交变应力作用下烟草细胞热力学相行为的研究

研究了交变应力对烟草愈伤组织细胞的影响, 采用本实验室研制的强声波发生装置来产生交变应力场, 并利用差式扫描量热仪（ＤＳＣ） 研究了不同强度和频率的交变应力作用后烟草细胞热力学相行为的变化。研究结果表明：交变应力的影响与应力的频率和强度密切相关,一定频率和强度范围内的交变应力能使得植物细胞的相变温度有明显的降低, 而过高频率的应力刺激则会使细胞相变温度升高。细胞热力学相变反映了细胞壁膜的流动性,相变温度变低表明细胞壁膜的流动性增强, 这必然为细胞的生长和分裂提供了便利的条件。 因此, 对于从细胞和分子水平研究交变应力对植物生长、发育的影响及其作用机理是一个很有意义的尝试     

声波刺激对猕猴桃愈伤组织ATP含量的影响

采用单因子实验设计,分别先固定作为交变应力的声波的频率或强度这两个参数中的一个,而改变另一个指标的值,用声波刺激木本植物中华猕猴桃(Actinidiachinensis)茎段的愈伤组织,并测定对比声波处理前后其ATP含量及变化情况。实验结果表明,声波对猕猴桃愈伤组织ATP的含量有着比较明显的增强或抑制的双重效应,适度的声场刺激将有利于提高植物的能量代谢水平,其中,最适的声波频率为1000Hz,最适声强为100dB左右 。     

声波刺激对中华猕猴桃ATP含量的影响

根据环境应力对植物应激效应的原理，运用声波刺激木质藤本植物中华猕猴桃茎段的愈伤组织，测定ATP含量及其变化情况，并通过与对照组(CK)相应值的比较，研究声波对植物能量代谢的影响。采用单因子实验设计，分别固定作为交变应力的声波的频率或强度这两个参数中的一个，而改变另一个指标的值，对实验材料进行处理，实验结果表明，声波对猕猴桃愈伤组织ATP的含量有着比较明显的增强或抑制的双重效应，适度的声场刺激将有利于提高植物的能量代谢水平，其中，最适的声波频率为1000Hz，而最适声强为100dB左右。     

声波刺激对菊花愈伤组织膜脂物理状态和膜脂代谢的影响

以蔗糖梯度法纯化的菊花 (Dendranthemamorifolium (Ramat.)Tzvel.)愈伤组织质膜微囊为材料 ,研究了声波刺激下质膜膜脂代谢和物理状态的变化。结果表明 ,一定强度 (10 0dB)和频率 (10 0 0Hz)的声波刺激使质膜磷酸二脂含量和二苯己三烯 (DPH)荧光偏振值降低 ,质膜光散射值、MC5 40荧光强度和磷酸单脂含量增加。表明一定强度和频率的声波刺激使质膜变疏松 ,膜的流动性增加 ,膜表面电荷密度和疏水性降低 ,膜脂合成代谢增加 ,分解代谢减弱。由此可见 ,膜脂物理状态和膜脂代谢对声波刺激极为敏感。     

声波刺激对菊花愈伤组织膜脂物理状态和膜脂代谢的影响

以蔗糖梯度法纯化的菊花(Dendranthema morifolium (Ramat.) Tzvel.)愈伤组织质膜微囊为材料,研究了声波刺激下质膜膜脂代谢和物理状态的变化.结果表明, 一定强度(100 dB)和频率(1 000 Hz)的声波刺激使质膜磷酸二脂含量和二苯己三烯(DPH)荧光偏振值降低,质膜光散射值、MC540荧光强度和磷酸单脂含量增加.表明一定强度和频率的声波刺激使质膜变疏松,膜的流动性增加,膜表面电荷密度和疏水性降低,膜脂合成代谢增加,分解代谢减弱.由此可见,膜脂物理状态和膜脂代谢对声波刺激极为敏感.     

