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Modulation of differentiation in vitro. II. Influence of cell spreading and surface events on myogenesis. 体外分化的调节。2细胞扩散和表面事件对肌发生的影响。
Pub Date : 1984-09-01 DOI: 10.1007/BF02618874
H Sénéchal, J P Wahrmann, D Delain, A Macieira-Coelho

We examined the influence of attachment and spreading on myogenesis by adding polylysine-covered beads at different times after plating the cells on a plastic substratum. We show that polylysine per se acting on the cell surface can modulate myogenesis independently of cell spreading. Thus cell shape would not be the limiting factor for the division and differentiation of L6 myoblasts. Multinucleation of the cells was found to be first enhanced by the addition of polylysine-covered beads to replicating myoblasts, although the final percentage of fusion attained by these cultures was lower than in the controls. A similar phenomenon was observed concerning myosin synthesis. No such effect could be observed when the beads were added to a nonfusing mutant or to fibroblasts. Our results show that this phenomenon is specific. We postulate that some of the surface molecules necessary for this process appear on myoblasts shortly before they fuse.

我们通过在不同时间添加聚赖氨酸覆盖的珠粒来检测细胞在塑料基质上的附着和扩散对肌肉发生的影响。我们表明,聚赖氨酸本身作用于细胞表面可以调节肌发生独立于细胞扩散。因此,细胞形态不是L6成肌细胞分裂和分化的限制因素。发现细胞的多核首先通过在复制的成肌细胞中添加聚赖氨酸覆盖的小珠来增强,尽管这些培养最终获得的融合百分比低于对照组。在肌球蛋白合成方面也观察到类似的现象。当这些小珠被添加到非融合突变体或成纤维细胞中时,没有观察到这种效果。我们的结果表明,这种现象是特定的。我们假设这个过程所必需的一些表面分子在成肌细胞融合前不久就出现了。
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引用次数: 4
A serum-free culture system for studying solute exchanges in the choroid plexus. 研究脉络膜丛溶质交换的无血清培养系统。
Pub Date : 1984-09-01 DOI: 10.1007/BF02618877
W F Agnew, R B Alvarez, T G Yuen, S B Abramson, D Kirk

Organ cultures of choroid plexus tissues from the lateral ventricle of juvenile rats have been maintained for periods up to 7 wk in a chemically defined, serum-free media. Of several media and various supplements evaluated, the best growth and survival was obtained with the Pasadena Foundation for Medical Research-4 media supplemented with three hormones: epidermal growth factor, insulin, and hydrocortisone. Autoradiographic studies demonstrated that the epithelial cells incorporated [3H]leucine and [3H]thymidine indicating active protein and DNA synthesis, respectively. The organ cultures were characterized by bulbous, vesicular outgrowths from the choroidal villi explants. The fluid-filled lumina of the vesicles reached diameters of 900 microns and were easily accessed by micropipettes. The walls of the vesicles were composed of single layers of epithelial cells in which the ultrastructural features in the in vivo tissue were well maintained. The in vivo polarity (apical end toward the media and basilar end of the cells toward the luminal cavity) was also maintained. This morphologically stable in vitro system seems to be a promising model for investigation of secretory mechanisms of choroidal tissue.

幼年大鼠侧脑室脉络膜丛组织的器官培养在化学定义的无血清培养基中维持了长达7周。在评估的几种培养基和各种补充剂中,帕萨迪纳医学研究基金会-4培养基中添加三种激素:表皮生长因子、胰岛素和氢化可的松,获得了最佳的生长和存活率。放射自显影研究表明,上皮细胞分别掺入[3H]亮氨酸和[3H]胸苷,表明活性蛋白和DNA合成。器官培养的特点是由脉络膜绒毛外植体生长出球根状、水疱状的外植体。囊泡内充满液体的腔直径达到900微米,用微移液器很容易接近。囊泡的壁由单层上皮细胞组成,其体内组织的超微结构特征保持得很好。细胞的体内极性(细胞的根尖端朝向介质,细胞的基底端朝向管腔)也保持不变。这种形态稳定的体外系统似乎是研究脉络膜组织分泌机制的一个有前途的模型。
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引用次数: 3
Comparative use of fructose and glucose in human liver and fibroblastic cell cultures. 果糖和葡萄糖在人肝脏和成纤维细胞培养中的比较应用。
Pub Date : 1984-09-01 DOI: 10.1007/BF02618875
B Delhotal, F Lemonnier, M Couturier, C Wolfrom, M Gautier, A Lemonnier

