雌激素对兔子宫内膜细胞EGF受体和细胞周期的调控

 由受体放射配基结合分析证明家兔子宫内膜细胞的EGF受体Kd值为0.53nmol/L,每个细胞的最大结合容量为1.11×10~4结合位点。10~(-10)mol/L雌二醇处理24h,细胞的最大结合容量增至2.75×10~4结合位点数/细胞,而Kd值无明显变化,可是,当10~(-5)mol/L雌二醇处理24h,细胞的EGF受体结合率,DNA合成速度率均下降。G_0/G_1期细胞比值明显下降,而G_2+M期和S期细胞明显上升。     

甲状旁腺素、表皮生长因子及维生素A酸对UMR106细胞EGF受体的调节作用

本文研究了EGF、PTH和RA对UMR106细胞EGF受体的调节作用。结果显示PTH能上调EGF的受体,UMR106细胞经bPTH(1-34)处理3天,EGF受体的相对结合率与对照比较提高了40.3％,每个细胞的EGF受体数目从7.22×10~3增加到1.44×10~4,Kd从2.02×10~(-11)增加到3.68×10~(-11)mol/L。而RA则能下调EGF受体,以RA处理3天,EGF受体数目从7.22×10~3下降到4.28×10~3,Kd则从2.02×10~(-11)增加到4.17×10~(-11)mol/L。提示PTH和RA可能通过调变其EGF受体而分别起到正性和负性生长调节作用。     

大鼠、小鼠胰蛋白酶的分离纯化及动力学性质

采用STI-Sepharose 4B亲和层析的方法,从鼠新鲜胰脏中分离得到纯的胰蛋白酶。大鼠胰蛋白酶的比活为24 615BAEEU/mg蛋白,总活性回收率47％,小鼠胰蛋白酶的比活为32 768BAEEU/mg蛋白,总活性回收率55％。经SDS-聚丙烯酰胺凝胶电泳鉴定,大鼠、小鼠胰蛋白酶均呈现单一蛋白带,两者的分子量都是24kD。用等电聚焦电泳测定,二者的等电点均为p19.5以上。对它们的动力学性质作了研究,大鼠胰蛋白酶的Km值为2.33×10~(-4)mol/L,K,值为0.92×10~(-5)mol/L,小鼠胰蛋白酶的Km值为5.60×10~(-4)mol/L,K:值为1.27×10~(-5)mol/L。     

玉米赤霉烯酮的放射免疫分析

将玉米赤霉烯酮转变成玉米赤霉烯酮-6’-羧甲氧肟,通过混合酸酐法将其与牛血清白蛋白结合并用以免疫兔获得抗体。抗体效价可达1:4×10~4,亲合常数为4.25×10~(10)L/mol,灵敏度提高为3.5pg。样品平均回收率达92％。批内与批间变异系数分别为6.1％和8.6％。     

顺铂与小鼠艾氏腹水肝癌细胞膜的相互作用

本文研究了顺铂对小鼠艾氏腹水肝癌细胞膜蛋白内源性荧光的淬灭作用和测定了其在膜上的结合量。结果表明顺铂能与癌细胞膜结合。按存在两类结合部位,得到表观结合常数和结合部位数为: K_1=1.35×10~5L/mol n_1=6.80×10~(-4)mol/g(protein) K_2=2.50×10~3L/mol n_2=1.92×10~(-3)mol/g(protein)     

阿魏酸在缺氧条件下促进人脐静脉内皮细胞血管形成的实验研究

目的:研究阿魏酸(ferulic acid,FA)在缺氧条件下对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)增殖、迁移和管腔样结构形成的影响。方法:原代培养人脐静脉内皮细胞,在缺氧实验条件下,细胞被分为7组,即1个对照组和6个实验组。对照组采用1%酒精处理,实验组用不同浓度(1×10~(-8)、1×10~(-7)、1×10~(-6)、1×10~(-5)、1×10~(-4)及1×10~(-3) mol/L)的阿魏酸处理。分别采用MTS法、划痕法、Matrigel法分析不同浓度阿魏酸处理对人脐静脉内皮细胞的增殖、迁移和管腔样结构形成的影响。结果:缺氧条件下,浓度为1×10~(-6)~1×10~(-4)mol/L的阿魏酸处理能明显促进HUVECs的增殖(P0.05),以1×10~(-5) mol/L处理的效果最好(P0.01);与对照组相比,1×10~(-6)mol/L(P0.05)、1×10~(-5) mol/L(P0.01)及1×10~(-4) mol/L(P0.01)阿魏酸处理均能明显促进HUVECs横向迁移,以1×10~(-5) mol/L处理迁移的细胞数量最多;1×10~(-8)~1×10~(-4) mol/L阿魏酸处理能不同程度地促进HUVECs管腔样结构的形成,以1×10~(-5) mol/L处理形成管腔样结构的数量最多(P0.01)。结论:阿魏酸在缺氧条件下能促进人脐静脉内皮细胞的增殖、迁移和管腔样结构形成。     

