体外培养的牛耳成纤维细胞的细胞周期研究

通过细胞体外培养,对牛耳成纤维细胞的周期分布进行了研究。不同代数的细胞在生长至70-85％汇合和血清饥饿72h两种状态的细胞周期分布无显著差异。对于体外培养的第8代细胞,生长至50-60％、70-85％和完全汇合时,细胞周期分布存在显著差异;而培养时间对血清饥饿培养和正常培养至充分汇合时的细胞周期分布无显著影响。结果表明,体外培养代数及培养方法不会明显影响细胞周期的分布,但同代细胞因处于不同生长阶段细胞周期分布会存在显著差异。     

克服昆明小鼠体外受精卵发育阻滞方法的研究

本研究应用CZB和WM培养液进行昆明小鼠体外受精胚胎的发育培养,建立了一个可行的胚胎体外培养的新方法,并通过改变培养液的成分及其含量,对胚胎发育的阻滞机理和突破方法进行了初步的探索。培养于WM中的受精卵发生阻滞,有48％停留于2细胞阶段;而CZB中的胚胎有81％发育为桑椹胚和囊胚。在WM中添加EDTA和谷氨酰胺得到了66％的囊胚;加大WM中乳酸钠和丙酮酸钠的比值未能克服发育的阻滞现象。实验结果表明,EDTA和谷氨酰胺在克服阻滞时具有协同作用。     

Cell membrane lipid molecular dynamics in a solenoid versus a magnetically shielded room

The generalized polarization function of the fluorescent probe 2-dimethylamino-6-lauroylnaphthalene has been used to evaluate the lipid dynamics in Friend erythroleukemia cell membrane. The values of this function varied during the culture growth cycle, showing decreased lipid dynamics 24–48 h from the cell seeding. When the cycle occurred in a solenoid producing a magnetic field of 70 μT at 50 Hz in addition to the 45 μT DC of the earth (short-term 4-day exposure), the membrane lipid dynamics during this same time-period decreased by about 10% (P < .04). After long-term (184 days) or extremely long-term (395 days) exposure of the cells to the magnetic field, little additional variation in the membrane lipid dynamics was observed, suggesting an adaptation phenomenon. A variation of membrane lipid dynamics was also observed due to in vitro cell differentiation (P < .02). Nevertheless, the exposure of both undifferentiating and differentiating cells to a highly attenuated magnetic field in a magnetically shielded room (20 nT DC plus 2.5 pT AC) did not induce any modification of membrane lipid dynamics. Bioelectromagnetics 19:107–111, 1998. © 1998 Wiley-Liss, Inc.     

人隐静脉血管内皮细胞的分离、培养及鉴定

利用冠脉搭桥术后遗弃的隐静脉段获取内皮细胞,采用消化酶消化收集内皮细胞,扩增、冻存、复苏,在体外建立内皮细胞系。此方法简便易行,能在体外获得大量生物学特性保持良好的内皮细胞,为临床血管内皮化研究提供新的细胞来源。     

Trypanosoma cruzi: Interaction with Vertebrate Cells. DNA Synthesis and Growth of Intracellular Amastigotes and their Relationship to Host Cell DNA Synthesis and Growth

SYNOPSIS DNA synthesis of intracellular Trypanosoma cruzi amastigotes, following the infection of bovine embryo skeletal muscle (BESM) cells, was studied by autoradiography. After penetration, there was a prereplicative lag period (∼12 h) followed by a synchronous round of DNA synthesis which was found to be independent of parasite number/BESM cell and the host cell DNA synthesis cycle. Parasite reproduction occurred, for the first time, at ∼ 21 h postinfection. It was concluded that T. cruzi trypomastigotes are in the G₁/G, phase of their cell division cycle and that after penetration parasite reproduction occurs independent of events controlling host cell DNA synthesis and growth. The early synchronous growth of intracellular amastigotes should facilitate further studies on the biochemical events controlling trypomastigote-to-amastigote transformation and amastigote reproduction. A further application is envisaged for studies on the mode of action of drugs with trypanocidal activity.     

