福司可林(FSK88)对HL60细胞抑增殖促分化的实验研究

研究了诱导分化剂FSK88对人白血病HL60细胞的抗肿瘤作用，将HL60细胞培养在含不同浓度FSK88的RPMI1640培养液中，测定FSK88对HL60细胞生长曲线、NBT阳性率、ANAE酶活性的影响，探讨了FSK88对HL60细胞裸鼠异种移植瘤体内抗肿瘤作用。结果表明，FSK88能诱导HL670细胞向单核／巨噬细胞方向分化，表现为生长抑制，NBT还原能力提高，酸性非特异性酯酶（ANAE）活性增加；流式细胞仪检测表明FSK88对HL60的诱导作用与细胞所处细胞周期的变化无明显相关性；FSK88可以有效的抑制白血病HL60的致瘤力，可见FSK88能抑制人白血病细胞的生长，促使其进入终末分化及凋亡。     

血管内皮生长因子反义核酸增强HL60和K562细胞对As_2O_3的敏感性

目的:探讨血管内皮生长因子(VEGF)反义核酸能否提高HL60和K562细胞对三氧化二砷(As2O3)的敏感性。方法:采用经筛选所得的最优反义核酸(A7),20个碱基经过全硫代修饰;以脂质体介导转染细胞,反义核酸和As2O3联合作用72h以后,用MTT法检测细胞生长情况,求IC50值;用ELISA法检测培养液中VEGF蛋白的浓度,用流式细胞仪检测细胞凋亡百分数。结果:VEGF反义核酸可显著降低HL60、K562细胞对As2O3的IC50值,下调VEGF蛋白的表达,增加As2O3诱导的HL60、K562细胞凋亡作用。结论:VEGF反义核酸具有增强HL60和K562细胞对As2O3的敏感性,增强As2O3诱导的HL60和K562细胞凋亡作用;提示内源性VEGF蛋白具有使细胞产生耐药性的作用。     

血管内皮生长因子反义核酸增强HL60和K562细胞对As2O3的敏感性细胞对As2O3的敏感性

目的:探讨血管内皮生长因子(VEGF)反义核酸能否提高HL60和K562细胞对三氧化二砷(As2O3)的敏感性.方法:采用经筛选所得的最优反义核酸(A7),20个碱基经过全硫代修饰;以脂质体介导转染细胞,反义核酸和As2O3联合作用72 h以后,用MTT法检测细胞生长情况,求IC50值;用ELISA法检测培养液中VEGF蛋白的浓度,用流式细胞仪检测细胞凋亡百分数.结果:VEGF反义核酸可显著降低HL60、K562细胞对As2O3的IC50值,下调VEGF蛋白的表达,增加As2O3诱导的HL60、K562细胞凋亡作用.结论:VEGF反义核酸具有增强HL60和K562细胞对As2O3的敏感性,增强As2O3诱导的HL60和K562细胞凋亡作用;提示内源性VEGF蛋白具有使细胞产生耐药性的作用.     

Fas在云芝糖肽诱导HL-60细胞凋亡中的作用

目的：探讨云芝糖肽（PSP）诱导HL-60细胞凋亡的Fas死亡受体信号转导途径．方法：观察PSP处理后HL-60细胞的形态变化,并采用流式细胞仪及免疫印迹检测细胞Fas,FADD蛋白的表达及Caspase-8凋亡酶的激活．结果：100,400g／mLPSP处理HL-60细胞48h后,细胞出现明显凋亡特征,同时伴随Caspase-8酶原被激活和Fas抗原的表达量增加,表现为剂量依赖关系．400g／mLPSP处理36h时Caspase-8酶原开始被剪切激活,48h时激活更为显著．各实验组FADD的表达量基本没有变化．结论：Fas死亡受体信号可能参与了PSP诱导HL-60细胞凋亡．     

