原癌基因c-erbB_2在原始卵泡启动生长中的作用

目的:探讨原癌基因c-erbB2在原始卵泡启动生长中表达变化及可能的作用。方法:选用2日龄SD大鼠卵巢在Waymouth培养体系中进行体外培养,用原位杂交、RT-PCR和免疫组化方法检测c-erbB2mRNA和蛋白在原始卵泡启动生长中及在表皮生长因子(EGF)作用下的表达情况,用Westernblot方法同步测定卵泡活化生长的重要标志物——增殖细胞核抗原(PCNA)和磷酸化细胞外信号调节激酶1/2(p-ERK1/2)的表达情况,并分析p-ERK1/2与c-erbB2mRNA表达变化的相关关系。结果:伴随原始卵泡启动生长过程,PCNA表达逐渐增加,EGF能促进原始卵泡的增殖和分化;原始卵泡中有c-erbB2mRNA及蛋白的表达,且随原始卵泡的启动生长及在EGF作用下表达增强;RT-PCR结果显示,c-erbB2mRNA表达在2日龄大鼠卵巢培养8d后与培养0d相比显著增加(0.297±0.018vs0.178±0.011,P0.05),并在EGF作用下进一步增强;p-ERK1/2含量的变化与c-erbB2mRNA表达的变化呈显著的正相关关系(rs=0.900,P0.05)。结论:c-erbB2在原始卵泡启动生长中起重要促进作用,并为介导EGF促进原始卵泡启动生长的关键信号分子;ERK-MAPK信号通路可能在介导c-erbB2调控原始卵泡生长中起作用。     

胰岛素样生长因子-Ⅰ(IGF-Ⅰ)、IGF结合蛋白-2和LH受体mRNA在卵泡闭锁过程中的表达及调节

研究了IGF-Ⅰ、IGF结合蛋白-2(IGFBP-2)和促黄体激素受体(LHR)mRNA在卵泡闭锁过程中的表达及调节.给26日龄大鼠注射15 IU PMSG,经检测,证实PMSG处理48 h后,一些小窦状卵泡的颗粒细胞已发生凋亡;96 h在排卵前卵泡中已可检测到凋亡细胞;120 h大多数的排卵前卵泡中均出现大量的凋亡细胞.48～120 h IGF-Ⅰ主要在窦前卵泡和小窦状卵泡表达;48与96 h,窦前与窦状卵泡的膜细胞均表达高水平的IGFBP-2.在48 h,颗粒细胞中有LHR的强信号,但在96和120 h,颗粒细胞的LHR表达减弱(P＜0.001).表皮生长因子(EGF)和IGF-Ⅰ均抑制窦前和窦状卵泡颗粒细胞凋亡.同时观察到EGF促进IGF-Ⅰ mRNA表达,IGF-Ⅰ刺激排卵前卵泡表达LHR mRNA.上述结果表明,各级卵泡的闭锁可能均受EGF和IGF-Ⅰ相互作用的调节.     

鸭卵泡及精巢中卵泡抑素和抑制素／活化素βB亚基mRNA表达的研究

卵泡抑素是与抑制素/活化素功能密切相关的一种糖蛋白激素,采用定量竞争RT-PCR技术对鸭各级卵泡及成熟与未成熟精巢中的卵泡抑素和抑制素/活化素βB亚基的mRNA表达丰度进行了研究,发现在上述组织中均有此两基因mRNA的表达,且只在小卵泡中表达较丰富.卵泡抑素在小黄卵泡(SYF)中表达量最高,以其mRNA的平均相对丰度为1.00,则F1(最大卵泡)、F2、F3、F4、F5、F6～8,LWF(大白卵泡)、TI(未成熟精巢)和TM(成熟精巢)中卵泡抑素mRNA的平均相对含量分别为0.0ll±0.004、0.019±0.006、0.02l±0.009、0.028±0.007、0.075±0.023、0.15±0.072、0.29±0.068、0.037±0.0l1和0.012±0.004.βB亚基也在小黄卵泡中表达量最高,以其mRNA的平均相对丰度为1.00,则F1、F2、F3、F4、F5、F6～8、LWF、TI和TM中B亚基mRNA的平均相对含量分别为0.009±0.003、0.013±0.005、0.019±0.007、0.023±0.006、0.29±0.084、0.84±0.093、0.031±0.008、0.38±0.072和0.046±0.013.结果显示卵泡抑素和βB亚基mRNA的表达模式是相似的,二者在小卵泡中均出现大量表达说明活化素B(βB-βB)的生物活性可能受卵泡抑素的紧密调节,二者共同在卵泡早期发育中起重要作用.     

