EGF对大鼠卵巢颗粒细胞增殖与分化影响的研究

许多研究发现,表皮生长因子(EGF)对生殖功能有重要的调节作用。本文用体外细胞培养的方法,研究了EGF对大鼠卵巢颗粒细胞增殖与分化的影响及其作用方式。结果如下:EGF可以明显抑制颗粒细胞DNA的合成,但促进孕酮的生成,后者是因为EGF能显著提高细胞内3β-羟甾脱氨酶(3β-HSD)的活性。放射受体分析表明,颗粒细胞上存在EGF的特异性受体,其K_d为1.83±0.3×10~(-8)mol/L,B_(max)为1.75±0.29×10~4个位点/细胞。卵巢免疫组化结果未发现颗粒细胞有EGF样免疫染色,而卵泡膜、黄体及间质内等均有阳性染色。以上结果提示,EGF可能通过旁分泌机制作用于颗粒细胞的EGF受体,从而调节细胞的生长和性激素的分泌,这对于颗粒细胞的成熟及卵泡的发育有着重要意义。     

ULTRASTRUCTURE AND FUNCTION OF GROWTH CONES AND AXONS OF CULTURED NERVE CELLS

Dorsal root ganglion nerve cells undergoing axon elongation in vitro have been analyzed ultrastructurally. The growth cone at the axonal tip contains smooth endoplasmic reticulum, vesicles, neurofilaments, occasional microtubules, and a network of 50-A in diameter microfilaments. The filamentous network fills the periphery of the growth cone and is the only structure found in microspikes. Elements of the network are oriented parallel to the axis of microspikes, but exhibit little orientation in the growth cone. Cytochalasin B causes rounding up of growth cones, retraction of microspikes, and cessation of axon elongation. The latter biological effect correlates with an ultrastructural alteration in the filamentous network of growth cones and microspikes. No other organelle appears to be affected by the drug. Removal of cytochalasin allows reinitiation of growth cone-microspike activity, and elongation begins anew. Such recovery will occur in the presence of the protein synthesis inhibitor cycloheximide, and in the absence of exogenous nerve growth factor. The neurofilaments and microtubules of axons are regularly spaced. Fine filaments indistinguishable from those in the growth cone interconnect neurofilaments, vesicles, microtubules, and plasma membrane. This filamentous network could provide the structural basis for the initiation of lateral microspikes and perhaps of collateral axons, besides playing a role in axonal transport.     

GROWTH AND ORGANIZED DEVELOPMENT OF CULTURED CELLS. V. THE GROWTH OF COLONIES FROM FREE CELLS ON NUTRIENT AGAR

When angiosperm cells are cultured in a liquid medium they may grow in the form of free, single cells and form small to large groups of cells. This has been shown in earlier papers. This paper deals with the growth of strains of cells (Daucus carota and Haplopappus gracilis cells were used), washed and filtered free from the larger groups, on nutrient agar media in Petri dishes, thus simulating familiar microbiological technique. Each discrete member of a suspension is referred to as a “unit.” On the order of 1–10% of the separate units of a suspension may be induced to grow into viable colonies, depending on the strain and the conditions employed. Whereas at least 30% of the free single carrot cells were shown to be capable of division, only up to about 4% continued their growth to form macroscopically visible colonies when they were widely dispersed. Coconut milk promotes the growth of carrot cells into colonies. Both coconut milk and napthaleneacetic acid, which interact synergistically, arc required for the growth of Haplopappus cells. Various techniques which affect viability (the frequency with which units grow into colonies) were investigated. The viability of carrot units was found (1) to increase with their density on the plates; (2) to decrease upon washing the suspensions prior to plating; (3) to increase with increasing initial size; and (4) to decrease to a vanishingly low value in rigorously filtered suspensions which consist principally of single cells, although the single cells were found to grow with appreciable frequency when the larger units were also present; and (5) to increase dramatically (100-fold) when a rigorously filtered suspension was plated on a medium upon which pieces of growing cultured tissue were placed. Thus, the induction of growth in free cells is enhanced, even in an environment nutritionally optimal for the growth of the larger cell masses, by as yet unknown factors which are contributed by the cells themselves, or by adjacent cells or groups of cells. It is suggested that within a group of cells growing in culture, and perhaps also in the organized growing regions of intact plants, the dividing cells are nourished or stimulated by adjacent but less frequently dividing cells. The implications of these results are discussed.     

