EGF促进小鼠卵母细胞体外减数分裂启动机制的研究

EGF和孕酮对小鼠卵母细胞减数分裂的重新启动具有促进作用,EGF的作用是通过促进颗粒细胞分泌孕酮实现的。使用孕酮合成关键酶3β-HSD的抑制剂Epostane可抑制EGF促进单层培养卵巢颗粒细胞的孕酮合成,从而降低EGF对卵母细胞的促进作用。Ca~(2 )参与了EGF和孕酮的促减数分裂重新启动作用。肝素可降低两者的作用。EGF和孕酮均可使单个卵丘颗粒细胞内的Ca~(2 )水平出现波动,并且EGF使卵丘细胞维持较高的Ca~(2 )水平。     

肝刺激因子对肝癌细胞增殖的调节作用

初断乳雄性ＳＤ大鼠的肝匀浆以超速离心和柱层析法分离纯化肝刺激因子（ＨＳＳ）,观察其对肝癌细胞增殖、细胞表皮生长因子（ＥＧＦ）受体表达及受体磷酸化的影响。结果表明,ＨＳＳ具有明显的促肝癌细胞分裂增殖能力,提高细胞周期中Ｓ期细胞所占比例。ＨＳＳ促肝癌细胞增殖作用与其促ＥＧＦ受体表达有关,表现为：（１）ＨＳＳ上调７０ｋＤ ＥＧＦ受体蛋白表达,此作用与ＥＧＦ合用后明显加强,即呈协同效应;（２）ＨＳＳ上调Ｅ     

一种紫菜多糖的制备及对淋巴细胞生长的影响

用DEAE－纤维素和SephadexG-200柱层析法分离纯化条斑紫菜的热水提取物,从中得到多糖PY2,并测出其分子量为2．0％10^4。用紫外和红外光谱对PY2的性质进行了鉴定。进一步测定了PY2对体外培养的小鼠骨髓细胞及淋巴细胞生长的影响。结果表明,PY2是一种少见的紫菜多糖,它不含有大多数紫菜侈糖具有的3,6－内醚－兰乳糖和硫酸基,它对小鼠脾脏淋巴细胞、胸腺淋巴细胞以及混合淋巴细胞的增殖有一定的抑制作用,而对骨髓细胞的增殖没有明显的影响。     

表皮生长因子对正常细胞与转化细胞染色质蛋白激酶活性的影响

本文研究了正常C3H/10T1/2CI8细胞(一种源于小鼠胚胎的成纤维细胞,简称NC3H/10)与用~3H-TdR转化的C3H/10T1/2CI8细胞(简称TC3H/10)染色质蛋白激酶(CAPK)的作用物特异性,发现这两种细胞的CAPK均能以外源性酸性酪蛋白为作用物,而不以外源性碱性组蛋白为作用物,由此推测CAPK的天然作用物主要为非组蛋白。观察了表皮生长因子(EGF)对培养细胞的CAPK活性的影响,EGF能使NC_3H/10和TC3H/10的CAPK活性分别增加105.6％(P<0.01)和50.7％(P<0.01),后者增加的幅度仅为前者的1/2,说明转化细胞CAPK活性对EGF刺激的敏感性较正常细胞低。EGF和1-油酰-2-乙酰-消旋-甘油(OAG)对无细胞体系的CAPK活性无直接影响,提示EGF可能通过核内受体或OAG以外的其它第二信使来影响CAPK的活性。     

鹿茸表皮生长因子

作者等通过应用一凝肢柱或DEAE凝胶柱和固定金属离子亲和凝腔柱(Lmmobilised Metal Affinity chromatography Column,IMAC)的组合,成功地从梅花鹿的鹿茸表皮层中分离出表皮生长因子(Epidermal Growth Factor, EGF)。此半纯化的EGF能刺激小鼠Balb/c 3T3的静态成纤维细胞对~3H-胸嘧啶的渗入,渗入量随EGF剂量的增加而升高。在鹿茸表皮层的组织中亦同时含有大量的EGF受体,而骨轴组织不舍有EGF,只含少量受体。     