人肝癌细胞表皮生长因子受体以及佛波酯对它的调变

利用放射受体结合测定证明了两株人体肝癌细胞株7402和7721细胞均有EGF受体的存在。受体数目分别为每细胞6.2×10~4和2.5×10~4,代表它们亲和力的解离常数K_D值分别为1.2 nmol/L和0.80 nmol/L。PMA处理对7721细胞的EGF受体及其亲和力均无明显影响;对7402细胞EGF受体却显示了调变作用。受体数目虽同样没有变化;但亲和力却随药物处理时间有一个下降、恢复的变化过程。在PMA处理1小时,EGF结合抑制达最大,此时受体的解离常数K_D值为3.0 nmol/L;在处理96小时,受体亲和力恢复并略有增高,此时K_D值为0.95 nmol/L。~3H-TdR参入实验表明,在PMA抑制7402细胞EGF受体亲和力的同时,细胞DNA的合成速率也相应下降。对于佛波酯等因子对EGF受体的调变作用,我们认为是属于生长因子启动细胞DNA合成进行细胞分裂的整个生物学过程中感受性因子对进行性因子的调节作用。     

催乳素及生长因子对体外培养的鸡卵泡膜细胞增殖的调节作用

目的和方法：本文利用建立的鸡卵泡膜细胞无血清培养,研究了一些激素及生长因子对鸡卵泡膜细胞增殖的调节作用。结果：ＰＲＬ能刺激体外培养的鸡卵泡膜细胞增殖,并具有剂量依从关系,其中１０００ｎｇ／ｍｌ的ＰＲＬ达到最大刺激效果,使膜细胞增殖３５５％;ＬＨ、ＥＧＦ、Ｉｎｓ、ＣＴ和Ｔｆ亦均能刺激鸡卵泡膜细胞增殖,且具有剂量依赖关系。     

利用GFP表达系统检测雌激素类化合物的研究

根据雌激素类化合物的转录调节原理,构建受雌激素应答元件（ERE）调控的3×ERE EGFP-N1重组报告基因载体,转染雌激素受体阳性的MCF-7乳腺癌细胞,分离出稳定转染的细胞克隆ERE-GFP-MCF7,将不同浓度的雌二醇（E2）及其他化合物加入稳定转染的细胞后检测GFP表达水平的变化。雌二醇（E2）以剂量依赖的方式诱导稳定转染细胞ERE-GFP-MCF7表达GFP,最大效应浓度为1×10－10mol/L ,EC50为1.5×10－11 mol/L;已知的植物雌激素大豆甙元和白藜芦醇同样以剂量依赖的方式诱导GFP的表达,EC50分别为2.4×10－7 mol/L和6.2×10－6 mol/L,而葡萄籽多酚没有明显的雌激素样活性。雌激素拮抗剂他莫西芬以剂量依赖的方式抑制雌二醇诱导GFP的表达,最大抑制浓度为1×10－7 mol/L。利用此细胞模型检测不同化合物诱导的GFP表达强度可快速检测雌激素受体的配体。     

转基因高表达卵泡抑素1对斑马鱼肌肉生长促进作用研究

抑肌素是转化生长因子β超家族成员,其主要功能是对肌肉的生长发育起负调控作用.在哺乳动物中,抑肌素信号是通过其特异性的与激活素受体ⅡB亚基结合而发挥作用.因此,其信号传递作用可以被其结合蛋白以及ActR ⅡB的结合蛋白卵泡抑素所抑制.在小鼠肌肉组织中,高表达卵泡抑素可以导致肌肉组织肌纤维的增生和肥大.为了研究鱼类卵泡抑素...     