EFFECTS OF ADENOSINE AND EPIDERMAL GROWTH FACTOR ON THE GROWTH OF MOUSE MAMMARY EPITHELIAL CELLS

The objective of this study was to determine if adenosine alters growth of mammary epithelium. Mouse mammary epithelial cells (NMuMG) were cultured in DMEM supplemented with 10% fetal calf serum. After serum starvation for 24h, EGF (0–100ng/ml) and/or adenosine (0–100μm ) was added. Adenosine at concentrations of 1, 10 or 100μm increased DNA synthesis significantly, when compared to control. Addition of epidermal growth factor (EGF) (10ng/ml) into 1 or 10μm adenosine showed the interaction in DNA synthesis between EGF and adenosine. A similar result was observed when 100μm adenosine added to various concentrations of EGF (0–100ng/ml). In the second mammary gland (thoracic) organ culture studies, mammary development scores were increased by adenosine (100μm ), EGF (100ng/ml) and adenosine plus EGF. These results indicate that the purine nucleoside adenosine stimulates mammary epithelial cell growth and interacts with EGF in DNA synthesis of mouse mammary epithelial cells.     

单相和两相发酵体系中Methylomonas Z201细胞的生长和环氧丙烷的合成

研究了单相和两相发酵体系中甲基单胞菌（Methylomonas）Z201细胞的生长和环氧丙烷的合成。在单相发酵体系中,底物丙烯和产物环氧丙烷抑制细胞生长,水相中环氧丙烷的浓度达到13mmol／L。在两相发酵体系中,十六烷作为生长底物甲烷以及反应底物丙烯和分子氧的“储器”,减小了丙烯对细胞生长的抑制作用,水相和十六烷相中环氧丙烷的浓度分别达到1．7mmol／L和2．6mmol／L。同休止细胞相比,单相和两相发酵体系中辅酶NADH的原位再生使生长细胞的操作稳定性显著提高,尤为两相体系为甚。     

异源PDGF-A链表达对CHO细胞生长和转化的作用

用DNA磷酸钙盐沉淀方法把含人PDGF(血小板衍生生长因子)A链cDNA的表达质粒pSV_2neo-A转染CHO细胞(中国仓鼠卵巢细胞),然后经G 418(400-800 μg/ml)筛选分离20个转染细胞株。选出其中At_1和Aot7细胞株所进行的实验结果表明,这些细胞的形态和生长行为均发生明显的变化,PDGF-A链mRNA的表达水平比CHO细胞明显增高,胞质有强阳性的PDGF荧光反应,显示有PDGF样蛋白的合成。这些细胞不但生长速率加快,有高密度持续生长的特性,而且能在软琼脂培基上形成大集落和在裸鼠体内接种形成纤维肉瘤,提示外源PDGF-A链基因的表达有使CHO细胞生长失控和发生细胞恶性转化的作用。     

GROWTH AND ORGANIZED DEVELOPMENT OF CULTURED CELLS. VI. THE BEHAVIOR OF INDIVIDUAL CELLS ON NUTRIENT AGAR

Freely suspended cells of a long continuously cultured strain of carrot were dispersed in nutrient agar medium in Petri dishes. After inoculation, cells in randomly chosen fields were photographed at low magnification. The same cells were photographed at intervals over the next 23 days. The fate of 678 units, 24% of which grew visibly, was determined from the photographic record. Cells and units displayed a marked degree of individuality. In some units, there was a progressive increase in cell number; in others, after a few divisions, growth ceased; in still other units, cells enlarged without dividing. Although the evidence of cell division in free cells is conclusive, its incidence and maintenance increased with the size of the initial unit. In general, growth started after an “induction period” of about 5 days, but some cells remained apparently unchanged considerably longer after inoculation, before they started to grow. Although single cells of all sizes were observed to grow by one means or another, it was generally the shorter ones that continued to grow into viable colonies. Elongated cells predominated in the carrot strains used; these grew into colonies first by septation, with little or no over-all increase in size, followed by a stage of cell enlargement. Growth by both cell enlargement and division then ensued. During growth by septation within single cells, some derived cells were seen to burst and die. The most frequent divisions were observed during septation when the average cell generation time was 24 hr. Lacking the controls that normally operate in the intact plant body, there was much variation in the forms assumed during growth, a number of which are illustrated. The importance of epigenetic factors, or external controls, that determine the growth of cells in the plant body is stressed.     