Analysis of the Cell Cycle and a Method Employing Synchronized Cells for Study of Protein Expression at Various Stages of the Cell Cycle

Study of protein expression during the cell cycle requires preparation of pure fractions of cells at various phases of the cell cycle. This was achieved by the development of methods for cell synchronization. Successful cell synchronization requires knowledge of the duration of all phases of the cell cycle. So, in the present review these interrelated problems are considered together. The first part of this review deals with basic methods employed for analysis of duration of cell cycle phases. The second summarizes data on treatments used for cell synchronization. Methods for calculation of percent of cells at various stages of the cell cycle in fractions of synchronized cells are considered in the third part. The fourth part of this review deals with a method of study of protein expression during the cell cycle by means of immunoblotting of synchronized cell fractions. In the Appendix, basic principles are illustrated with practical examples of analysis of the cell cycle, synchronization, and study of expression of some proteins at various stages of the cell cycle using synchronized XL2 (Xenopus laevis) cells.     

Proliferative growth of neonatal cerebellar cells in culture: Regulation by male and by maternal serum in late gestation

Neonatal cerebellar cells were utilized as a model system to examine the effect of 20 day pregnant rat serum on proliferative growth in the CNS. Cells were prepared by mechanical dissociation and cultured as mixed cells or populations enriched in astrocytes or oligodendrocytes. Cell proliferation was estimated by measurement of DNA, protein, and/or mitochondrial reductase activity (MTT). When mixed cells were incubated with 10% male rat serum, both total DNA and protein content increased after 6 days of culture. By contrast, neither of these parameters were altered in cultures incubated with 10% pregnant serum. When cells were incubated with either male or pregnant sera, changes in MTT activity paralleled changes in protein content. Graded concentrations of pregnant serum (5–20%) added to mixed cell cultures produced consistently lower MTT values when compared with identical concentrations of male serum. In addition, MTT activity was diminished in both astrocytes and oligodendrocytes incubated with graded concentrations of pregnant sera when compared with similar concentrations of non-pregnant sera. When potential effects of these different sera on the cell cycle were examined, an increase in the number of cells in the S and G2/M phase was similar, and DNA doubling began to increase at 96 hrs in the presence of either male or 20 day pregnant sera. Thus the inhibition of cell growth by pregnant serum was not likely a result of either cytotoxicity or a delay of entry of cells into the cell cycle. To examine whether this inhibition of cell growth may reflect the effect of pregnant serum on endogenous growth factor production, we tested the production of IGF-II by cerebellar cells. Production of an endogenous source of IGF-II was apparent using an RNAse protection assay and was noted using Slot Blot analysis of mRNA extracted at sequential times during cell incubation. Mixed cell cultures also secreted immunologically defined IGF-II. These observations are consistent with the previous demonstration that the fraction of pregnant serum which bound IGF-II also inhibited cell growth. The inhibitory effect of pregnant serum was diminished by preincubating aliquots of sera with graded concentrations of IGF-I prior to adding sera to tissue culture medium. Pregnant serum inhibition was also diminished by prolonging incubation times beyond 6 days. The blunting of pregnant serum inhibition may have been consequent to either a continuing production of endogenous growth factors or to the potential emergence of resistant cells due to prolonged tissue culture incubation. Since cells studied in a primary culture of limited duration may more accurately reflect the physiologic properties of this tissue, the model presented herein could provide a new approach to study brain development.West Side Medical CenterNorthwestern University medical School     

Activity of 1,2-benzisoxazole-3-one and indole-2,3-dione on plant regeneration in vitro and on cell elongation