大黄蒽醌衍生物对酪氨酸酶的抑制作用

大黄素对酪氨酸酶有显著的竞争性抑制作用,K_i值为1.51×10~(-4)mol,50％抑制的药物浓度为36.6μg/ml;大黄酸的抑制作用较弱,芦荟大黄素几乎无抑制作用。氯化铜(3.3×10~(-7)mol/L)、半胱氨酸(3.3×10~(-7)mol/L)和牛血清白蛋白(1.0mg/ml)对大黄素抑制酪氨酸酶有较强的拮抗作用,恢复率分别为60.0％、45.7％和61.1％。大黄素能与牛血清白蛋白非特异性结合形成复合物,引起吸收光谱红移55毫微米。大黄蒽醌衍生物对酪氨酸酶的抑制作用可能是大黄抗黑色素瘤的作用机制之一。     

植物及动物钙调素抗体免疫反应特性的比较研究

用酶联免疫吸附测定和胶体金免疫电镜定位技术对三种钙调素抗体与植物和动物钙调素的免疫反应特性进行了比较研究。结果表明,在与小麦钙调素的相对亲和力中,抗小麦钙调素抗体大于抗 DNP 修饰猪脑钙调素抗体,抗牛脑钙调素抗体则很弱。抗小麦钙调素抗体与小麦钙调素的 K_D(解离常数)值为2.50×10~(-9)mol/L;抗 DNP 修饰猪脑钙调素抗体与小麦钙调素的 K_D 值为2.82×10~(-8)mol/L,而它与牛脑钙调素的 K_D 值为1.90×10~(-)mol/L。定位玉米根尖细胞钙调素,抗小麦钙调素抗体比抗 DNP 修饰猪脑钙调素抗体有更高的标记密度.定量小麦钙调素,抗小麦钙调素抗体比抗 DNP 修饰猪脑钙调素抗体有较高的检测灵敏度。     

表皮生长因子和前列腺素E1对小鼠精原细胞增殖的促进作用

利用生殖细胞-体细胞无血清共培养模型研究了表皮生长因(EGF)和前列腺素E1(PGE1)对小鼠A型精原细胞增殖的影响.A型精原细胞在ITS培养液(添加胰岛素、转铁蛋白和亚硒酸钠的DMEM)中培养24 h后进行c-kit、EGF、表皮生长因子受体(EGFR)、环氧化酶-1(COX-1)及环氧化酶-2(COX-2)的免疫细胞化学检测,72 h后测定其形成集落教的情况.结果显示,A型精原细胞呈c-kit阳性,EGF、EGFR、COX-1及COX-2主要表达于精原细胞.EGF(10-7～10-6mol/L)或PGE,(10-8.10一mol/I_.)均可显著促进精原细胞集落的形成.此外,前列腺素(PG)受体拮抗剂SCl9220(10-6～10-5mol/L)可抑制PGE1对精原细胞的促增殖作用,COX-1抑制剂SC560(10-7～10-5mol/L)和COX-2抑制剂NS398(10-7～10-5mol/L)能抑制EGF促进精原细胞增殖的作用.因此,EGF可通过促进局部PG的产生而刺激精原细胞的增殖.     

