核仁抗原在白血病诊断中的初步观察

用抗核仁抗原NAg-1的抗血清对各种白血病人骨髓组织进行免疫荧光染色.结果表明,急性白血病患者的NAg-1阳性细胞率显著高于正常人及各种非白血病贫血患者.在处于完全缓解的白血病患者中,具有较高的NAg-1阳性率者(>10％)比较低者(<10％)更易复发.     

全反式维甲酸对人白血病细胞系HL-60细胞增殖的影响

目的:观察全反式维甲酸(ATRA)对体外培养人白血病细胞系HL-60细胞增殖的影响。方法:体外培养人白血病细胞HL-60细胞,将不同浓度的ATRA作用于HL-60细胞分别12、24h和48h后,MTT法检测ATRA对HL-60的增殖影响,免疫组织化学染色增生细胞核抗原(PCNA)检测HL-60细胞生长活性。结果:MTT法显示10-9mmol/L ATRA作用12h后能抑制HL-60细胞的增殖(P<0.05),并呈剂量和时间依赖性。ATRA能浓度依赖性地抑制HL-60细胞表达PCNA(P<0.05)。结论:ATRA能够显著抑制人白血病细胞HL-60细胞的增殖。     

NB4和HL - 60细胞诱导的脐血T细胞TCR Vβ基因谱系分析

目的：了解经NB4细胞和HL-60细胞体外诱导后脐血T细胞的TCR Vβ亚家族限制性利用和克隆性增殖情况。方法：采用混合淋巴细胞／肿瘤细胞培养（MLTC）方法,用经丝裂霉素灭活的NB4细胞和HL-60细胞体外诱导脐血T细胞增殖,利用RT-PCR分析经MLTC前后T细胞的24个TCR Vβ亚家族的利用情况,并用基因扫描分析其克隆性特点。结果：诱导前脐血T细胞表达20个TCR Vβ亚家族,诱导后的T细胞所表达的TCR Vβ亚家族随培养时间增加而逐渐减少,且诱导组呈现出克隆趋势或寡克隆增殖特点。两种细胞诱导后10d均可见Vβ1的克隆增殖出现变化,此外,NB4诱导的T细胞出现Vβ15,而HL-60诱导的T细胞则出现Vβ23的克隆增殖情况。结论：NB4细胞和HL-60细胞可诱导脐血TCR VβT细胞克隆性增殖,此优势表达的克隆性T细胞可能与特异性抗原介导的细胞免疫反应有关。     

中华补血草多酚诱导白血病细胞HL-60凋亡的研究

采用MTT法研究了中华补血草根多酚提取物对HL-60细胞的抑增殖效果;AO/EB染色、DNA片段化检测和Annexin V-FITC流式细胞术等方法检测了多酚提取物对HL-60细胞诱导凋亡结果.Western blotting检测HL-60细胞株中Bcl-2、Bax蛋白的表达量.结果表明,中华补血草多酚能明显抑制HL-60细胞增殖,诱导HL-60细胞凋亡并具有质量浓度依赖性;Western blotting检测经药物处理的细胞内Bax蛋白显著升高,而Bcl-2蛋白表达水平明显降低,差异具有统计学意义(P<0.05),其凋亡机制可能与下调Bcl-2蛋白、上调Bax蛋白表达量相关.     

三氧化二砷联合Trolox对人早幼粒白血病细胞HL-60增殖和凋亡的影响

利用MTT法、台盼蓝排染法、Hoechst33258荧光染色法、瑞氏-姬姆萨染色法以及单细胞凝胶电泳法检测了三氧化二砷(As2O3)单独作用及联合Trolox后对人早幼粒白血病细胞HL-60的增殖抑制、凋亡诱导作用及其DNA损伤作用.结果表明,1μmol.L-1As2O3作用即可抑制HL-60细胞的增殖,联合100μmol.L-1Trolox对HL-60细胞增殖抑制作用较As2O3单独作用时明显增强.高浓度的As2O3(4μmol.L-1)作用24h可引起HL-60细胞的凋亡,与Trolox联合作用,在低浓度(1μmol.L-1)下即可引起细胞凋亡.联合100μmol.L-1Trolox可以显著增强As2O3引起的HL-60细胞的DNA损伤.这种损伤可能导致细胞周期阻滞,从而在生长曲线中体现出对HL-60细胞的增殖抑制作用.     