EGF在新生大鼠原始卵泡生长启动中的作用

以灵敏的增殖细胞核抗原(PCNA)为指标,研究了表皮生长因子(EGF)和FSH对新生大鼠原始卵泡生长启动的作用.结果表明,注射EGF两天,卵巢中有较多的卵泡颗粒细胞(GC)开始增殖由扁平变为立方形,到第4天时GC的增殖状况和层数更加明显;而FSH对卵泡启动在早期无明显作用,直到第7天时才有明显结果.原位杂交显示,抑制素αmRNA从出生后第5天开始表达,FSH受体(FSHR)从第6天开始表达,随后逐渐增强.提示EGF而不是FSH对原始卵泡的生长启动可能起一定的作用,抑制素也可能参与卵泡的早期发育过程.     

血清对全反式视黄酸抑制肺癌细胞生长的影响

 研究不同浓度的血清对全反式视黄酸 (ATRA)抑制肺癌细胞生长的影响 .当细胞培养在 10 %血清中 ,ATRA不能抑制肺癌细胞生长 ,但是当细胞培养在 1%血清中 ,ATRA能够有效地抑制肺癌细胞生长 .视黄酸受体RARβ介导视黄酸的抗癌作用 .Northern印迹分析表明 ,在高浓度血清中AT RA不能诱导RARβ表达 ,但在低浓度血清中ATRA可以诱导RARβ表达 ,并且瞬时转染和CAT测定证实是通过激活RABβ启动子转录活性而诱导RARβ表达的 .孤生受体Nur77受到血清生长因子刺激后会大量表达 ,具有抗视黄酸活性的作用 .肺癌细胞培养在低浓度血清中 ,Nur77mRNA低水平表达和Nur77蛋白不表达 .然而在高浓度血清中 ,Nur77mRNA和蛋白高水平表达 .另外 ,在无血清条件下 ,EGF也可以诱导Nur77表达 .结果提示 ,血清中的生长因子可能拮抗ATRA抑制肺癌细胞生长的作用 ,其作用途径可能是通过刺激细胞中Nur77表达 ,或者通过下调RARβ启动子的转录活性而抑制RARβ的表达     

利用FRET技术在活细胞内观察EGF对PKA作用的时空成像

cAMP依赖的蛋白激酶(protein kinase A,PKA)在细胞生长与分化过程中扮演重要角色,特别是在调节Ras信号通路引起的细胞增殖效应中起着重要作用。为了在活细胞内动态观察表皮生长因子(epidermal growth factor,EGF)对PKA的作用,采用一种可以检测PKA酶活性的报告蛋白(A-kinase activity reporter,AKAR)——这种报告蛋白是利用荧光共振能量转移(fluorescence resonance energy transfer,FRET)原理设计的,使其在人类肺癌细胞(ASTC-a-1)中稳定表达。加入EGF刺激因子后,随时间变化的成像分析显示出在活细胞生理条件下被EGF作用的PKA酶活性变化的时空信息。这些资料为EGF作用PKA提供了直接的实时证据。     

Activin-A up-regulates type I activin receptor mRNA levels in human immortalized extravillous trophoblast cells.

Activin is known to play an important regulatory role in reproduction, including pregnancy. To further examine the role and signaling mechanism of activin in regulating placental function, the steady-state level of activin type I receptor (ActRI) mRNA in immortalized extravillous trophoblasts (IEVT) cells was measured using competitive PCR (cPCR). An internal standard of ActRI cDNA for cPCR was constructed for the quantification of ActRI mRNA levels in IEVT cells. ActRI mRNA levels were increased in a dose-dependent manner by activin-A with the maximal effect observed at the dose of 10 ng/ml. Time course studies revealed that activin-A had maximal effects on ActRI mRNA levels at 6 hours after treatment. The effects of activin-A on ActRI mRNA levels was blocked by follistatin, an activin binding protein, in a dose-dependent manner. In addition, inhibin-A inhibited basal, as well as activin-A-induced ActRI mRNA levels. These findings provide evidence, for the first time, that activin-A modulates ActRI mRNA levels in human trophoblast cells.     