DNA REPLICATION: DIRECTION AND RATE OF CHAIN GROWTH IN MAMMALIAN CELLS

Using pulse labeling techniques with [³H]thymidine or [³H]cytidine, combined with DNA fiber autoradiography, we have investigated the direction and rate of DNA chain growth in mammalian cells. In general, chain elongation proceeds bidirectionally from the common origin of pairs of adjacent replication sections. This type of replication is noted whether the DNA is labeled first with [³H]thymidine of high specific activity, followed by [³H]thymidine of low specific activity or the sequence is reversed. Approximately one-fifth of the growing points have unique origins and in these replication units, chain growth proceeds in one direction only. Fluorodeoxyuridine and hydroxyurea both inhibit DNA chain propagation. Fluorodeoxyuridine exerts its effect on chain growth within 15–23 min, while the effect of hydroxyurea is evident within 15 min under conditions where the endogenous thymidine pool has been depleted by prior treatment with fluorodeoxyuridine. Puromycin has no effect on chain growth until 60 min after addition of the compound, even though thymidine incorporation is more than 50% reduced within 15 min. After 2 h of treatment with puromycin, the rate of chain growth is reduced by 50%, whereas thymidine incorporation is reduced by 75%. Cycloheximide reduces the rates of DNA chain growth and thymidine incorporation 50% within 15 min, and, on prolonged treatment, the decrease in rate of chain growth generally parallels the reduction in thymidine incorporation.     

GROWTH AND ORGANIZED DEVELOPMENT OF CULTURED CELLS. VII. CELLULAR VARIATION

Variation in long-continued cultures of Haplopappus gracilis and Daucus carota has been investigated. A strain of carrot tissue was isolated that grew with a compact habit, in contrast to the highly friable habit of the parent strain. Its dividing cells were arranged quite differently than in the parent strain. Earlier work had shown that Haplopappus cultures could be reversibly altered in their pigmentation and form, by changing the culture medium. This was confirmed, and it was further shown that pronounced changes in nitrogenous compounds also occurred in response to factors in the medium. However, strains of Haplopappus were isolated which differed persistently from the parent strain, even when they were maintained under the same conditions. The variant strains, grown in the same medium, showed differences in their content of nitrogenous compounds. Stock cultures also changed spontaneously with time with respect to their content of nitrogenous substances. Acriflavine, at low concentration, inhibited the growth and formation of colonics by cells plated on nutrient agar, but, by prolonged exposure to sublethal amounts of the drug, resistant strains were isolated. Certain of the spontaneous variant strains were found to differ from each other and from the parent strain in their chromosome complements in ways that are described and to which the observed changes in morphology and metabolism of the cultures may be attributed. The variations that may occur in the free cells in culture are contrasted with the greater uniformity of the cells as they exist in the plant body.     

4-PA对BEL-7402细胞的生长调控和诱导凋亡作用的研究

应用ＭＴＴ、流式细胞术研究了新型肿瘤诱导分化剂４－ＰＡ对人肝癌细胞系ＢＥＬ－７４０２细胞的生长调控、诱导调亡作用。结果表明：４－ＰＡ对ＢＥＬ－７４０２细胞增殖抑制的ＩＣ５０为４－９ｍｍｏｌ／Ｌ,随着作用时间的增加,ＩＣ５０增加。作用效果迅速,在０．５小时就有效地抑制了细胞增殖,抑制率随４－ＰＡ浓度的增加和作用时间的处长而增加,在达到最大抑制率后出现饱和。流式细胞仪对细胞周期和细胞调亡的分析发现：４     

GROWTH AND ORGANIZED DEVELOPMENT OF CULTURED CELLS IV. THE BEHAVIOR OF THE NUCLEUS

Mitra , J., Marion O. Mapes , and F. C. Steward . (Cornell U., Ithaca, New York.) Growth and organized development of cultured cells. IV. The behavior of the nucleus. Amer. Jour. Bot. 47(5) : 357—368. Illus. 1960.–The nuclei and the chromosomes of carrot cells have been examined at various stages throughout the following sequence: (1) growth of a tissue culture from a preformed explant of secondary phloem from the carrot root; (2) growth and multiplication of carrot cells freely suspended in a liquid medium; (3) growth and re-formation of organs (roots) and whole plants (including flowers) from cells in the freely suspended state. The cells of the carrot are normally diploid (2n = 18), the cells which develop in the explant are also diploid, and the cells of the re-formed organs, and even the flowers developed upon plants grown from cells, are also normal and diploid; normal meioses also occur. Nevertheless, the wide range in growth and form of the freely suspended cells is accompanied by a rich diversity of cytological conditions; these include tetraploid and highly polyploid nuclei which divide, haploidy and such chromosomal aberrations as di- and even tri-centric bridges. Two division figures showing chromosome numbers at different levels of ploidy were seen within the confines of one large cell, and, in another, 2 adjacent division figures were observed with chromosome numbers lower than diploid. Small thick-walled, densely protoplasmic cells divide to form bi- and tetra-nucleate conditions, and in a giant cell a highly multinucleate condition has been seen. Despite this, however, all the regenerated roots and plants yet examined are normally diploid. The implications of these events are discussed.     