血管内皮生长因子反义核酸增强HL60和K562细胞对As2O3的敏感性

目的：探讨血管内皮生长因子(VEGF)反义核酸能否提高HL60和K562细胞对三氧化二砷(As2O3)的敏感性。方法：采用经筛选所得的最优反义核酸(A7)，20个碱基经过全硫代修饰；以脂质体介导转染细胞，反义核酸和AsO3联合作用72h以后，用MTT法检测细胞生长情况，求IC50值；用ELISA法检测培养液中VEGF蛋白的浓度，用流式细胞仪检测细胞凋亡百分数。结果：VEGF反义核酸可显降低HL60、K562细胞对As2O3的IC50值，下调VEGF蛋白的表达，增加As2O3诱导的HL60、K562细胞凋亡作用。结论：VEGF反义核酸具有增强HL60和K562细胞对As2O3的敏感性，增强As2O3诱导的HL60和K562细胞凋亡作用；提示内源性VEGF蛋白具有使细胞产生耐药性的作用。     

c-myc oncogene protein synthesis is independent of the cell cycle in human and avian cells

Several lines of evidence suggest a role for the myc oncogene in cell proliferation. Most recently, mitogenic stimulation of quiescent lymphoid, fibroblast and epithelial cells has been demonstrated to lead to a sharp increase in c-myc RNA levels. To determine how c-myc expression is linked to the cell proliferative cycle, we have used centrifugal elutriation to enrich for populations of avian and human cells at different stages of the cell cycle. Centrifugal elutriation is a counterflow centrifugation method that separates cells on the basis of volume, a parameter correlating well with progression through the cell cycle. Using myc-specific anti-peptide antibodies, we show here that the synthesis, half-life and modification of c-myc proteins are constant throughout the cell cycle of normal and transformed cells.     

血管内皮生长因子基因反义核酸设计及其对HL60细胞生长的影响

目的:用计算机设计筛选出血管内皮生长因子(VEGF)的高效反义核酸;用实验方法研究VEGF反义核酸对HL60细胞生长的影响。方法:用RNAstructure(version3 7)软件,选择总自由能(Overall△G37)的相对低的反义核酸,共计7条,长度18～20核苷酸,全硫代修饰;细胞培养72h,采用胎盼蓝拒染法观察存活细胞,用ELISA法检测培养液中VEGF蛋白水平,分析反义核酸对HL60细胞的作用。结果:筛选出6条反义药物对HL60细胞生长有明显抑制作用,其中4条优于阳性对照组(A2);A7组细胞生长抑制率达41 74%。培养液中VEGF蛋白表达抑制率达47 81%。Overall△G37与反义核酸活性密切相关(r=-0 842,P<0 01)。结论:计算机辅助设计有助于获得更好的反义药物,VEGF反义核酸可抑制HL60细胞生长及VEGF蛋白表达,VEGF可能成为白血病治疗的新靶点。     

银杏叶多糖对人脐静脉内皮细胞与HL-60细胞粘附的影响

根据人脐静脉血管内皮细胞(ECV-304)的显微和超微结构,研究了银杏叶多糖(GBLP)对ECV-304细胞和HL-60细胞粘附作用的影响.结果表明,正常生长的ECV-304细胞呈不规则形,细胞质丰富,细胞核呈圆形或椭圆形.电镜下,可见细胞表面有绒毛,细胞内具有Weibel-Palade小体.当ECV-304细胞受凝血酶(thrombin)刺激后,与HL-60细胞的粘附能力明显提高;而当用200 μg/ mL和500 μg/ mL剂量的GBLP处理ECV-304细胞后,与HL-60细胞的粘附率明显下降.这表明200 μg/ mL和500 μg/ mL剂量的GBLP对人脐静脉内皮细胞与HL-60细胞粘附有抑制作用.     

Wangzaozin A调节NADPH氧化酶源性活性氧诱导HL-60细胞分化

利用台盼蓝排染法、吉姆萨染色及流式细胞术对对映-贝壳杉烷二萜wangzaozin A影响人早幼粒白血病细胞HL-60生长、细胞形态、NBT还原能力、细胞吞噬及细胞表面抗原CD11b表达进行了检测.结果显示,0.2~0.8μmol/L wangzaozin A抑制HL-60细胞生长,0.6和0.8μmol/L处理组细胞显示了明显的G1期周期阻滞.随着wangzaozin A浓度升高及处理时间延长,细胞核质比减小,肾形核、杆状核和分叶核细胞增多及胞质中颗粒状物质增加;同时,细胞NBT还原能力、吞噬能力及细胞表面抗原CD11b表达显著增强,表明wangzaozin A可诱导HL-60细胞向成熟粒细胞分化.进一步利用荧光探针DCF检测显示0.6和0.8μmol/L wangzaozin A处理细胞12h后细胞内ROS显著升高;抗氧化剂NAC及NADPH氧化酶抑制剂APO显著抑制wangzaozin A诱导HL-60细胞上调CD11b表达,表明wangzaozin A可通过上调NADPH氧化酶源性的活性氧浓度诱导HL-60细胞分化.     