参杞合剂对人鼻咽癌细胞CNE细胞周期及凋亡的影响

目的研究参杞合剂(SQ)在体外对CNE细胞周期及凋亡的影响。方法应用MTT法观察参杞合剂对细胞的抑制作用,流式细胞术观察不同浓度参杞合剂作用不同时间后CNE细胞周期的改变,电镜结合DNA电泳分析参杞合剂诱导凋亡的作用。结果SQ对CNE细胞生长有明显抑制作用,且其作用强度呈现出对浓度和时间的依赖性。CNE细胞在SQ作用下随着时间的延长和浓度的增加,G0/G1期比率下降,S期比率升高,出现S期阻滞。0.0625 g.生药/ml的SQ作用48 h后诱导出凋亡,凋亡率随着浓度的增加、时间的延长而增加,电镜下可见典型凋亡小体。琼脂糖凝胶电泳呈现出凋亡特征性的DNA条带。结论参杞合剂可直接杀伤肿瘤细胞,其机制可能通过阻滞细胞周期S期,诱导肿瘤细胞凋亡实现的。     

钙调素对细胞周期的调节

RC3细胞是一种用真核表达载体1~(CaM)转染NIH 3T3细胞建成的可调钙凋素(Calmodulin,CaM)高表达细胞模型。通过分子杂交及蛋白免疫印迹方法证实在地塞米松(Dexamethasome,DXM)作用下,RC3细胞可高表达CaM。CaM的过表达使G_1期细胞减少,S期细胞增加;CaM拮抗剂三氟拉嗪(trifluoperazine,TFP)则使G_1期细胞增加,S期细胞减少。高表达CaM使细胞分裂指数提高,G_2期细胞减少,有丝分裂前期细胞增加,M中期细胞比例下降。而TFP处理则使分裂指数下降,G_2期细胞增加,M前期细胞减少,M中期细胞增加。实验结果表明CaM在G_1/S、G_2/M和M中期/M后期3个位点上对细胞周期进行调控;通过加速G_1至S期,G_2至M期和M中期至M后期的进程,使细胞倍增时间缩短,促进细胞增殖。本工作表明,RC3细胞作为CaM表达可调细胞模型,是研究细胞周期调控的有力工具。     

胡桃楸提取液对肿瘤细胞细胞周期的影响

目的考察胡桃楸提取液对Hela和K562细胞周期的影响。方法用流式细胞仪分析胡桃楸提取液对Hela、K562细胞细胞周期的影响。结果胡桃楸提取物体外作用于Hela细胞可引起细胞周期在S期的停滞,这种效果随药物浓度和作用时间的增加而增加,胡桃楸提取物体外作用于K562细胞,可引起细胞周期在G1期的停滞,随药物浓度的增加而增加。结论胡桃楸提取液对Hela细胞的生长抑制作用可能通过S期阻滞实现,对K562细胞抑制作用可能通过G1期阻滞实现。     

竹红菌甲素对体外培养细胞光敏作用的实验研究

本文用免疫荧光细胞化学、流式荧光分析等方法研究了光敏剂H_A对培养的HeLa细胞的光敏化作用。结果表明,H_A光敏化作用使细胞形态发生变化,细胞表面微绒毛丧失,细胞增殖受到抑制,上述变化随光照剂量增加而加剧,其中对肿瘤细胞的抑制作用较正常二倍体细胞更大,细胞群体中G_1期细胞下降,S期细胞增多。H_A的光敏化作用还使胞质微管变稀疏,微管中心的亮荧光近乎消失。以上实验说明,H_A的光敏作用对培养细胞的作用引起细胞膜、细胞骨架的损伤,使细胞周期部分阻断于S期,从而抑制了细胞的增殖。     

D类细胞周期素在细胞周期中的作用

细胞周期进程由周期性表达的细胞周期素和细胞周期素信号性激酶推动完成,CyclinD在G1期中期开始表达,是最早合成的细胞周期素,它与Cdk4或Cdk6形成的活性复合物,不仅可促进pRb的磷酸化,也可隔离P27对cyclinE-Cdk2活性的抑制,使细胞顺利越过G1期进入S期,除此之外,cyclinD过度表达还可抑制细胞增殖,在细胞分化和衰老过程中发挥重要作用。     