The effect of fructose as a substitute for glucose in cell culture media was investigated in human skin fibroblast and liver cell cultures. Cells were grown for between 2 and 10 days in identical flasks in four different media, containing 5.5 mmol X 1-1 and 27.5 mmol X 1-1 glucose and fructose, respectively. In the presence of fructose, cell growth was stimulated, but less in liver cells than fibroblasts. At Day 6, increases were observed in [3H]thymidine incorporation, protein levels, and amino acid consumption, and a reduction was noted in ATP levels. In media containing 5.5 mmol X 1-1 glucose or fructose, consumption of fructose was four times lower than that of glucose at Day 3 and did not rise until Day 6. In fructose media, the lactate production was very low (four to five times less than that of glucose) and the pH values were always higher. Some findings were different for the fibroblasts and liver cells, owing to the specific characteristics of these two cell types in culture; this applied especially to the effects of glucose and fructose concentrations of 27.5 mmol X 1-1. Several possible explanations for the stimulation of cell growth in fructose medium were discussed.

在人皮肤成纤维细胞和肝细胞培养中,研究了果糖在细胞培养基中替代葡萄糖的效果。在相同的烧瓶中,细胞在四种不同的培养基中生长2至10天,分别含有5.5 mmol X -1和27.5 mmol X -1葡萄糖和果糖。在果糖的存在下,细胞生长受到刺激,但肝细胞比成纤维细胞少。在第6天,观察到[3H]胸苷掺入,蛋白质水平和氨基酸消耗增加,并且注意到ATP水平降低。在含有5.5 mmol X -1葡萄糖或果糖的培养基中,果糖的消耗量在第3天比葡萄糖低4倍,直到第6天才上升。在果糖培养基中,乳酸产量非常低(比葡萄糖少4 - 5倍),pH值总是较高。在培养过程中,由于成纤维细胞和肝细胞的特性不同,结果有所不同;这尤其适用于葡萄糖和果糖浓度为27.5 mmol X -1时的影响。讨论了果糖培养基中刺激细胞生长的几种可能的解释。
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引用次数: 9
Fish cell lines: establishment and characterization of nine cell lines from salmonids. 鱼类细胞系:九种鲑鱼细胞系的建立和鉴定。
Pub Date : 1984-09-01 DOI: 10.1007/BF02618871
C N Lannan, J R Winton, J L Fryer

Nine permanent cell lines have been established from five species of salmonids native to America's Pacific Northwest. With the exception of a hepatoma from an adult trout, the lines were derived from normal tissues of embryonic or juvenile fish. Cells were routinely grown in Eagle's minimum essential medium with 10% fetal bovine serum. Optimum growth temperatures for these lines ranged from 21 to 24 degrees C. All survived storage for at least 1 yr at -65 degrees C and at least 5 yr in liquid nitrogen. Six of the lines were demonstrably free of any microbial contamination but mycoplasmas were found in three. Eight of the lines were heteroploid. The morphology of only one was fibroblastic. All the lines effectively replicated one or more of the common salmonid viruses. Isozyme patterns were consistent with those of the species of origin. These cell lines have significant application in fish virology.

从美国西北太平洋地区的五种鲑鱼中建立了九种永久性细胞系。除了来自成年鳟鱼的肝癌外,这些细胞系都来自胚胎或幼鱼的正常组织。细胞在含有10%胎牛血清的Eagle最小基本培养基中常规生长。这些品系的最佳生长温度为21 ~ 24℃,在-65℃和液氮条件下至少能存活1年。其中6条线明显没有任何微生物污染,但3条线发现了支原体。其中8个系为异倍体。只有一个细胞形态为成纤维细胞。所有的菌株都有效地复制了一种或多种常见的沙门氏菌病毒。同工酶模式与起源种一致。这些细胞系在鱼类病毒学研究中具有重要的应用价值。
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引用次数: 188
Membrane potentials, electrolyte contents, cell pH, and some enzyme activities of fibroblasts. 膜电位、电解质含量、细胞pH和成纤维细胞的一些酶活性。
Pub Date : 1984-09-01 DOI: 10.1007/BF02618872
Y C Yen-Chow, S Y Chow, W S Jee, D M Woodbury