植物生长激素的细胞凝集效应

凝集素的发现已近100年.最初H.Stillmark发现蓖麻籽提取液可使红细胞凝聚.后来发现不但在植物种子中,而且在植物的其他部位如马铃薯块茎、番茄汁、延命菊叶子、某些葫芦科植物的韧皮部以及病毒、细菌、真菌、无脊椎动物和脊椎动物各类组织如兔肝,小鼠腺胸、牛肺、人肝中都含有凝集素·其中以豆科植物凝集素的含量最丰富,如大豆、刀豆蛋白质中凝集素占1.5—3％. 凝集素能凝集红血球、淋巴细胞、成纤维细胞、癌细胞、精子和细菌,有的还有清除血中糖蛋白,促细胞分裂,免疫和毒性等作用.鉴于豆科植物凝集素含量高、分布面广,并能凝集、识别根瘤菌使其便于侵入根部促进细胞分裂形成根瘤;而2,4-D也能凝集、诱导根瘤菌使其侵入由2,4-D促细胞分裂形成的根瘤原     

肝刺激因子对人肝癌细胞BEL -7402p21~(ras)表达的影响

从雄性初断乳SD大鼠肝匀浆中提取肝刺激因子 (HSS)并加以部分纯化 ,观察其促人肝癌细胞增殖活性及对p2 1ras蛋白表达的影响。结果表明 :(1)HSS具有明显的促人肝癌细胞增殖活性 ,其分子量为 14～ 2 0kD ;(2 )HSS可提高p2 1ras蛋白表达 ,具有时间 效应关系 ,并与EGF呈协同作用 ;(3)HSS调节p2 1ras蛋白表达具有剂量 效应关系 ,且呈现出饱和性。鉴于我们已报道HSS上调EGF受体蛋白和基因表达这一事实 ,本实验结果进一步说明 ,HSS促人肝癌肝细胞增殖与其调节EGF受体介导的信号分子传导过程相关。     

成纤维细胞表皮生长因子的胞吞和向细胞核转移研究

本文以C3H小鼠胚胎正常成纤维细胞株C3H10T1/2C18(简称NC3H10)及其由氚标记脱氧胸苷(~3H-TdR)恶性转化的细胞株(简称TC 3H 10)为对象,研究了表皮生长因子(EGF)在受体介导下的胞吞和向细胞核转移现象。~(125)I-EGF 与细胞膜上的EGF 受体结合后,胞吞和向细胞核转移呈时间依赖性。两种细胞的EGF 的胞吞和向细胞核转移率接近。但是NC 3 H 10细胞~(125)I-EGF 胞知和向细胞核转移的绝对量明显高于TC 3 H 1 0细胞(p<0.05)。SDS-PAGE 电泳表明向细胞核转移的~(125)I-EGF 是未被降解的完整分子,溶酶体抑制剂NH_4Cl 对~(125)I-EGF 向细胞核转移有明显促进作用(p<0.05)。这些结果表明受体介导下的EGF 胞吞和向细胞核转移可能与EGF 的细胞核内凋节作用密切相关。     

尖吻蝮(Agkistrodon acutus)蛇毒的分离及其生物活性的研究

用二乙氨乙基纤维素柱层析法将尖吻蝮蛇毒分成15个组分,并对粗蛇毒及各分离组分进行了各种生物活力的测定,结果表明:1.粗蛇毒中存在有精氨酸酯酶,蛋白水解酶、碱性磷酸酶,磷酸二酯酶,5′—磷酸二酯酶,5′—核苷酸酶,ADP酶、ATP酶,L—氨基酸氧化酶及磷酸酯酶A等10种酶活力;但不存在胆碱酯酶及核糖核酸酶的活力;2.蛋白水解酶,5′—核苷酸酶及ADP酶普遍存在于粗蛇毒经分离后的各组分中;3.精氨酸酯酶存在于组分6—13中,以第8组分的活性最高;4.第1及5—13组分显示有较高的凝血活性,第1—2组分及9—15组分显示有出血毒,第12(小鼠死亡率2/5)及第13组分(小鼠死亡率3/5)显示有较高的毒性;第1—2,9—15组分显示有纤溶活性。     

人重组卵泡刺激素和表皮生长因子对性未成熟小鼠卵母细胞和颗粒细胞复合体体外发育的作用(英文)