nm23-H1基因与人早幼粒白血病细胞HL-60增殖的相关性分析

目的探讨nm23-H1基因的表达与白血病细胞HL-60增殖之间的关系。方法：以25ng／mL阿糖胞苷处理HL-60细胞，MTT法测定细胞生长抑制率，NBT还原比色法判断细胞分化状况，RT-PCR检测nm23-H1基因表达的变化；构建nm23-H1基因的真核表达质粒pEGFP-N1-nm23-H1，转染HL-60细胞，通过细胞生长曲线和血清依赖性实验检测nm23-H1基因的过表达对HL-60细胞生长的影响。结果：小剂量Ara-C对HL-60细胞的生长呈时间依赖性抑制，作用4d后细胞NBT还原能力增强且nm23-H1基因的表达下调；转染nm23-H1基因的HL-60细胞生长加快、血清依赖性下降。结论：Ara-C对HL-60细胞增殖的抑制作用与下调nm23-H1基因的表达有-定关系；nm23-H1基因在HL-60细胞中的过表达有促细胞增殖的作用，即增高了HL-60细胞的恶性程度。     

异叶败酱中三萜化合物常春藤皂苷元对人早幼粒白血病细胞HL-60的增殖抑制、周期阻滞及凋亡诱导作用

利用倒置显微镜观察法、台盼蓝排染法、Hoechst33258荧光染色法、DNA ladder电泳以及流式细胞分析技术,检测了从甘肃产异叶败酱(Patrinia heterophylla Bunge.)中分离得到的三萜化合物常春藤皂苷元(HG)对人早幼粒白血病细胞HL-60的增殖抑制、周期阻滞及凋亡诱导作用.结果表明,常春藤皂苷元在较低浓度(10～40 μmol·L-1)条件下,时HL-60细胞增殖具有良好的抑制作用;在较高浓度(40～50μmol·L-1)条件下,对HL-60细胞具有致死作用;同时,常眷藤皂苷元对HL-60细胞的G1期阻滞和凋亡诱导作用极可能是其实现增殖抑制和致死作用的主要途径.     

二烯丙基二硫诱导人白血病细胞系HL-60细胞凋亡的研究

目的研究二烯丙基二硫(diallyl disulfide,DADS)诱导人急性髓性白血病细胞系HL-60细胞凋亡和对细胞周期的影响。方法采用MTT、流式细胞仪等方法检测DADS对HL-60的增殖抑制,诱导凋亡以及细胞周期分布的影响。结果20μΜDADS作用HL-60细胞48h,MTT显示DADS对HL-60细胞有明显的抑制增殖作用;流式细胞仪分析结果显示DADS对HL-60细胞有明显的G2/M期阻滞作用;用丫啶橙染色法观察到不同阶段的凋亡细胞形态学改变,且DADS呈时间依赖性诱导HL-60细胞凋亡。结论DADS引起HL-60细胞G2/M期阻滞并诱导凋亡可能是其抗肿瘤的机制之一。     

邻苯二甲酸二丁酯（DBP）诱导肿瘤细胞凋亡机制的初步研究

邻苯二甲酸二丁酯（DBP）在体外对肺巨细胞癌细胞（PG）和人早幼粒白血病细胞（HL-60）的增殖有明显的抑制作用;经DBP处理过的PG细胞,DNA断裂,在琼脂糖凝胶电泳上呈现出特异的断裂带;流式细胞术分析表明,DBP能提高PG细胞的凋亡率,并且随作用时间的延长而增大;斑点杂交的结构显示,被DBP处理过的HL-60细胞中某些原癌基因、抑癌基因和肿瘤抑制基因的mRNA表达水平都有所下降,这提示DBP对HL-60细胞增殖抑制作用的机制之一可能是通过抑制这些癌基因和突变的抑癌基因的表达来实现的。     

小剂量阿糖胞苷体外诱导HL-60细胞凋亡的作用

阿糖胞苷是白血病的重要药物,其小剂量化疗仍用于临床治疗白血病,并取得了一定的疗效,但其作用机制尚未完全阐明,可能与药物诱导细胞分化或凋亡有关.文中用TUNEL法和流式细胞仪分析小剂量Ara-C(10-8M)对HL-60细胞周期和细胞凋亡的影响,用免疫组化法和原位杂交法分别检测P53蛋白和p53mRNA、bcl-2mRNA表达水平的改变,以探讨bcl-2和p53基因表达与小剂量阿糖胞苷诱导HL-60细胞凋亡的关系.结果表明:小剂量Ara-C能诱导HL-60细胞凋亡,其分子机制可能与P53蛋白、p53mRNA表达增加和bcl-2mRNA表达降低有关.     