The resting membrane potential of the cultured fibroblasts derived from rabbit subcutaneous tissues was -10.2 +/- 0.20 mV (n = 390). This potential was affected by the potassium concentration in the culture medium, but not by other chemical or hormonal preparations, such as dibutyryladenosine 3',5'-cyclic monophosphate (0.5 to 5.0 mmol/l), sodium fluoride (10(-5) to 10(-4) M), hydrocortisone (10(-7) to 10(-6) M), parathyroid extract (0.5 to 1.0 U/ml), or thyrotrophin (5 to 10 mU/ml). The Na+, K+, and Cl- concentrations of the cultured fibroblasts were 35.4, 85.7, and 22.6 mmol/l cell water, respectively. The water and protein contents of these cells were 82.1 and 9.18 g/100-g cells, respectively. The intracellular pH of fibroblasts as determined by [14C] dimethyloxazolidine-2, 4-dione, and 3H2O ranged between 6.9 and 7.1 when the pH of the culture medium was maintained at 7.4. The activities of Na+, K+-, HCO3(-)-, and Ca++, Mg++-ATPases in these cultured cells were 19.0 +/- 2.1, 13.6 +/- 2.1, and 6.6 +/- 1.2 nmol pi/mg protein per minute, respectively, and the carbonic anhydrase activity was 0.054 U/mg protein. Calculations based on the values for the membrane potential and the electrolyte concentrations observed in this study indicate that Na+, K+, Cl-, and H+ are not distributed according to their electrochemical gradients across the cell membrane. Na+, Cl-, and H+ are actively transported out of the cells and K+ into the cells.

兔皮下组织成纤维细胞静息膜电位为-10.2 +/- 0.20 mV (n = 390)。这种电位受培养基中钾浓度的影响,但不受其他化学制剂或激素制剂的影响,如3',5'-环单磷酸二丁基腺苷(0.5至5.0 mmol/l),氟化钠(10(-5)至10(-4)M),氢化可的松(10(-7)至10(-6)M),甲状旁腺提取物(0.5至1.0 U/ml)或促甲状腺激素(5至10 mU/ml)。Na+、K+和Cl-浓度分别为35.4、85.7和22.6 mmol/l细胞水。细胞含水量为82.1 g/100 g,蛋白质含量为9.18 g/100 g。当培养基pH保持在7.4时,用[14C]二甲基氯恶唑烷- 2,4 -二酮和3H2O测定成纤维细胞内pH值在6.9 ~ 7.1之间。Na+、K+-、HCO3(-)-和Ca++、Mg++- atp酶的活性分别为19.0 +/- 2.1、13.6 +/- 2.1和6.6 +/- 1.2 nmol pi/ Mg蛋白/ min,碳酸酐酶活性为0.054 U/ Mg蛋白/ min。根据本研究中观察到的膜电位值和电解质浓度的计算表明,Na+、K+、Cl-和H+并不是按照它们在细胞膜上的电化学梯度分布的。Na+、Cl-和H+被主动地运出细胞,K+被主动地运入细胞。
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引用次数: 5
Sensitivity of human pancreatic carcinoma cell line (MIA PaCa-2) to Bisantrene and Theprubicin in vitro. 人胰腺癌细胞系(MIA PaCa-2)对双蒽醌和普鲁比星的体外敏感性。
Pub Date : 1984-09-01 DOI: 10.1007/BF02618873
G Fountzilas, L O Lim, A A Yunis

We tested the effect of Bisantrene (BS) and Theprubicin (THP-ADR) on cell growth of a human pancreatic carcinoma cell line (MIA PaCa-2). After 1 h exposure ID50 of BS or THP-ADR was 3 X 10(-7) and 5 X 10(-8) M, respectively. Increasing the exposure time from 1 h to continuous exposure for 5d resulted in 11-fold decrease in ID50 for BS and a 6-fold decrease for THP-ADR. Both drugs inhibited [14C]thymidine incorporation to the same extent and caused an accumulation of cells into G2 + M phase of the cell cycle.

