紫杉醇对小鼠T细胞的影响

为研究抗癌药紫杉醇(PTX)对T细胞行为的影响及其分子机制,利用流式细胞术分析多克隆刺激剂ConA刺激下T细胞行为:羧基荧光素乙酰乙酸琥珀酰亚胺酯(CFSE)标记技术分析T细胞增殖相关指数;碘化丙锭染色分析细胞周期分布;AnnexinV-PI染色检测T细胞凋亡;荧光标记的单克隆抗体染色检测T细胞活化表达CD25的百分率。结果显示.PTX对ConA刺激下小鼠T细胞增殖具有明显的抑制作用,且呈剂量依赖性。该浓度范围PTX阻滞T细胞于G2/M期,诱导凋亡及抑制CD25表达。25nmol/L的PTX与10nmol/L的环孢素A(CsA)具有明显的协同抑制效应。以上结果表明PTX可明显抑制多克隆刺激剂ConA诱导的T细胞体外增殖,是G2/M期阻滞、凋亡诱导和CD25表达抑制等多种机制共同作用的结果。     

人不同来源T细胞对丝裂原、PMA和A23187的反应性比较

本文比较了胎儿胸腺细胞、正常成人外周血Ｔ细胞及儿童扁桃体Ｔ细胞对丝裂原ＰＷＭ、ＰＨＡ和ＣｏｎＡ、ＰＫＣ激活剂ＰＭＡ和Ｃａ２＋导入剂Ａ２３１８７的反应性。结果表明：胎儿胸腺细胞对ＰＷＭ的反应对ＰＨＡ和ＣｏｎＡ的反应性很弱；正常成人外周血Ｔ细胞对ＰＨＡ的反应性最强，对ＰＷＭ和ＣｏｎＡ的反应性较弱；儿童扁桃体Ｔ细胞对３种丝裂原的反应性均很强；ＰＭＡ单独作用能强烈诱导儿童扁桃体Ｔ细对正常成人外周血Ｔ细胞及胸腺细胞作用较弱；ＰＷＭ和ＰＭＡ对胎儿胸腺细胞及正常成人外周血Ｔ细胞均有明显协同作用，但对儿童扁桃体Ｔ细胞无协同作用；Ａ２３１８７单独作用对３种Ｔ细胞的增殖作用均很弱，Ａ２３１８７对ＰＷＭ诱导的增殖均有部分抑制作用。这些结果对于认识处于不同发育阶段及不同部位的的活化途径和功能有一定意义。     

A Combination of Calcium Ionophore and 12-O-Tetradecanoyl-Phorbol-13-Acetate (TPA) Stimulates the Growth of Purified Resting B Cells

In this study we investigated whether the calcium ionophores A23187 and ionomycin can act synergistically with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to stimulate the growth of resting B lymphocytes purified from human tonsil cells. Ionomycin, A23187, and TPA added separately to cultures at doses of 0.4-1.6 micrograms/ml, 0.2-0.8 micrograms/ml, and 0.05-0.25 ng/ml respectively, did not induce DNA synthesis in resting B lymphocytes. In contrast, calcium ionophores at concentrations of 0.4-1.6 micrograms/ml ionomycin and 0.2-0.8 micrograms/ml A23187, in the presence of 0.05-4 ng/ml TPA, induced marked DNA synthesis and B-cell proliferation, as shown by analyses of incorporation of [3H]thymidine, growth kinetics, and the percentage of cells in the S and G2 + M phases of the cell cycle. These results show that the synergistic effects of calcium ionophores and TPA can bypass the requirement for antigen and exogenous growth factors in B-cell activation. These observations are similar to those obtained from studies of T lymphocytes by other workers.     