Enhancement of polyoma virus middle T antigen tyrosine phosphorylation by epidermal growth factor

Polyoma virus codes for three proteins involved in host cell transformation: the large, middle and small T antigens. Middle T antigen is a major transforming protein which is responsible for the induction of the phenotype of transformed cells and, without it, transformation does not occur (reviewed in refs 1-4). Middle T antigen alone can transform established cell lines, although large, and possibly small, T antigens are also required for the full expression of the phenotype of transformed cells in media with a low concentration of serum. A subfraction of middle T antigen is associated with a protein kinase activity which phosphorylates middle T antigen in vitro on tyrosine. There is a strong correlation between the level of this kinase activity and the degree of expression of the phenotype of transformed cells. We report here that epidermal growth factor (EGF) stimulates tyrosine phosphorylation of middle T antigen, suggesting the possibility that mitogenic growth factor(s) regulates this phosphorylation activity.     

Superantigen implicated in dependence of HIV-1 replication in T cells on TCR V beta expression.

In the pathogenesis of AIDS it is not yet understood whether the small fraction of CD4+ T cells (approximately 1%) infected with the human immunodeficiency virus (HIV) are randomly targeted or not. Here we present evidence that human CD4 T-cell lines expressing selected T-cell antigen receptor V beta gene products can all be infected in vitro with HIV-1, but give markedly different titres of HIV-1 virion production. For example, V beta 12 T-cell lines from several unrelated donors reproducibly yielded up to 100-fold more gag gene product (p24gag antigen) than V beta 6.7a lines. This is consistent with a superantigen effect, because the V beta selectivity was observed with several divergent HIV-1 isolates, was dependent on antigen-presenting cells and on major histocompatibility complex (MHC) class II but was not MHC class II-restricted. The in vivo significance of these findings is supported by the preferential stimulation of V beta 12+ T cells by freshly obtained irradiated antigen-presenting cells from some HIV-1-seropositive but not HIV-1-negative donors. Moreover, cells from patients positive for viral antigen (gp120) were enriched in the V beta 12 subpopulation. V beta 12+ T cells were not deleted in AIDS patients, however, raising the possibility that a variety of mechanisms contribute to T-cell depletion. Our results indicate that a superantigen targets a subpopulation of CD4+ cells for viral replication.     

A second human interleukin-2 binding protein that may be a component of high-affinity interleukin-2 receptors

Although activated human T and B lymphocytes express both high-affinity and low-affinity membrane receptors for interleukin-2 (IL-2), the structural features that distinguish these receptors have remained unresolved. The high-affinity receptors appear to mediate IL-2 induced T cell growth and internalization of IL-2, whereas no function has yet been ascribed to the low-affinity receptors. The Tac antigen is an IL-2 binding protein of relative molecular mass 55,000 (Mr 55K) that participates in the formation of both high- and low-affinity receptors. But Tac complementary DNA transfection and membrane fusion studies have suggested that additional T-cell components are required to produce high-affinity IL-2 receptors. In this study, we report the identification of a second human IL-2 binding protein that (1) has an Mr of approximately 70K, (2) lacks reactivity with the anti-Tac antibody, (3) binds IL-2 with intermediate affinity and (4) is present on the surface of resting T cells, large granular lymphocytes (natural killer cells), and certain T and B cell lines in the absence of the Tac antigen. Chemical crosslinking of 125I-labelled IL-2 bound to high-affinity IL-2 receptors produces labelling of both the p70 protein and the Tac antigen and the anti-Tac antibody blocks the crosslink detection of both of these proteins. Expression of Tac cDNA in a T cell line expressing the p70 protein, but lacking both Tac and high-affinity receptors, results in the reconstitution of high-affinity IL-2 receptors in these cells. Together, these findings suggest that the high-affinity human IL-2 receptor may be a membrane complex composed of at least the p70 protein and Tac antigen.     

Nucleolar proteome dynamics

The nucleolus is a key organelle that coordinates the synthesis and assembly of ribosomal subunits and forms in the nucleus around the repeated ribosomal gene clusters. Because the production of ribosomes is a major metabolic activity, the function of the nucleolus is tightly linked to cell growth and proliferation, and recent data suggest that the nucleolus also plays an important role in cell-cycle regulation, senescence and stress responses. Here, using mass-spectrometry-based organellar proteomics and stable isotope labelling, we perform a quantitative analysis of the proteome of human nucleoli. In vivo fluorescent imaging techniques are directly compared to endogenous protein changes measured by proteomics. We characterize the flux of 489 endogenous nucleolar proteins in response to three different metabolic inhibitors that each affect nucleolar morphology. Proteins that are stably associated, such as RNA polymerase I subunits and small nuclear ribonucleoprotein particle complexes, exit from or accumulate in the nucleolus with similar kinetics, whereas protein components of the large and small ribosomal subunits leave the nucleolus with markedly different kinetics. The data establish a quantitative proteomic approach for the temporal characterization of protein flux through cellular organelles and demonstrate that the nucleolar proteome changes significantly over time in response to changes in cellular growth conditions.     