Antigen-induced apoptotic death of Ly-1 B cells responsible for autoimmune disease in transgenic mice.

Studies on transgenic mice expressing immunoglobulins against self-antigens have shown that self-tolerance is maintained by active elimination (clonal deletion), functional inactivation (clonal anergy) of self-reactive B cells, or a combination of both. We have established and characterized a transgenic mouse line expressing an anti-erythrocyte autoantibody. In contrast to other autoantibody transgenic lines, about 50% of the animals of this transgenic line suffer from autoimmune disease, indicating a loss of self-tolerance. Here we show that peritoneal Ly-1 B cells (also known as B-1 cells) are responsible for this autoimmune disease in our transgenic mice. A few self-reactive Ly-1 B cells that have somehow escaped the deletion mechanism expand in the peritoneum because of the absence of self-antigen. These Ly-1 B cells are eliminated in vivo by apoptosis once exposed to self-antigen. On the basis of these results we propose a novel autoantibody production mechanism whereby self-reactive B cells sequestered in compartments free of self-antigens may survive, proliferate and be activated for generation of pathogenic autoantibodies in autoimmune diseases.     

Haemagglutinin mutations responsible for the binding of H5N1 influenza A viruses to human-type receptors

H5N1 influenza A viruses have spread to numerous countries in Asia, Europe and Africa, infecting not only large numbers of poultry, but also an increasing number of humans, often with lethal effects. Human and avian influenza A viruses differ in their recognition of host cell receptors: the former preferentially recognize receptors with saccharides terminating in sialic acid-alpha2,6-galactose (SAalpha2,6Gal), whereas the latter prefer those ending in SAalpha2,3Gal (refs 3-6). A conversion from SAalpha2,3Gal to SAalpha2,6Gal recognition is thought to be one of the changes that must occur before avian influenza viruses can replicate efficiently in humans and acquire the potential to cause a pandemic. By identifying mutations in the receptor-binding haemagglutinin (HA) molecule that would enable avian H5N1 viruses to recognize human-type host cell receptors, it may be possible to predict (and thus to increase preparedness for) the emergence of pandemic viruses. Here we show that some H5N1 viruses isolated from humans can bind to both human and avian receptors, in contrast to those isolated from chickens and ducks, which recognize the avian receptors exclusively. Mutations at positions 182 and 192 independently convert the HAs of H5N1 viruses known to recognize the avian receptor to ones that recognize the human receptor. Analysis of the crystal structure of the HA from an H5N1 virus used in our genetic experiments shows that the locations of these amino acids in the HA molecule are compatible with an effect on receptor binding. The amino acid changes that we identify might serve as molecular markers for assessing the pandemic potential of H5N1 field isolates.     

Proteolytic turnover of the Gal4 transcription factor is not required for function in vivo

Transactivator-promoter complexes are essential intermediates in the activation of eukaryotic gene expression. Recent studies of these complexes have shown that some are quite dynamic in living cells owing to rapid and reversible disruption of activator-promoter complexes by molecular chaperones, or a slower, ubiquitin-proteasome-pathway-mediated turnover of DNA-bound activator. These mechanisms may act to ensure continued responsiveness of activators to signalling cascades by limiting the lifetime of the active protein-DNA complex. Furthermore, the potency of some activators is compromised by proteasome inhibition, leading to the suggestion that periodic clearance of activators from a promoter is essential for high-level expression. Here we describe a variant of the chromatin immunoprecipitation assay that has allowed direct observation of the kinetic stability of native Gal4-promoter complexes in yeast. Under non-inducing conditions, the complex is dynamic, but on induction the Gal4-promoter complexes 'lock in' and exhibit long half-lives. Inhibition of proteasome-mediated proteolysis had little or no effect on Gal4-mediated gene expression. These studies, combined with earlier data, show that the lifetimes of different transactivator-promoter complexes in vivo can vary widely and that proteasome-mediated turnover is not a general requirement for transactivator function.