Phosphatase Inhibitors and Premature Chromosome Condensation in Human Peripheral Lymphocytes at Different Cell-Cycle Phases

The cytogenetical reaction of human peripheral lymphocytes to okadaic acid and calyculin A was examined. Calyculin A could induce PCC about 20 times more effectively than okadaic acid. Their mechanisms of PCC induction were judged similar by their dose-dependent manner and chromosome morphology. Contrary to earlier studies suggesting that chemicals could not induce PCC in G1 cells where little cyclin B is present, the present study showed that calyculin A could induce PCC in lymphocytes not only at S and G2/M but also at the second G1 phase after PHA stimulation in vitro. PCC was induced slightly in lymphocytes both at G0 and the first in vitro G1 phase even when the calyculin A concentration increased one hundred fold. It was found that calcium ionophore A23187 increased frequencies of G0-PCC induced by calyculin A, although a further refinement is necessary to obtain a suitable morphology of G0-PCC for cytogenetic studies.     

Calcium ionophore A23187 as a secretagogue for rat mast cells: Does it bypass inhibition by calcium flux blockers?

It is widely accepted that an increase in cytoplasmic calcium activity is a stimulus-secretion coupling event required to initiate secretion in mast cells and basophils. This study is concerned with the notion that inhibition of secretion by certain agents thought to be calcium influx inhibitors, including dibutyryl cyclic AMP (db-cAMP), quercetin, verapamil and ruthenium red can be bypassed with the calcium-carrying ionophore A23187. The importance of the bypass concept lies in its implication that, in the absence of A23187, these agents inhibit secretion solely by impairing physiologic calcium gating.We used a sensitive new technique of doubly labeling mast cells with¹⁴C-serotonin and⁵¹Cr to monitor secretion and cytolysis respectively. We looked for doses of inhibitors which inhibited anti-IgE-induced secretion but had no effect on A23187-induced secretion, as predicted by the bypass hypothesis. db-cAMP significantly inhibited anti-IgE-induced secretion in only half of our experiments, but consistently and substantially augmented A23187-induced secretion. From these results, it appears that db-cAMP acts via multiple pathways, and that potentiation of A23187-induced secretion may, under certain circumstances, cancel the inhibitory effects of db-cAMP. Ruthenium red inhibited anti-IgE-induced secretion but not A23187-induced secretion only at very high A23187 concentrations. The likelihood of a stoichiometric mutual inactivation between ruthenium red and A23187 suggests that an excess of A23187 could relieve ruthenium red inhibition by binding to, and thereby inactivating, the drug. Verapamil and quercetin inhibited both anti-IgE-induced and A23187-induced secretion at the same doses.In conclusion, A23187 antagonizes inhibition of mast cell secretion by db-cAMP and ruthenium red under certain conditions. Our data make it unlikely, however, that such antagonism results from a simple bypass mechanism.     

Superantigen and endotoxin synergize in the induction of lethal shock

Endotoxin (lipopolysaccharide; LPS) and superantigens (exotoxins) have been identified as potent inducers of lethal shock. While endotoxin primarily interacts with CD 14 receptors on macrophages, superantigens like the staphylococcal enterotoxin B (SEB) preferentially activate T cells. Both cell types are triggered to release pro-inflammatory cytokines that in turn induce lethal shock. We analyzed whether endotoxin and superantigen interact during the induction phase of lethal shock. We report that LPS and SEB operate synergistically. Lethal doses of both inducers were reduced 100-fold when given in combination. The induced serum levels of tumor necrosis factor, interleukin-6, and interferon-γ (IFN-γ) were elevated and remained high for a prolonged period. Moreover, synergistic action of LPS and SEB induced lethal toxic shock even without presensitization of mice with D -galactosamine (D -GalN). Opposed to D -GalN-pretreated mice, mice injected with LPS and SEB showed less liver damage, but rather apoptosis of epithelial cells in the bowel. Cyclosporin A and treatment with anti-IFN-γ monoclonal antibody blocked the synergistic action of LPS and SEB, indicating that T cell-derived IFN-γ is the mediator of the observed synergism. Concomitant injection of LPS and SEB had no influence on SEB-induced T cell deletion and anergy induction. Since Gram-positive and Gram-negative bacteria can be recovered from septic blood samples, the synergistic action of endotoxin and superantigens might be relevant during lethal septicemia.     