We tested the morphogenetic and cell elongating activity of 1,2-benzisoxazole-3-one, a compound similar to 1,2-benzisoxazole-3-acetic acid but lacking the lateral carbon chain. For comparison, we tested also the activity of indole 2,3-dione, having the same indolic ring as indole 3-acetic acid but no lateral carbon chain. The tests were made on the regeneration of tomato (Lycopersicon esculentum Miller var. Alice) from cotyledons and on pea (Pisum sativum L. var. Alaska) stem elongation. We found that 1,2 benzisoxazole-3-one retains part of the high shoot inducing activity of 1,2-benzisoxazole-3-aceticacid, while indole-2,3-dione is inactive. Both compounds have no effect on root induction or cell elongation. It seems therefore that the activity of 1,2 benzisoxazole-3-acetic acid is partly related to the structure of its ring, and that also in this respect 1,2 benzisoxazole-3-acetic acid differs from other auxinlike compounds.Abbreviations BOA 1,2-benzisoxazole-3-acetic acid - BOO 1,2-benzisoxazole-3-one - IAA in-dole-3-acetic acid     

Synchronization of some human cell strains by serum and calcium starvation

Summary A technique was investigated for producing parasynchronous growth of some established, aneuploid human cell strains. Removal of both serum and calcium from exponentially growing monolayer cells tended to inhibit their growth. After 20 hr, a high percentage of the cell population was arrested in or near mitosis. Readdition of serum and calcium caused parasynchronous growth of the cells of three human strains studied. All three strains incorporated tritiated thymidine maximally 10 to 15 hr after serum and calcium were added, and cell numbers increased rapidly 17 to 25 hr after the growth medium was reconstituted. Population-doubling ranged from 80% to 100% of the theoretical. The yield of parasynchronous cells is high with this technique and may produce a significant amount of nontemporally distorted biological material upon which direct biochemical analysis can be performed at various times within the generation cycle.     

Anabolic-androgenic steroids: In cell culture

Summary Testosterone and related steroids at physiological concentrations positively stimulate in cell culture a number of reactions in a variety of tissues from different species of animals. Cells maintained in cell culture provide a means to study toxic effects in target organs and also the mechanism of action of these steroids.     

Screening of five specific cell cycle inhibitors using a T Cell lymphoma cell line synchrony/release assay

To obtain different cell populations at specific cell cycle stages, we used a cell culture synchronization protocol. Effects of five different cell cycle inhibitors acting throughout the cell cycle were examined by DNA flow cytometric analysis of a synchrony/release lymphoma cell line (CEM). The screening synchronized protocol showed that staurosporine, mimosine and aphidicolin are reversible G1 phase inhibitors that act at different times. Staurosporine acted in early G1, exhibited the strongest cytotoxic effect, and induced apoptosis. Mimosine and aphidicolin acted in late G1 and at the G1/S boundary, respectively. Hydroxyurea arrested CEM cells in early S phase, but later than the aphidicolin arrest point. Nocodazole synchronized CEM cells in M phase. All the inhibitors examined in this study can be used to synchronize cells at different phases of the cell cycle and were reversible with little toxicity except for staurosporine which is highly toxic. Because the regulatory mechanism of the cell cycle is disrupted by their effects on protein synthesis, however, these drugs must be used with caution.     

小鼠原生殖细胞体外培养及其应用研究

原生殖细胞（ｐｒｉｍｏｒｄｉａｌｇｅｒｍｃｅｌｌ,ＰＧＣ）是胚胎生殖谱系最原始形式的细胞,在体胚胎迁移期ＰＧＣ增殖极为旺盛。体外培养的小鼠迁移期ＰＧＣ在饲养层细胞和三种生长因子（干细胞生长因子、碱性成纤维细胞生长因子及白血病抑制因子）的共同作用下,可发展为长期增殖并维持不分化状态的胚胎性干细胞,即胚胎生殖细胞（ｅｍｂｒｙｏｎｉｃｇｅｒｍｃｅｌｌ,ＥＧ）,具全能性发育潜能。ＥＧ建系成功对于研究生殖细胞发育以及寻找新的转基因动物操作的有效载体具有重要价值。     

Cytosolic thymidine kinase activity in cultured human fibroblasts from individuals with galactokinase deficiency

