and non-raft membrane proteins mediates the necessary redistribution of specialized molecules for cell migration.     

Inhibition of epidermal growth factor/transforming growth factor-alpha-stimulated cell growth by a synthetic peptide

Estrogen-stimulated growth of the human mammary adenocarcinoma cell line MCF-7 is significantly inhibited by monoclonal antibodies to the epidermal growth factor (EGF) receptor that act as antagonists of EGF's mitogenic events by competing for high-affinity EGF receptor binding sites. These antibodies likewise inhibit the EGF or transforming growth factor-alpha (TGF-alpha)-stimulated growth of these MCF-7 cells. An analogous pattern of specific EGF or TGF-alpha growth inhibitory activity was obtained using a synthetic peptide analog encompassing the third disulfide loop region of TGF-alpha, but containing additional modifications designed for increased membrane affinity [( Ac-D-hArg(Et)2(31),Gly32,33]HuTGF-alpha(31-43)NH2). The growth factor antagonism by this synthetic peptide was specific in that it inhibited EGF, TGF-alpha, or estrogen-stimulated growth of MCF-7 cells but did not inhibit insulin-like growth factor-1 (IGF-1)-stimulated cell growth. Altogether, these results suggest that a significant portion of the estrogen-stimulated growth of these MCF-7 cells is mediated in an autocrine/paracrine manner by release of EGF or TGF-alpha-like growth factors. The TGF-alpha peptide likewise inhibited EGF- but not fibroblast growth factor (FGF)- or platelet-derived growth factor (PDGF)-stimulated growth of NIH-3T3 cells in completely defined media; but had no effect on growth or DNA synthesis of G0-arrested cells, nor did it effect growth of NR-6 cells, which are nonresponsive to EGF. Although this synthetic peptide did not directly compete with EGF for cell surface receptor binding, it exhibited binding to a cell surface component (followed by internalization), which likewise was not competed by EGF. The peptide did not directly inhibit EGF-stimulated phosphorylation of the EGF receptor, nor did it inhibit phosphorylation of an exogenous substrate, angiotensin II, by activated EGF receptor. The TGF-alpha peptide did, however, affect the structure of laminin as manifested by laminin self-aggregation; this affect on laminin may, in turn, have a modulatory effect on EGF-mediated cell growth.