Modulation of transforming growth factor alpha-dependent expression of epidermal growth factor receptor gene by transforming growth factor beta, triiodothyronine, and retinoic acid

We have investigated the actions of transforming growth factor (TGF) type alpha on epidermal growth factor (EGF) receptor mRNA expression in MDA-468 human mammary carcinoma cells in serum-free media. We found that exposure of MDA-468 cells to TGF alpha results in elevated levels of EGF receptor mRNA. This increase in mRNA accumulation showed time and dose dependence. Addition of TGF beta 1 enhanced the accumulation of EGF receptor mRNA induced by TGF alpha in a time- and dose-dependent manner. We also found that triiodothyronine at physiological concentrations exerts synergistic control on the action of TGF alpha alone, or in association with TGF beta 1, on EGF receptor mRNA expression. Similarly, retinoic acid treatment also enhanced in a time- and dose-dependent manner the TGF alpha-dependent response of EGF receptor mRNA and acted synergistically with TGF beta 1. The results described here suggest that optimum regulation of EGF receptor gene expression by TGF alpha is a complex process involving synergistic interactions with heterologous growth factors and hormones.     

AngⅡ和PKC对心肌细胞AngⅡ 1型受体的转录调节

利用体外培养的心肌细胞,观察血管紧张素Ⅱ（ＡｎｇⅡ）和蛋白激酶Ｃ（ＰＫＣ）在诱导ＡｎｇⅡ１型受体（ＡＴ１）基因表达及蛋白质代谢中的作用．研究结果表明：ＡｎｇⅡ可诱导ＡＴ１ｍＲＮＡ水平一过性下调,呈时间及剂量依赖性,１０ｎｍｏｌ／ＬＡｎｇⅡ刺激细胞６ｈ,引起ＡＴ１ｍＲＮＡ水平降低幅度最大,降至对照的５１．６％±９．５％,然后逐渐回升,２４ｈ恢复至对照水平．３０μｍｏｌ／ＬＨ－７（ＰＫＣ抑制剂）能阻断ＡｎｇⅡ诱导的ＡＴ１ｍＲＮＡ水平的下调．０．３μｍｏｌ／Ｌ的ＰＭＡ（ＰＫＣ激活剂）单独应用可诱导ＡＴ１ｍＲＮＡ水平下调达对照的４３％±８％,加入ＡＴ１拮抗剂ＤＭＰ８１１及Ｄｕｐ７５３均可阻断ＡｎｇⅡ诱导的ＡＴ１ｍＲＮＡ水平的下调．１０ｎｍｏｌ／Ｌ的ＡｎｇⅡ刺激心肌细胞９６ｈ可使蛋白含量降低至对照的７３．４％±５．６％,而加药持续刺激１４４ｈ可使蛋白含量较对照增加３３．８％±６．３％,Ｈ－７不能阻断ＡｎｇⅡ诱导的蛋白含量降低,但可有效地抑制蛋白含量的增加．以上结果提示：ＡｎｇⅡ对心肌细胞ＡＴ１基因的转录和细胞的蛋白代谢有调节作用,而ＰＫＣ则参与了ＡｎｇⅡ的这种调节作用     

促性腺激素释放激素及多巴胺对斜带石斑鱼生长激素分泌及其mRNA表达的调控

研究斜带石斑鱼生长激素分泌及其mRNA表达的调控规律对于性别分化的控制、临床药物的选择,以及石斑鱼的增养殖等均具有重要的理论意义和实践意义。本文应用静态孵育系统,采用放射免疫测定法和化学发光液相杂交实验,研究GnRH和DA对斜带石斑鱼GH分泌、GHmRNA合成的调控作用。100nmol／LsGnRH作用斜带石斑鱼脑垂体碎片1也4h,明显促进GH的释放和GHmRNA的合成,并具有时间依存性;10nmol／L～1μmol／LsGnRH作用1h能明显促进斜带石斑鱼脑垂体释放GH,促进GHmRNA的合成,表现出明显的剂量效应。100nmol／L、1μmol／LmGnRH作用1h以一定的剂量依存方式促进GH的释放、促进GHmRNA的合成,但mGnRH的效应比相应剂量的sGnRH的作用弱。APO为DA受体的非选择性激动剂,不同剂量APO对斜带石斑鱼脑垂体碎片的作用结果显示,10nmol／L-1μmol／L APO以剂量依存方式促进斜带石斑鱼脑垂体碎片释放GH、促进GHmRNA的合成：1μmol／LAPO作用12h以上明显促进GH的释放和GHmRNA的合成,并随时间的延长而增加。与sGnRH对斜带石斑鱼GH释放、GHmRNA合成的作用相比,APO的作用较弱。本文研究结果证实GnRH和DA能促进斜带石斑鱼脑垂体GH释放和GHmRNA合成。     