DIVISION AND GROWTH OF SINGLE MESOPHYLL CELLS ISOLATED ENZYMATICALLY FROM TOBACCO LEAVES

Single mesophyll cells isolated enzymatically from tobacco leaves divided and grew into aggregates of several cells in defined media. Morphological changes accompanying the division and the growth suggested that these cells are in the course of dedifferentiation. Some of the potentialities of these cells as an experimental material for developmental and genetical studies are discussed.     

LIF基因转染的ES细胞生长与分化特性的研究

我们将人D型LIF cDNA以正反两种方向分别克隆到载体pKCR 3,并引入neo~r基因,构建成pSVLD( )和pSVLD(-)质粒,按磷酸钙沉淀法分别转染ES-5胚胎干细胞,经G418和不同浓度LIF条件培液共同筛选、Nor-thern和Southern分析以及ES-5细胞集落分化抑制能力测定,建立了过度表达分泌LIF的ESL( )细胞株和表达外源反义LIF RNA的ESL(-)细胞株。我们发现,ESL( )A2细胞能够在无外源LIF常规培液下至少传13代以上,仍能正常生长和传代,并保持与ES-5细胞同样的体外生长的特征性形态,以及具有干细胞特点和发育多潜能性,表明过度表达LIF确实能使ES细胞完全脱离对外源LIF条件培液的依赖性;而表达反义LIP RNA的ESL(-)细胞对培液中LIF浓度的依赖性明显升高,也更易分化,说明ES细胞内源LIF基因的表达水平虽低,但对于抑制ES细胞的分化仍可能是必需的。形态学观察发现,体外悬滴培养中经10~(-6)mol/L RA诱导后,过度表达LIF并未产生抑制ESL( )A2细胞分化的现象,和亲本ES-5细胞比较,也未发现其明显改变了10~(-6)mol/L RA对ESL( )A2细胞诱导分化的方向;而相同条件下,表达外源反义LIF RNA,则使ESL(-)B5细胞更易于向形态明确的细胞包括成纤维样和梭样细胞分化。上述细胞株的建立,提供了一个研究在不添加LIF的常规培液中生长的ES细胞或表达外源反义LIF RNA的ES细胞的生长分化的模型。     

低氧对NG108－15细胞生长及分化的影响

神经母细胞瘤和神经胶质瘤细胞融合的克隆细胞系ＮＧ１０８－１５细胞在含分化剂双丁基环化单磷酸腺苷（ｄＢｆｃＡＭＰ）的培养液培养后分化,成为具神经细胞特征的细胞。本实验利用四唑盐（ＭＴＴ）微量比色法,并结合焦油紫染色,测定和观察细胞的生长及分化状况,研究了低氧（２％Ｏ２＋９３％Ｎ２＋５／ＣＯ２）对未分化的,分化中的和已分化完成的ＮＧ１０８－１５细胞的影响。获得的主要结果是;低氧明显降低未分化细胞增殖和存活率,使分化完成的细胞大量死亡;低氧影响ＮＧ１０８－１５细胞的分化,使细胞在分化中出现体积膨大,突起短等异常特征,经焦油紫染色,胞质中无尼氏体（即不着色）的细胞增多。低氧是否可能使未分化ＮＧ１０８－１５细胞向更多地表达胶质细胞特征的方向分化？将是一个十分有趣的问题。     

4-PA对BEL-7402细胞的生长调控和诱导凋亡作用的研究

应用ＭＴＴ、流式细胞术研究了新型肿瘤诱导分化剂４－ ＰＡ 对人肝癌细胞系ＢＥＬ－ ７４０２ 细胞的生长调控、诱导凋亡作用。结果表明： ４ － ＰＡ 对ＢＥＬ－ ７４０２ 细胞增殖抑制的ＩＣ５０ 为４ －９ｍ ｍｏｌ／Ｌ,随着作用时间的增加,ＩＣ５０ 增加。作用效果迅速,在０ ．５ 小时就有效地抑制了细胞增殖,抑制率随４－ ＰＡ 浓度的增加和作用时间的延长而增加,在达到最大抑制率后出现饱和。流式细胞仪对细胞周期和细胞凋亡的分析发现：４ － ＰＡ 对细胞增殖期（Ｓ期、Ｇ２ 期） 有较强的抑制和逆转作用,使细胞群截止在Ｇ１ 期。随着作用时间的延长和４ － ＰＡ 浓度的增加,细胞凋亡的比例增加,凋亡细胞多来自于细胞增殖期（Ｓ 期、Ｇ２ 期） 。诱导凋亡和使细胞截止于细胞静息期是４ － ＰＡ 抑制细胞增殖的主要途径。