PROLIFERATION RATE OF HAEMOPOIETIC STEM CELLS AFTER DAMAGE BY SEVERAL CYTOSTATIC AGENTS

The haemopoietic tissue of mice was damaged by different cell-cycle-stage specific and cell-cycle-stage non-specific cytostatic agents. The proliferation rate among the surviving pluripotential stem cells, i.e. those cells forming colonies in spleens of lethally irradiated mice (CFUs), was then investigated. The results suggest that, at least in the CFUs population, the cells which synthesize DNA in the S phase of the cell cycle inhibit the entry of the non-proliferating G₀ cells into cell cycle. This evidence was based on the ability of three cytostatic agents, hydroxyurea, cytosine arabinoside and methotrexate, which are toxic specifically to the S phase cells to increase the proliferation in the CFUs population. This increase was quite out of proportion to the small amount of damage they caused to the population. Colchicine, which kills cells in mitosis, and ionizing irradiation, damaging cells in all stages, proved to be much weaker stimulators of proliferation. It has been suggested that a mechanism for the control of cellular proliferation might be based on the negative feedback in the cell cycle. In this feedback control loop the cells which are preparing for cell division in the S phase of the cell cycle inhibit the entry of the non-proliferating G₀ cells into cell cycle.     

Increased mitochondrial uptake of rhodamine 123 by CDDP treatment.

Rhodamine 123 (R 123) is a positively charged dye at physiological pH that accumulates specifically in the mitochondria of living cells without cytotoxic effect. In the present study, the uptake of R 123 by EL-4 lymphoma cells in culture with anticancer agents was measured by flow cytometry. Changes in R 123 uptake during the cultivation period were compared with cell distribution at different phases of the cell cycle. According to the increase in the proportion of S phase cells, mitochondrial synthesis increased, giving rise to a maximal fluorescence intensity of about 1.3-fold. Synchronous cultures showed the same relationship between increased mitochondrial uptake of R 123 and the S phase fraction as was observed in normal cultures. After treatment with 10(-3) M 5-fluorouracil (5-FU) for 1 h, EL-4 cells showed an increased binding of R 123 per cell followed by an accumulation of early S phase cells transiently. However, uptake of R 123 decreased 24 h later. On the contrary, after treatment with 10 micrograms/ml of cis-diamminedichloroplatinum (CDDP), a G2 + M block was observed from 12 h of reseeding and accumulation of the G2 + M cells continued. In this case, high uptake of R 123 continued during the observation period. From these results, mitochondrial synthesis seemed to increase according to the increment in proportion of S phase when the acceleration of the cell cycle turnover was augmented or the cycle was blocked in S phase by 5-FU. CDDP inhibited the cell division at G2 + M phase and caused increased R 123 fluorescence per cell. The stainability of R 123 may indicate the activity of cell division and may be a good way of evaluating the efficacy of antitumor drugs on the cells.     

Actin filament reorganization in HL-60 leukemia cell line after treatment with G-CSF and GM-CSF

Currently, information regarding the influence of growth factors on the cytoskeleton, including G-CSF and GMCSF, remains limited. In the present study we show alterations in F-actin distribution and cell cycle progression in HL-60 promyelocytic leukemia cells, resulting from treatment with these cytokines in vitro. We found that both agents caused F-actin reorganization. Although multiple potential effects of various growth factors have been described previously, in our experimental conditions, we observed some rather subtle differences between the effects of G-CSF and GM-CSF on studied cells. The presence of these cytokines in the cell environment caused not only increased F-actin labeling in the cytoplasm, but also a weaker intensity of peripheral ring staining in comparison with control cells. In spite of the fact that HL60 cells exposed to G-CSF and GM-CSF contained different F-actin structures such as aggregates and F-actin network, the rate of actin polymerization was not significantly enhanced. Moreover, alterations were mainly related to considerable changes in the relative proportion of these different structures, what might be reflected by specific features of the differentiation process, with regard to the kind of stimulating factor used. Thus, reorganization of F-actin and other results obtained in our experimental conditions, might reflect unique characteristics of the differentiation process in HL-60 cells, involving low apoptosis frequency, the G1 to S phase transition in the cell cycle, as well as possible alternative ways of the cell death.     