The effect of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) on granulocyte macrophage-colony stimulating factor (GM-CSF) production by neuronal precursor cells

AIMS: To determine whether granulocyte macrophage-colony stimulating factor (GM-CSF) production by neuronal precursor (NT2) cells can be regulated by IL-1beta and TNF-alpha. BACKGROUND: We have previously demonstrated GM-CSF expression by neurons of the developing human brain, as well as by NT2 cells. IL-1beta and TNF-alpha upregulate GM-CSF production in glial cells, but GM-CSF regulation in neurons is as yet undefined. We hypothesized that IL-1beta and TNF-alpha would increase GM-CSF mRNA and protein production in NT2 cells. METHODS: The effect of IL-1beta and TNF-alpha on GM-CSF production was assessed by dose response (0 to 2,000 U/ml), and time course (0 to 48 hours incubation) experiments. GM-CSF mRNA and protein production were assessed by quantitative RT-PCR and by ELISA. The effect of these cytokines on cell turnover was determined by BrdU incorporation. RESULTS: IL-1beta increased GM-CSF mRNA and protein expression by NT2 cells. This effect was time and dose dependent, and the effective dose ranging from (20-200 U/ml). TNF-alpha increased GM-CSF mRNA expression to a lesser extent than did IL-1beta (maximal stimulation at 200 U/ml), and a minimal increase in net protein accumulation was noted. Neither cytokine increased NT2 cell turnover. CONCLUSIONS: IL-1beta and TNF-alpha both increase GM-CSF mRNA expression by NT2 cells, but only IL-1beta increases net GM-CSF protein accumulation.     

Modulation of oncogene expression by epidermal growth factor and gamma-interferon in A431 squamous carcinoma cells

Epidermal growth factor (EGF) acts, in a dose dependent manner, as both a mitogen and an inhibitor of growth of the A431 squamous carcinoma cell line. gamma-interferon (IFN) also inhibits A431 cell growth. The dual effects of EGF on A431 growth and expression of the oncogenes, EGF receptor (EGFR) and Ha-ras, were evaluated with or without gamma-IFN. A mitogenic level (10pM) of EGF had no effect on expression of EGFR 10 kb mRNA or protein. gamma-IFN combined with 10pM EGF caused an initial drop in EGFR mRNA not reflected at the protein level; at 72 hours, the level of EGFR 10kb mRNA rose and inhibition of cell growth was observed. Treatment with a cytostatic amount (10nM) of EGF resulted in decreased expression of EGFR 10kb mRNA and protein within 24 hours; combined treatment with gamma-IFN caused rapid cell death. Expression of Ha-ras mRNA paralleled that of EGFR mRNA upon treatment with 10pM EGF and/or gamma-IFN, but differed with 10nM EGF.     

The role of peroxisome proliferator-activated receptor-beta/delta in epidermal growth factor-induced HaCaT cell proliferation

Epidermal growth factor (EGF) has been shown to be a potent mitogen for epidermal cells both in vitro and in vivo, thus contributing to the development of an organism. It has recently become clear that peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) expression and activation is involved in the cell proliferation. However, little is known about the role of PPARβ/δ in EGF-induced proliferation of HaCaT keratinocytes. In this study, HaCaT cells were cultured in the presence and absence of EGF and we identified that EGF induced an increase of PPARβ/δ mRNA and protein level expression in time-dependent and dose-dependent manner, and AG1487, an EGF receptor (EGFR) special inhibitor, caused attenuation of PPARβ/δ protein expression. Electrophoretic mobility shift assay (EMSA) revealed that EGF significantly increased PPARβ/δ binding activity in HaCaT keratinocytes. Antisense phosphorothioate oligonucleotides (asODNs) against PPARβ/δ caused selectively inhibition of PPARβ/δ protein content induced by EGF and significantly attenuated EGF-mediated cell proliferation. Treatment of the cells with L165041, a specific synthetic ligand for PPARβ/δ, significantly enhanced EGF-mediated cell proliferation. Finally, c-Jun ablation inhibited PPARβ/δ up-regulation induced by EGF, and chromatin immunoprecipitation (ChIP) showed that c-Jun bound to the PPARβ/δ promoter and the binding increased in EGF-stimulated cells. These results demonstrate that EGF induces PPARβ/δ expression in a c-Jun-dependent manner and PPARβ/δ plays a vital role in EGF-stimulated proliferation of HaCaT cells.     