研究了双蒽醌(BS)和普比星(THP-ADR)对人胰腺癌细胞系(MIA PaCa-2)细胞生长的影响。暴露1 h后,BS和THP-ADR的ID50分别为3 × 10(-7)和5 × 10(-8) M。将暴露时间从1小时增加到连续暴露5d,导致BS的ID50降低11倍,THP-ADR降低6倍。两种药物抑制[14C]胸腺嘧啶掺入的程度相同,并导致细胞积聚进入细胞周期的G2 + M期。
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引用次数: 1
Cultured hepatoma cells for the study of enzyme regulation: induction of ornithine decarboxylase by insulin and asparagine. 培养肝癌细胞的酶调节研究:胰岛素和天冬酰胺诱导鸟氨酸脱羧酶。
Pub Date : 1984-09-01 DOI: 10.1007/BF02618878
V R Potter, T R Evanson, D P Gayda, J A Gurr

The induction and decay of ornithine decarboxylase (ODC) by insulin and asparagine in cultures of H4-II-EC3 (H35) hepatoma cells was studied in a modified Waymouth medium in the presence of fetal bovine serum (FBS) and in serum-free media. The insulin response was enhanced by the presence of asparagine although the effect of asparagine was not so much on the initial increase as it was on a slowing of the decline after the maximum was reached at 6 to 8 h after the supplements were added together with fresh medium. In all cases the initial ODC activity was zero at zero time for addition of media and supplements, and, after reaching the maximum, activity declined to near zero by 24 h. Fetal bovine serum gave induction that followed a similar time course but was inferior to the combination of insulin plus asparagine and, in fact, FBS inhibited the latter response. Putrescine (the product formed from ornithine by ODC), at 10(-5) M, markedly inhibited the induction of ODC by insulin or FBS, but the inhibition was less when asparagine was present.

研究了胰岛素和天冬酰胺对H4-II-EC3 (H35)肝癌细胞培养中鸟氨酸脱羧酶(ODC)的诱导和降解作用。天冬酰胺的存在增强了胰岛素的反应,尽管天冬酰胺的作用与其说是对最初的增加,不如说是在补充物与新鲜培养基一起添加后6至8小时达到最大值后的下降减缓。在所有情况下,添加培养基和补充剂的初始ODC活性在零时间为零,并且在达到最大值后,活性在24小时内下降到接近零。胎牛血清的诱导过程类似,但不如胰岛素加天冬酰胺的组合,事实上,FBS抑制了后者的反应。腐胺(ODC由鸟氨酸形成的产物)在10(-5)M时,明显抑制胰岛素或FBS诱导的ODC,但当有天冬酰胺存在时,抑制作用较弱。
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引用次数: 1
Changes in serum influence the fatty acid composition of established cell lines. 血清变化影响已建立细胞系的脂肪酸组成。
Pub Date : 1984-09-01 DOI: 10.1007/BF02618879
L L Stoll, A A Spector

The fatty acid composition of different kinds of commercially available serum used to supplement cell culture media differs widely. As compared with fetal bovine serum, horse and bovine calf serum have a very high content of linoleic acid (18:2) and are low in arachidonic acid (20:4). (Fatty acids are abbreviated as number of carbon atoms:number of double bonds). Swine serum contains substantial amounts of both 18:2 and 20:4. Only fetal bovine serum contains more than 1% docosahexaenoic acid (22:6). Considerable differences in fatty acid composition occur when cells are grown in media containing any of these different serum supplements. The 18:2 and 20:4 content of 3T3 mouse fibroblast phospholipids is highest when the medium contains horse serum, intermediate with bovine calf serum, and lowest with swine or fetal bovine serum. Likewise, the highest phospholipid 18:2 content in Madin-Darby canine kidney cells (MDCK) occurs when the medium contains horse serum. With MDCK cells, however, growth in swine serum produces the highest 20:4 content. The 3T3 cell phospholipids accumulate more than 1% 22:6 only when the medium contains fetal bovine serum, whereas in no case do the MDCK cell phospholipids accumulate appreciable amounts of 22:6. The fact that the cellular fatty acid composition is likely to change should be taken into account when changes are contemplated in the serum used to grow established cell lines.

不同种类的市售血清用于补充细胞培养基的脂肪酸组成差别很大。与胎牛血清相比,马和牛犊牛血清的亚油酸含量很高(18:2),花生四烯酸含量很低(20:4)。(脂肪酸缩写为碳原子数:双键数)。猪血清含有大量的18:2和20:4。只有胎牛血清含有超过1%的二十二碳六烯酸(22:6)。当细胞在含有任何这些不同血清补充剂的培养基中生长时,脂肪酸组成会发生相当大的差异。3T3小鼠成纤维细胞磷脂的18:2和20:4含量以马血清最高,牛血清次之,猪和胎牛血清最低。同样,当培养基中含有马血清时,Madin-Darby犬肾细胞(MDCK)中磷脂18:2含量最高。然而,MDCK细胞在猪血清中生长产生最高的20:4含量。只有当培养基中含有胎牛血清时,3T3细胞磷脂积累超过1% 22:6,而MDCK细胞磷脂在任何情况下都不会积累可观的22:6。当考虑用于培养已建立的细胞系的血清发生变化时,应考虑到细胞脂肪酸组成可能发生变化的事实。
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引用次数: 64
On disturbance of homeostasis in organ-cultured tissue. 器官培养组织内稳态紊乱的研究。
Pub Date : 1984-08-01 DOI: 10.1007/BF02619609
K Norrby, S Bergström, P Druvefors