卵泡刺激素(FSH)对有腔卵泡和排卵前卵泡的促生长作用已被普遍接受,但关于其对腔前卵泡发育的作用报道结果不尽相同。关于表皮生长因子(EGF)对腔前卵泡的作用尚不确切。本研究目的在于探讨人重组卵泡刺激素(rechFSH)和EGF对早期卵泡发育的作用。利用胶原酶消化法从12日龄的小鼠卵巢中分离得到卵母细胞-颗粒细胞复合体(OGCs)(Fig.1)。体外每孔30~40个培养物并分别添加胎牛血清(FBS)、rechFSH和EGF。培养物每4天测量卵母细胞和OGCs直径,并每天照相。结果显示rechFSH显著促进小鼠OGCs及其卵母细胞的体外发育,这一作用可被EGF进一步增强(p<0.05)(Fig.2)。但到第八天培养结束时,培养后的OGCs卵母细胞要显著小于体内同期生长对照组(p<0.05)(Fig.3)。说明FSH和EGF在卵泡早期发育中起重要作用。     

Evidence that epidermal growth factor is present in swiftlet's (Collocalia) nest

1. An epidermal growth factor (EGF)-like activity was detected and partially purified from swiftlet's nest extract. 2. The partially purified EGF-like activity was able to (a) generate competitive binding curves parallel to the standard curves in radioreceptor assay and (b) stimulate thymidine incorporation in quiescent culture of 3T3 fibroblasts and the latter activity can be suppressed by mouse EGF antibody. 3. Partial characterization of the EGF-like activity in terms of pI, molecular weight and its behavior on gel filtration column suggest that it bears similar physical properties to the EGFs isolated from the mouse and the shrew.     

Factors in the rat submaxillary gland that stimulate growth of cultured glioma cells: identification and partial characterization

The effect of rat submaxillary extract on the growth of rat C6 glioma cells in serum-free culture has been examined. Extracts (10-15 microgram/ml) of submaxillary glands from both male and female rats markedly enhanced the growth of serum-deprived C6 cells and, in combination with insulin, transferrin, and NIH-LH (a source of fibroblast growth factor), were able to stimulate C6 cell growth to an extent comparable to that achieved with an optimal amount of fetal calf serum. The mitogenic activity of rat submaxillary extracts was found to be heat-labile, acid-stable, and partially inactivated by protease and 2-mercaptoethanol. Under our assay conditions, biologically active preparations of purified mouse submaxillary gland epidermal growth factor (EGF) or nerve growth factor (NGF) were not mitogenic for C6 cells, nor was the mitogenic activity of rat submaxillary extracts inhibited by antiserum to these mouse submaxillary gland growth factors. These results suggest that the active component(s) of rat submaxillary extracts is unrelated to either EGF or NGF. The growth-enhancing effect also appears unrelated to esteropeptidase activity present in these extracts since the mitogenic activity was unaffected by several protease inhibitors. Moreover, two purified mouse submaxillary gland arginylesteropeptidases, EGF-binding protein and gamma-subunit of 7 S NGF, were unable to elicit a comparable growth response even when added to cell culture medium at unreasonably high concentrations. The C6 cell mitogenic activity of crude submaxillary extracts could be separated into two biologically similar components by either gel filtration on Sephadex G-100, preparative isoelectric focusing in a pH gradient of 3-10, or adsorption to DEAE-cellulose followed by elution with a sodium chloride gradient. One of the active components was acidic in nature and had an apparent molecular weight of 40,000, while the other was near neutral in charge and possessed a molecular weight of approximately 20,000. The relationship between these two C6 cell mitogenic components and the rat submaxillary gland component responsible for stimulating Balb/c-3T3 cell growth in serum-free, factor supplemented medium (McClure et al., 1979, J. Cell Biol. 83:96a) is also discussed.     

Characterization of polyclonal antibodies that distinguish acidic and basic fibroblast growth factors by using western immunoblotting and enzyme-linked immunosorbent assays