黄芪甲苷对人骨髓间充质干细胞体外增殖及细胞因子表达的影响

目的：探讨黄芪甲苷对人骨髓间充质干细胞体外增殖及细胞因子表达的影响。方法：采用Percoll密度离心法和贴壁法分离纯化hBMSC;流式细胞术检测hBMSC表面标志;MTT法检测不同浓度黄芪甲苷干预72h后hBMSC的增殖情况;Real-timePCR法检测经黄芪甲苷干预后hBMSC对SCF、VEGF、SDF-1、GM-CSFmRNA的表达水平。结果：成功分离培养出laBMSC;不同浓度（20、40、80、160、320mg／mL）的黄芪甲苷可促进hBMSC增殖（P〈0．05）,其中160mg／mL组促增殖最明显;黄芪甲苷可促进hBMSC对SCF、VEGF、SDF-1mRNA的表达,而GM—CSFmRNA表达无明显变化。结论：黄芪甲苷可促进hBMSC体外增殖,可能与其促进hBMSC对SCF、VEGF、SDF-1mRNA表达有关。     

Regulation of the expression of type 1 plasminogen activator inhibitor in Hep G2 cells by epidermal growth factor

To identify factors potentially influencing expression of type 1 plasminogen activator inhibitor (PAI-1), we characterized the human tissue-specific distribution of PAI-1 mRNA and the influence of epidermal growth factor (EGF) on expression of steady state levels of PAI-1 mRNA and secretion of PAI-1 by Hep G2 cells. Two species of PAI-1 mRNA (3.2 and 2.2 kilobases) were detected, and the ratio of the two varied among tissues (3 to 5:1) in contrast to the 1:1 ratio detected in Hep G2 cells. Expression of PAI-1 mRNA was inversely related to the distribution of tissue-type plasminogen activator mRNA (2.3 kilobases). Nu-Serum, a growth media supplement, increased steady state levels of PAI-1 mRNA 5-fold within 3 h. Factors responsible were found to be trypsin-sensitive and dialysis-resistant. Antisera to EGF attenuated Nu-Serum-induced increases of PAI-1 mRNA by 57%, suggesting that EGF or EGF homologous peptides contributed to the response. EGF elicited increases of PAI-1 mRNA levels in a dose-dependent manner. Induction was rapid (7-fold at 3 h with 5 ng/ml) and complete within 10 h. The response was not attenuated by cycloheximide (25 micrograms/ml). Factor X and glyceraldehyde-3-phosphate dehydrogenase mRNA did not increase. Increased levels of PAI-1 antigen were detected in conditioned media of Hep G2 cells by 4 h and were maximal at 8 h (6-fold). We conclude that the expression of PAI-1 mRNA is tissue-specific and regulated by epidermal growth factor in Hep G2 cells.     

黄芪甲苷对人骨髓间充质干细胞体外增殖 及细胞因子表达的影响*

摘要目的：探讨黄芪甲苷对人骨髓间充质干细胞体外增殖及细胞因子表达的影响。方法：采用Percoll 密度离心法和贴壁法分离 纯化hBMSC;流式细胞术检测hBMSC 表面标志;MTT 法检测不同浓度黄芪甲苷干预72 h后hBMSC 的增殖情况;Real-time PCR 法检测经黄芪甲苷干预后hBMSC对SCF、VEGF、SDF-1、GM-CSF mRNA 的表达水平。结果：成功分离培养出hBMSC;不同 浓度（20、40、80、160、320 mg / mL）的黄芪甲苷可促进hBMSC增殖(P<0.05),其中160 mg/mL 组促增殖最明显;黄芪甲苷可促进 hBMSC对SCF、VEGF、SDF-1 mRNA的表达,而GM-CSF mRNA 表达无明显变化。结论：黄芪甲苷可促进hBMSC 体外增殖,可 能与其促进hBMSC 对SCF、VEGF、SDF-1 mRNA表达有关。