Sodium arsenite induces chromosome endoreduplication and inhibits protein phosphatase activity in human fibroblasts

Arsenic, strongly associated with increased risks of human cancers, is a potent clastogen in a variety of mammalian cell systems. The effect of sodium arsenite (a trivalent arsenic compound) on chromatid separation was studied in human skin fibroblasts (HFW). Human fibroblasts were arrested in S phase by the aid of serum starvation and aphidicolin blocking and then these cells were allowed to synchronously progress into G2 phase. Treatment of the G2-enriched HFW cells with sodium arsenite (0–200 μM) resulted in arrest of cells in the G2 phase, interference with mitotic division, inhibition of spindle assembly, and induction of chromosome endoreduplication in their second mitosis. Sodium arsenite treatment also inhibited the activities of serine/threonine protein phosphatases and enhanced phosphorylation levels of a small heat shock protein (HSP27). These results suggest that sodium arsenite may mimic okadaic acid to induce chromosome endoreduplication through its inhibitory effect on protein phosphatase activity. © 1995 Wiley-Liss, Inc.     

C225单用及与DDP联用对HeLa细胞的抑制作用与机制研究

探讨C225单用及与DDP联用对人子宫颈癌HeLa细胞的体外抑制作用及其机制。分别用C225(100、300μg/ml)与DDP(0.1、0.5、1μg/ml)以单用和联用处理HeLa细胞72 h后,MTT法检测对HeLa细胞生长的抑制作用及协同效应;FCM技术检测C225(300μg/ml)与不同工作浓度DDP单用及联用72 h后对细胞周期及凋亡的影响;并用HOE-CHEST33258染色技术进行细胞凋亡的形态学检测。结果C225能明显抑制HeLa细胞增殖,与DDP联合具有协同作用或相加作用;C225能使细胞周期阻滞于G1期,并诱导细胞凋亡;与DDP联用后凋亡率更高,使细胞周期进一步阻滞于G2期,同时降低S期细胞比例。荧光显微镜下可见实验对照组所发荧光较弱,无凋亡小体出现,而各药物干预组均出现一定数量的凋亡细胞,并随药物浓度的增加而增多,其中尤以联合用药组最为明显。两药单用及联用均对HeLa细胞的体外生长具有显著抑制作用,C225可通过将细胞周期阻滞于G0-G1期及诱导细胞凋亡发挥其抑瘤效应;两药间存在协同作用,机制可能与C225增加HeLa细胞对DDP的敏感性有关。     

细胞外信号调节激酶1/2的阻断剂PD98059对小鼠淋巴细胞增殖的影响

目的探讨细胞外信号调节激酶1/2(ERK1/2)信号通路的特异性阻断剂PD98059对小鼠淋巴细胞体外增殖的影响。方法分离小鼠淋巴结细胞,藉羧基荧光素乙酰乙酸(CFDA-SE)染色,用多克隆刺激剂刀豆A蛋白(ConA)或者佛波醇酯(PDB)加离子霉素(Ion)刺激,以流式细胞术分析PD98059对淋巴细胞增殖的影响;利用碘化丙锭(PI)染色分析PD98059对细胞周期的影响。结果CFDA-SE染色分析显示,当浓度为5、10、20、30、40μmol/L时,PD98059对ConA诱导的淋巴细胞增殖具有明显的抑制作用,并呈现明显的剂量依赖关系(r=0.985,P<0.01)。选择最佳浓度30μmol/L的PD98059,观察其对不同时间淋巴细胞增殖的影响,结果发现,随着时间的延长,PD98059对淋巴细胞增殖的抑制作用明显降低(P<0.01),但抑制率随时间的延长而明显升高。细胞周期分析进一步表明,PD98059可阻止ConA刺激的淋巴细胞进入S期和G2/M期,而亚二倍体峰变化并不明显。PD98059对PDB Ion刺激的淋巴细胞细胞周期的影响与ConA刺激相似,不同的是S期和G2/M期的变化较ConA的作用更明显。结论PD98059对小鼠淋巴细胞的增殖有明显地抑制作用,并可阻止其进入S期和G2/M期,提示ERK1/2信号通路的活化在淋巴细胞增殖中起重要作用。