Rabbit polyclonal antibodies were raised against ovalbumin conjugates of purified bovine brain acidic fibroblast growth factor (aFGF) and a synthetic peptide containing the N alpha-terminal 1-24 amino acid sequence of bovine basic fibroblast growth factor (bFGF). These antibodies were used to specifically detect 1-ng quantities of aFGF and bFGF by using enzyme-linked immunosorbent assay (ELISA) and Western immunoblot procedures. Antibodies raised against aFGF recognized bovine brain aFGF and bovine recombinant aFGF but very poorly recognized recombinant bFGF or purified porcine or bovine pituitary bFGF with ELISA and Western immunoblot procedures. Antibodies raised against bFGF (1-24) recognized purified bovine, porcine, and recombinant human bFGF but only very poorly recognized aFGF with ELISA and Western immunoblot procedures. In vitro addition of anti-bFGF antibodies was able to partially neutralize bFGF-stimulated 3H-thymidine incorporation by COMMA-D mouse mammary epithelial cells while having no effect on aFGF or epidermal growth factor (EGF) stimulation. In vitro addition of anti-aFGF antibodies had no effect on bFGF- or EGF-stimulated 3H-thymidine incorporation, but surprisingly, had a potentiating effect on aFGF stimulation. Antibodies against aFGF immobilized on protein A-Sepharose were able to specifically and completely remove mitogenic activity from solutions containing aFGF but had no effect on removal of mitogenic activity from control solutions containing bFGF or EGF. Similarly, immobilized anti-bFGF antibodies completely removed mitogenic activity from solutions of bFGF, but not aFGF or EGF controls. These antibodies have been useful for the identification and characterization of growth factors from tissue and recombinant sources.     

Fibroblast mitogens in swine milk include an epidermal growth factor-related peptide

Mammary secretions obtained from lactating sows can support in vitro growth of mammalian cells when added to Dulbecco's Modified Eagle Medium. In order to identify the growth factors present within, sow milk was fractionated and fractions having mitogenic activity were identified by their ability to stimulate DNA synthesis in density-arrested, quiescent (murine) AKR-2B fibroblasts. A prominent mitogenic activity (peak III) was observed in the 3,000-5,000 Mr range. This activity was partially purified by (1) preparative Sephadex G-200 chromatography, (2) gel-filtration in Sephadex G-50 columns and (3) DEAE-cellulose anion exchange chromatography. The last step resolved peak III activity into at least two distinct components. One component was highly-purified by use of reverse-phase high-performance liquid chromatography (RP-HPLC). This activity was identified as an epidermal growth factor (EGF)-related peptide based on its inactivation by polyclonal antibody (IgG fraction) specific for murine EGF (mEGF) and proteolytic agents. The other component is unrelated to EGF. Using cloned mEGF cDNA as a probe, the presence of EGF-related mRNA in lactating mammary tissues and newborn pig small intestine was also demonstrated. These factors may contribute to the preferential growth of gastrointestinal tissues in neonatal pigs.     

Isolation and partial characterization of canine epidermal growth factor/urogastrone.

Canine epidermal growth factor (EGF)/urogastrone was partially purified from dog urine by fractional precipitation with (NH4)2SO4, ion-exchange chromatography with DEAE-cellulose DE-52, gel filtration with Sephadex G-50, and a second DE-52 chromatography, to yield receptor-competing activity equivalent to 13 micrograms of standard mouse EGF/litre of starting urine. The purification was monitored by a competitive radioreceptor assay using fixed monolayers of A431 cells. The partially purified canine EGF/urogastrone demonstrated a growth-stimulating activity in 3T3 mouse fibroblast cells as potent as mouse EGF. Analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed one major peptide component with an Mr similar to that of mouse EGF, and two minor peptides of slightly higher Mr. The major peptide component was isolated after reduction and its amino acid composition was determined.     

Regulation of growth of LNCaP human prostate tumor cells by growth factors and steroid hormones

The mitogenic activity of several growth factors on androgen responsive LNCaP human prostate tumor cells was studied. A two-fold stimulation of cell proliferation was observed after a culture period of 6 days in 1 ng EGF/ml, 10 ng TGF-alpha/ml or 20 ng basic FGF/ml. TGF-beta (0.02 ng/ml), which did not affect cell proliferation when added alone to the culture medium, inhibited the EGF- and TGF-alpha-induced growth. The synthetic androgen R1881 (0.1 nM) stimulated cell proliferation three-fold and increased the number of EGF receptors from 11500 to 28500 sites/cell. One of the mechanisms involved in androgen action on these cells is therefore an increased EGF receptor expression and increased sensitivity to EGF. TGF-beta did not directly affect androgen-responsive growth but inhibited the synergistic effect of EGF. A considerable expression of TGF alpha (precursors) could be demonstrated on the cells by immunohistochemical staining. However the staining intensity was not affected by androgens. These results make it less likely that androgen-responsive growth is mediated by regulation of secretion of an EGF- or TGF alpha-like activity, which in turn acts in an autocrine manner to stimulate growth. Estrogens, progestagens and antiandrogens do not inhibit androgen responsive growth of LNCaP cells but have striking growth stimulatory effects, increase EGF receptor level and increase acid phosphatase secretion. LNCaP cells contain a modified androgen receptor system with respect to both steroid specificity and antiandrogen sensitivity. It has recently been shown that the stimulatory effects are due to a mutated amino acid in the steroid binding domain of the androgen receptor.     