吗啡和血管紧张素Ⅱ对大鼠脑突触小体Ca~(2 )摄取的拮抗效应

行为实验已多次证明,脑室注射血管紧张素Ⅱ(AⅡ)可以对抗吗啡的镇痛作用,但机制不明。吗啡阻止神经末梢钙摄取被认为是其镇痛的机理之一,因此本工作研究了AⅡ和吗啡对大鼠脑突触小体~(45)Ca摄取的作用及相互关系。结果表明,吗啡(10~(-8)—10~(-6)mol/L)对~(45)Ca摄取有明显的抑制作用,10~(-7)mol/L时抑制41％(P<0.001),该效应可被吗啡受体阻断剂纳洛酮(10~(-6)mol/L)完全翻转。与吗啡的作用相反,AⅡ(10~(-8)—110~(-6)mol/L)可促进突触小体对~(45)Ca的摄取,10~(-7)mol/L时增加75％(P<0.001),该效应可被AⅡ受体阻断剂Saralasin(10~(-6)mol/L)完全翻转。将不同剂量的AⅡ(10~(-8)—10~(-6)mol/L)和10~(-8)mol/L吗啡与突触小体共同孵育,则吗啡抑制~(45)Ca摄取的作用被完全翻转。以上结果表明,AⅡ促进脑突触小体Ca~(2 )摄取,对抗了吗啡抑制Ca~(2 )摄取的作用,可能是AⅡ抗吗啡镇痛的机制之一。     

TGFα、EGF对绵羊卵母细胞成熟的影响

采用地衣红染色和免疫荧光的方法,观察了培养在基础培养液加BSA、血清、BSA EGF和BSA TGFα四组成熟培养液中绵羊卵母细胞的核成熟状态和α-微管蛋白分布,以及成熟培养后皮质颗粒(CG)的分布情况。结果表明培养22h的上述各组卵母细胞的核成熟率分别为63.5%、75.2%、73.1%、69.8%,处于第一次减数分裂末期的比率分别为27.0%、16.3%、15.9%、16.9%,EGF、TGFα和血清的添加明显提高了核的成熟率(P<0.05),显著减少了处于第一减数分裂末期的比例(P<0.05);α-微管蛋白的正常率(66.6%、66.6%、73.6%)也显著高于BSA组(43.3%)(P<0.05);CG发生迁移较好的卵母细胞比率分别为33.9%、58.8%、54.7%、47.9%,与BSA组相比,EGF和血清的添加明显促进了CG向皮质区的迁移(P<0.05)。实验表明TGFα和EGF均促进了绵羊卵母细胞成熟过程中从第一减数分裂末期向第二减数分裂中期的转变,并且能够替代血清中的某些成分促进和改善体外成熟卵母细胞核成熟的质量,EGF比TGFα更能促进绵羊卵母细胞胞质的成熟。     

The chemical synthesis and binding affinity to the EGF receptor of the EGF-like domain of heparin-binding EGF-like growth factor (HB-EGF).

Heparin-binding EGF-like growth factor (HB-EGF), which belongs to the EGF-family of growth factors, was isolated from the conditioned medium of macrophage-like cells. To investigate the effect of N- and C-terminal residues of the EGF-like domain of HB-EGF in the binding affinity to the EGF receptor on A431 cell. We synthesized HB-EGF(44-86) corresponding to the EGF-like domain of HB-EGF and its N- or C-terminal truncated peptides. Thermolytic digestion demonstrated three disulfide bond pairings of the EGF-like domain in HB-EGF is consistent with that of human-EGF and human-TGF-alpha. HB-EGF(44-86) showed high binding affinity to EGF-receptor, like human-EGF. The truncation of the C-terminal Leu86 residue from HB-EGF(44-86), HB-EGF(45-86) or HB-EGF(46-86) caused a drastic reduction in the binding affinity to the EGF receptor. These results suggest that the EGF-like domain of HB-EGF plays an important role in the binding to the EGF receptor, and its C-terminal Leu86 residue is necessary for binding with the EGF-receptor. In addition, the deletion of the two N-terminal residues (Asp44-Pro45) from HB-EGF(44-86) caused a 10-fold decrease in relative binding affinity to the EGF receptor. This indicates that the two N-terminal residues of the EGF-like domain of HB-EGF are necessary for its optimal binding affinity to the EGF receptor.     

雌激素拮抗剂对胎盘EGF受体及其基因表达的影响

中期孕鼠在他莫昔芬作用下,其颌下腺,血清中ＥＧＦ含量下降,胎盘中ＥＧＦ受体结合位点数下降以及它的ｍＲＮＡ表达受到抑制,再次证实了他莫昔芬抑制雌激素诱导ＥＧＦ受体ｍＲＮＡ的表达。从而使ＥＧＦ受体结合位点数减少,因此,他莫昔芬对孕鼠胚胎生长发育有不可忽视的影响。     

Presence and localization of epidermal growth factor (EGF)- and EGF-receptor-like immunoreactivity in Tetrahymena

The presence of EGF- and EGF-receptor-like immunoreactivity in Tetrahymena was studied with the help of FITC-labelled monoclonal antibodies, using flow cytometry and confocal microscopy. Tetrahymena has endogeneous EGF and treatment with it significantly increases the hormone content of the cells. The hormone is diffusely localized, particularly in the cytopharynx, where it forms a structure larger than 10 microm. EGF-receptors are also demonstrable, particularly in the cortical region in connection with cilia, and EGF treatment significantly enhances cortical fluorescence. The relationships between these observations and literary data on the effects of EGF in Tetrahymena are discussed.     