The structural genes for human galactokinase (GALK) and the human cytosolic form of thymidine kinase (TK1) are located on 17q21–q22. These two loci are tightly linked, and studies on Chinese hamster cell lines have shown that the expression of TK1 and GALK genes may alter simultaneously. We investigated the possibility of a dependent mutation of TK1 and GALK genes in cultured fibroblasts obtained from two patients homozygous for the GALK^G-deficient gene. Since we showed that the TK1 level varies as a function of the passage and the growth rate of a given strain, our experiments were performed on nonstored skin fibroblasts, between the third and the fifth passage for both controls and patients. We found that TK1 levels in GALK-deficient cells were almost 75% of those observed in control strains with a similar growth rate. Previous results in the literature have shown a pronounced decrease in TK1 activity in three GALK-deficient fibroblastic strains. We suggest that these disparities of TK1 levels in GALK-deficient fibroblasts may be related either to genetic heterogeneity of GALK deficiency or to differences in culture conditions. This work was supported in part by grants from La CNAMTS and l’Université de Paris-Sud (AI 86 10).     

L929细胞镉中毒模型的建立

以L929细胞为研究对象,建立镉中毒体外模型,研究镉中毒对成纤维细胞生长发育的影响.本研究使用含有终浓度为100、50、25和5μmol·L-1氯化镉的DMEM培养基培养L929细胞,培养后0、6、12、24、36、48 h时分别观察细胞形态学变化,并应用MTT(四甲基偶氮唑盐)比色法来测定细胞的活性,研究结果表明,与正常组比较,当镉浓度大于50 μmol·L-1时,细胞形态损伤严重,细胞活性明显下降(p＜0.01);当镉浓度为5 μmol·L-1时,细胞未出现明显损伤,细胞活性无统计学差异(p＞0.05);时间效应观察结果显示,当镉作用36 h以上,细胞出现严重损伤,较多细胞悬浮于细胞培养基中,细胞活性明显下降(p＜0.01).结果表明,镉的终浓度为25 μmol·L-1,与L929细胞共同作用24 h为建立镉中毒体外模型的最佳条件.     

Kinetics of Thymidine Uptake,Its Phosphorylation,and Incorporation into DNA in the Sugar-Beet Cell Culture

Thymidine uptake, its phosphorylation, and incorporation into DNA were studied in a fast-growing sugar-beet (Beta vulgaris L.) cell suspension. A high rate and specificity of thymidine uptake were observed: total uptake reached a steady-state level for 3 min. The average kinetic constants for thymidine uptake were calculated for eight-day-old cells at 20°C as K _M = 25 M and V _max = 11.6 pmol/(min mg fr wt). The values of K _M for thymidine phosphorylation in vivo (53.2 M) and K _M for thymidine kinase (EC 2.7.1.21), which we purified earlier from broad bean seedlings, were of the same order of magnitude. The kinetics of thymidine phosphorylation in vivo displayed two distinct phases, which were determined by external thymidine concentration. Above 100 M thymidine, the rate of the process tended to rise, indicating the possible involvement of another mechanism for thymidine phosphorylation, most likely with the participation of nonspecific nucleoside phosphotransferase (NPT; EC 2.7.1.77). A further stage of thymidine salvage, its incorporation into DNA, occurred with a high affinity for thymidine phosphates; K _M = 2.8 M. The presence of other nucleosides (uridine or a high concentration of adenosine) in the medium markedly inhibited thymidine uptake. Nevertheless, these nucleosides did not diminish the percentage of thymidine phosphates of total thymidine uptake, which pointed to the specificity of thymidine phosphorylation and the insignificance of NPT activity. The analogue of thymidine, 5"-amino-2",5"-dideoxythymidine, known as thymidine kinase inhibitor, had no effect on thymidine uptake. The data presented provide evidence that the main route of the thymidine salvage in fast-growing sugar-beet suspension engages thymidine kinase, and NPT is activated only when nucleosides flood the cell.