Identification of a 15,000-molecular-weight form of immunoreactive transforming growth factor alpha in extracts of porcine pituitary

Two different mitogenic activities were identified from extracts of porcine pituitary by using COMMA-D mouse mammary epithelial cells in a serum-free 3H-thymidine incorporation assay. Porcine pituitaries were extracted in phosphate-buffered saline (pH 7.4) and 25-80% (NH4)2SO4 pellets were dialyzed and chromatographed by using DEAE-Sepharose chromatography (pH 8.0), resulting in two peaks (I and II) of mitogenic activity. Peak I represented a recovery of 73% of the units of mitogenic activity present in crude extract of pituitary while only 1.25% of the activity was recovered in peak II. Peak I was further purified by using CM-Sephadex and heparin-Sepharose chromatographies and yielded a mitogen that was able to elicit one-half-maximal stimulation of 3H-thymidine incorporation by COMMA-D cells at 48 pg/ml. As expected with pituitary as the tissue source, peak I was confirmed to be basic fibroblast growth factor (bFGF) by using specific antibodies in enzyme-linked immunosorbent assay and Western immunoblotting procedures. Peak II was further purified by using chromatofocusing (pH 7.3-5.0), reverse-phase, and cation-exchange HPLCs. The mitogenic activity eluted at pH 6.3 from chromatofocusing, migrated as a 13-kDa molecule on gel filtration HPLC, and did not bind to heparin-Sepharose under conditions which bound fibroblast growth factors. The material purified from peak II and rat synthetic transforming growth factor alpha (TGF alpha) competed in a parallel fashion with 125I-epidermal growth factor for receptor binding with A431 human epidermal carcinoma cells. In addition, the mitogen purified from peak II showed a single immunoreactive band migrating at 15 kDa when specific antiserum against TGF alpha was used in a Western immunoblotting procedure. The data suggest that in addition to the well-documented presence of bFGF, normal adult porcine pituitaries contain a 15-kDa form of immunoreactive TGF alpha that binds to EGF receptors and is mitogenic for mammary epithelial cells.     

Brucella abortus lipopolysaccharide is mitogenic for spleen cells of endotoxin-resistant C3H/HeJ mice.

Preparations of lipopolysaccharide (LPS) from rough and smooth strains of Brucella abortus were mitogenic for spleen cells of athymic nude mice, C3H/HeAU mice, and the endotoxin-resistant C3H/Hej mice. The mitogenic response induced by crude smooth-LPS (f5) was greater than that produced by purified smooth-LPS (f5p); however, the dose-response curves were similar for both preparations. The mitogenic activity of mouse spleen cells to both f5 and f5p was higher than that produced by stimulation with purified rough-LPS. The dose-response curves with rough-LPS were also qualitatively different from those produced with the preparations of smooth-LPS.     

Plasminogen activator is an apparent lymphocyte mitogen

Culture fluids of avian sarcoma virus (ASV)-transformed but not normal chicken embryo cells frequently elicited a mitogenic response in normal avian and murine lymphocytes. We examined the possibility that plasminogen activator (PA) might be responsible for the observed mitogenic effect. PA activity, present in culture medium, was correlated positively with lymphocyte mitogenic capacity. Treatment of cells with phorbol myristate acetate, which elevates PA levels, increased mitogenesis. Similar treatment with dexamethasone, which inhibits PA biosynthesis and/or secretion, reduced lymphocyte mitogenic activity. Addition to culture fluids of either benzamidine or diisopropylfluorophosphate, both specific PA inhibitors, blocked lymphocyte proliferative responsiveness to culture fluids. In contrast, neither epsilon-amino-caproic acid nor trasylol, which inhibits plasmin esterase activity but not PA, abrogated lymphocyte responsiveness. Furthermore, purified urokinase, an enzyme of similar substrate specificity to PA, had lymphocyte stimulatory activity. These results strongly suggest that PA can function as a lymphocyte mitogen.