The intact membranous rat mesentery was cultured in Eagle's minimum essential medium containing no serum or only low concentrations of serum. The procedure is in some important respects superior to previous organ culture techniques. To estimate the extent of disturbance of homeostasis of the tissue in culture, the spontaneous mast-cell histamine release was quantitated after preculture preparation of the specimens and after different intervals in culture. Also, the proliferation of fibroblasts and mesothelial cells that predominate in the mesentery was assessed at 48 h by cytofluorometric quantitation of DNA in single-tissue cells. Spontaneous histamine release was time dependent during cultivation, amounting to ca. 50% at 48 h, and was affected by the medium used for moistening the tissue before cultivation. Culturing also brought about great spontaneous increase in the proliferation of fibroblasts and mesothelial cells, the rate being related to the concentration of serum. Addition of the mast-cell secretagogues 48/80 or polymyxin B at 1 h caused rapid release of 50 to 60% of the histamine and was followed by augmented proliferation in the serum-containing media. The spontaneous increase of cell proliferation in tissue culture may be causally related to mast-cell secretion. Further studies are needed to define factors influencing the spontaneous mast-cell secretion and the mast-cell-dependent mitogenesis in normal tissue cells.

完整的膜大鼠肠系膜在Eagle的最低必要培养基中培养,不含血清或只有低浓度的血清。该方法在某些重要方面优于以往的器官培养技术。为了估计组织在培养中稳态的紊乱程度,在标本的预培养准备和不同的培养间隔后,对肥大细胞自发组胺释放进行了定量。此外,在48小时内,通过对单个组织细胞的DNA进行细胞荧光定量,评估了在肠系膜中占主导地位的成纤维细胞和间皮细胞的增殖情况。在培养过程中,自发组胺释放与时间有关,在48 h时约为50%,并受培养前用于湿润组织的培养基的影响。培养也使成纤维细胞和间皮细胞的增殖自发增加,其增殖速率与血清浓度有关。在1小时内加入肥大细胞分泌剂48/80或多粘菌素B,可快速释放50%至60%的组胺,随后在含血清的培养基中增殖增强。组织培养中细胞增殖的自发增加可能与肥大细胞分泌有因果关系。正常组织细胞中影响肥大细胞自发分泌和肥大细胞依赖性有丝分裂发生的因素有待进一步研究。
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引用次数: 0
Culture conditions induce the appearance of immortalized C3H mouse cell lines. 培养条件诱导C3H小鼠永生化细胞系的出现。
Pub Date : 1984-08-01 DOI: 10.1007/BF02619607
L Curatolo, E Erba, L Morasca

Mouse fibroblasts were cultured by three different procedures: (a) changing the 0.2 ml/cm2 of growth medium every 2nd d and seeding 1 X 10(5) cells/cm2 after confluency; (b) changing the 0.4 ml/cm2 of growth medium only at subculture performed at confluency by a 1:2 split and keeping the bottles incubated on a rocking platform; (c) the same as Method b but keeping the bottles stationary throughout culture. By Method a no lines were immortalized over 36 experiments whereas Method b gave 1/4 immortalized lines and Method c gave 10:12 immortalized lines. Cells always went into crisis at the 9th to 11th doubling. Immortalized lines had a tetraploid DNA content.

采用三种不同的方法培养小鼠成纤维细胞:(a)每2 d更换0.2 ml/cm2的生长培养基,汇合后播种1 X 10(5)个细胞/cm2;(b)仅在传代时以1:2的比例更换0.4 ml/cm2的培养基,并将瓶子放在摇摆平台上孵育;(c)与方法b相同,但在整个培养过程中保持瓶固定。方法a经过36次实验,没有获得永生化系,方法b获得1/4永生化系,方法c获得10:12永生化系。细胞总是在第9到11倍时陷入危机。永生化系的DNA含量为四倍体。
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引用次数: 9
期刊
In Vitro
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