Cell Cycle Dynamics of Cultured Coral Endosymbiotic Microalgae (Symbiodinium) Across Different Types (Species) Under Alternate Light and Temperature Conditions

Dinoflagellates of the genus Symbiodinium live in symbiosis with many invertebrates, including reef‐building corals. Hosts maintain this symbiosis through continuous regulation of Symbiodinium cell density via expulsion and degradation (postmitotic) and/or constraining cell growth and division through manipulation of the symbiont cell cycle (premitotic). Importance of premitotic regulation is unknown since little data exists on cell cycles for the immense genetic diversity of Symbiodinium. We therefore examined cell cycle progression for several distinct SymbiodiniumITS2‐types (B1, C1, D1a). All types exhibited typical microalgal cell cycle progression, G₁ phase through to S phase during the light period, and S phase to G₂/M phase during the dark period. However, the proportion of cells in these phases differed between strains and reflected differences in growth rates. Undivided larger cells with 3n DNA content were observed especially in type D1a, which exhibited a distinct cell cycle pattern. We further compared cell cycle patterns under different growth light intensities and thermal regimes. Whilst light intensity did not affect cell cycle patterns, heat stress inhibited cell cycle progression and arrested all strains in G₁ phase. We discuss the importance of understanding Symbiodinium functional diversity and how our findings apply to clarify stability of host‐Symbiodinium symbioses.     

Quantitative Analysis of Centromeric FISH Spots during the Cell Cycle by Image Cytometry

Two-color fluorescence in situ hybridization (FISH) with chromosome enumeration DNA probes specific to chromosomes 7, 11, 17, and 18 was applied to CAL-51 breast cancer cells to examine whether the fluorescence intensity of FISH spots was associated with cell cycle progression. The fluorescence intensity of each FISH spot was quantitatively analyzed based on the cell cycle stage determined by image cytometry at the single-cell level. The spot intensity of cells in the G₂ phase was larger than that in the G_0/1 phase. This increased intensity was not seen during the early and mid S phases, whereas the cells in the late S phase showed significant increases in spot intensity, reaching the same level as that observed in the G₂ phase, indicating that alpha satellite DNA in the centromeric region was replicated in the late S phase. Thus, image cytometry can successfully detect small differences in the fluorescence intensities of centromeric spots of homologous chromosomes. This combinational image analysis of FISH spots and the cell cycle with cell image cytometry provides insights into new aspects of the cell cycle. This is the first report demonstrating that image cytometry can be used to analyze the fluorescence intensity of FISH signals during the cell cycle.     

Effects of low frequency electromagnetic field on proliferation of human epidermal stem cells: An in vitro study

To investigate the effects of low frequency electromagnetic fields (EMF) on the proliferation of epidermal stem cells, human epidermal stem cells (hESC) were isolated, expanded ex vivo, and then exposed to a low frequency EMF. The test and control cells were placed under the same environment. The test cells were exposed for 30 min/day to a 5 mT low frequency EMF at 1, 10, and 50 Hz for 3, 5, or 7 days. The effects of low frequency EMF on cell proliferation, cell cycle, and cell‐surface antigen phenotype were investigated. Low frequency EMF significantly enhanced the proliferation of hESC in the culture medium in a frequency‐dependent manner, with the highest cell proliferation rate at 50 Hz (P < 0.05). Exposure to a low frequency EMF significantly increased the percentage of cells at the S phase of the cell cycle, coupled with a decrease in the percentage of cells in the G1 phase (P < 0.05) but the effect was not frequency dependent. The percentage of CD29⁺/CD71^? cells remained unchanged in the low frequency EMF‐exposed hESC. The results suggested that low frequency EMF influenced hESC proliferation in vitro, and this effect was related to the increased proportion of cells at the S phase. Bioelectromagnetics 34:74–80, 2013. © 2012 Wiley Periodicals, Inc.     