IGF and FGF cooperatively establish the regulatory stem cell niche of pluripotent human cells in vitro

Distinctive properties of stem cells are not autonomously achieved, and recent evidence points to a level of external control from the microenvironment. Here, we demonstrate that self-renewal and pluripotent properties of human embryonic stem (ES) cells depend on a dynamic interplay between human ES cells and autologously derived human ES cell fibroblast-like cells (hdFs). Human ES cells and hdFs are uniquely defined by insulin-like growth factor (IGF)- and fibroblast growth factor (FGF)-dependence. IGF 1 receptor (IGF1R) expression was exclusive to the human ES cells, whereas FGF receptor 1 (FGFR1) expression was restricted to surrounding hdFs. Blocking the IGF-II/IGF1R pathway reduced survival and clonogenicity of human ES cells, whereas inhibition of the FGF pathway indirectly caused differentiation. IGF-II is expressed by hdFs in response to FGF, and alone was sufficient in maintaining human ES cell cultures. Our study demonstrates a direct role of the IGF-II/IGF1R axis on human ES cell physiology and establishes that hdFs produced by human ES cells themselves define the stem cell niche of pluripotent human stem cells.     

A telomerase component is defective in the human disease dyskeratosis congenita

The X-linked form of the human disease dyskeratosis congenita (DKC) is caused by mutations in the gene encoding dyskerin. Sufferers have defects in highly regenerative tissues such as skin and bone marrow, chromosome instability and a predisposition to develop certain types of malignancy. Dyskerin is a putative pseudouridine synthase, and it has been suggested that DKC may be caused by a defect in ribosomal RNA processing. Here we show that dyskerin is associated not only with H/ACA small nucleolar RNAs, but also with human telomerase RNA, which contains an H/ACA RNA motif. Telomerase adds simple sequence repeats to chromosome ends using an internal region of its RNA as a template, and is required for the indefinite proliferation of primary human cells. We find that primary fibroblasts and lymphoblasts from DKC-affected males are not detectably deficient in conventional H/ACA small nucleolar RNA accumulation or function; however, DKC cells have a lower level of telomerase RNA, produce lower levels of telomerase activity and have shorter telomeres than matched normal cells. The pathology of DKC is consistent with compromised telomerase function leading to a defect in telomere maintenance, which may limit the proliferative capacity of human somatic cells in epithelia and blood.     

Two forms of transforming growth factor-beta distinguished by multipotential haematopoietic progenitor cells

Type-beta transforming growth factors (TGF-beta s) are polypeptides that act hormonally to control proliferation and differentiation of many cell types. Two distinct homodimeric TGF-beta polypeptides, TGF-beta 1 and TGF-beta 2 have been identified which show approximately 70% amino-acid sequence similarity. Despite their structural differences, TGF-beta 1 and TGF-beta 2 are equally potent at inhibiting epithelial cell proliferation and adipogenic differentiation. The recent immunohistochemical localization of high levels of TGF-beta in the bone marrow and haematopoietic progenitors of the fetal liver has raised the possibility that TGF-beta s might be involved in the regulation of haematopoiesis. Here we show that TGF-beta 1, but not TGF-beta 2, is a potent inhibitor of haematopoietic progenitor cell proliferation. TGF-beta 1 inhibited colony formation by murine factor-dependent haematopoietic progenitor cells in response to interleukin-3 (IL-3) or granulocyte-macrophage colony stimulating factor (GM-CSF), as well as colony formation by marrow progenitor cells responding to CSF-1 (M-CSF). The progenitor cell lines examined were approximately 100-fold more sensitive to TGF-beta 1 than TGF-beta 2, and displayed type-I TGF-beta receptors with affinity approximately 20-fold higher for TGF-beta 1 than TGF-beta 2. These results identify TGF-beta 1 as a novel regulator of haematopoiesis that acts through type-I TGF-beta receptors to modulate proliferation of progenitor cells in response to haematopoietic growth factors.