Analysis of cell cycle activity and population dynamics in heterogeneous plant cell suspensions using flow cytometry

Flow cytometry was used to measure cell cycle parameters in Solanum aviculare plant cell suspensions. Methods for bromodeoxyuridine (BrdU) labeling of plant nuclei were developed so that cell cycle times and the proportion of cells participating in growth could be determined as a function of culture time and conditions. The percentage of cells active in the cell cycle at 25 degrees C decreased from 52% to 19% within 7.6 d of culture; presence of a relatively large proportion of non-active cells was reflected in the results for culture growth. While the maximum specific growth rate of the suspensions at 25 degrees C was 0.34 d-1 (doubling time: 2.0 d), the specific growth rate of active cells was significantly greater at 0.67 d-1, corresponding to a cell cycle time of 1.0 d. A simple model of culture growth based on exponential and linear growth kinetics and the assumption of constant cell cycle time was found to predict with reasonable accuracy the proportion of active cells in the population as a function of time. Reducing the temperature to 17 degrees C lowered the culture growth rate but prolonged the exponential growth phase compared with 25 degrees C; the percentage of cells participating in the cell cycle was also higher. Exposure of plant cells to different agitation intensities in shake flasks had a pronounced effect on the distribution of cells within the cell cycle. The proportion of cells in S phase was 1.8 times higher at a shaker speed of 160 rpm than at 100 rpm, while the frequency of G0 + G1 cells decreased by up to 27%. Because of the significant levels of intraculture heterogeneity in suspended plant cell systems, flow cytometry is of particular value in characterizing culture properties and behavior.     

REGENERATIVE PROLIFERATION OF MOUSE EPIDERMAL CELLS FOLLOWING APPLICATION OF A SKIN IRRITANT (CANTHARIDIN)

The strong skin irritant cantharidin dissolved in benzene was applied to the back of hairless mice. Single cell suspensions of epidermal basal cells were obtained and flow microfluorometric measurements of cellular DNA content were made. Smears were made for autoradiography, and the [³H]TdR labelling index (LI) and mean grain count (MGC) were assessed up to 3 days after cantharidin application. Three successive peaks of cells with S phase DNA content accompanied by three LI peaks were observed. The first two peaks were follwed by peaks of cells in G₂ phase, indicating that after the acute cell injury caused by cantharidin the cells traversed the cell cycle in partial synchrony through two subsequent cell cycles, each of 10–12 hr duration. During this phase of rapid proliferation the LI reached the proportion of cells in S phase, contrary to what is observed in untreated mouse epidermis, where the labelled cells contribute to about half the proportion of cells with S phase DNA content. The first two peaks of cells in S phase and LI coincided with an increased MGC, whereas the third peak was accompanied by a MGC significantly below control values. This indicates that this latter peak is due to a longer DNA synthesis time rather than to a partially synchronized and increased cell proliferation. The duration of the G₁, S and G₂ phases seems to be reduced initially in rapidly proliferating epidermis.     

EFFECTS OF HYDROXYUREA ON THE KINETICS OF COLONY FORMING UNITS OF BONE MARROW IN THE MOUSE

The number of nucleated bone marrow cells, the number of CFU and the number of DNA-synthesizing cells in the mouse were studied after injection of hydroxyurea. It was found that one injection provokes a partial synchronization of surviving cells and probably stimulates the transition of CFU from the quiescent to the cycling state. the changes of the proportion of CFU in the S phase makes it possible to estimate approximately a cell cycle duration of about 12 hr.