Ecdysteroids in adult males and females of Drosophila melanogaster

Ecdysteroid titres in whole flies and different tissues of adult male and female Drosophila were determined at various times after eclosion using a radioimmunoassay. The ecdysteroid titre decreased as the flies matured after eclosion. The differences in titre between males and females can be accounted for by their difference in body weight. The ecdysteroids were found to be distributed throughout several tissues. At eclosion not all of the ecdysteroid complement present could be accounted for by that found localised in tissues. After maturation of the flies the ecdysteroids in various tissues can account for the majority of that detected in whole-fly extracts. Ecdysteroids were produced during in vitro culture of various tissues, but the quantities detected were low by comparison with ring glands of wandering 3rd-instar larvae. Neither the ovaries nor the abdominal body walls (fat body) seem to be a major source of hormone, and they are only able to convert minute quantities of ecdysone to the biologically active form, 20-hydroxyecdysone, in vitro. The amounts of 20-hydroxyecdysone present were measured using high performance liquid chromatography and radioimmunoassay. We tentatively suggest that the differential experession of the yolk-protein-genes in the fat bodies of males and females does not result from differences in hormone titres between them.     

Effect of epidermal growth factor on preimplantation development and its receptor expression in porcine embryos.

The present study aimed to determine the influence of exogenous epidermal growth factor (EGF) on in vitro preimplantation porcine embryo development and its mRNA expression for EGF receptor (EGFR). Oocytes were aspirated from abattoir ovaries, selected and cultured in defined, protein-free media for 44 hr before in vitro fertilization (IVF). Thirty-six hours after IVF, two-cell stage embryos were selected and treated or cultured until embryo treatment. In experiment 1, compact morulae were selected on day 4 after IVF and randomly allocated into 5 groups: NCSU 23 with PVA as group 1; NCSU 23 with PVA and 0.1 ng/ml, 1.0 ng/ml, 10.0 ng/ml EGF as group 2, 3, 4, respectively; NSCU 23 with 0.4% BSA as group 5. In experiment 2, treatment groups were the same as in experiment 1 except that 0.1% crystallized BSA was added to both washing media and all treatment groups instead of PVA. In experiments 3 and 4, two-cell stage embryos were treated and cultured in the same experimental design as experiments 1 and 2, respectively. RT-PCR was used to detect the mRNA expression of EGF receptor in compact morulae and blastocysts. The PCR products were subjected to direct DNA sequencing. There was no significant improvement in the development rate of embryos from compact morulae to blastocysts in the presence of various EGF concentrations (0.1, 1.0, 10.0 ng/ml) versus without EGF addition. They were all significantly lower than those embryos cultured in the continuous presence of 0.4% BSA. However, when a reduced concentration (0.1%) of crystallized BSA was added to all the treatment groups, a significantly lower rate of embryo development was observed in control media (NCSU23 with 0.1% crystallized BSA) compared with those developed in culture media with 0.4% BSA. With the addition of EGF at 10 ng/ml (with 0.1% BSA), embryo development rates were significantly improved over the control group (P < 0.05) and were as good as those rates in 0.4% BSA culture group. When embryos were selected and treated from the 2-cell stage, they did not develop to blastocyst stages after five more days' culture without any protein (BSA) or growth factor addition. When 0.1% BSA was included in the media, blastocyst formation rates were significantly improved by EGF addition at the concentration of both 1.0 or 10 ng/ml (P < 0.05) as compared to 0.0 or 0.1 ng/ml. EGFR mRNA was detected in both compact morulae and blastocyst stages of porcine embryos and confirmed by direct DNA sequencing. Our results indicate that IVM-IVF porcine embryo developmental rates could be improved by the addition of EGF in the culture media with the presence of a reduced amount of defined BSA (>97% albumin). However, EGF alone was not able to elicit any stimulatory effects on embryo development in the absence of protein supplementation. Further studies are needed to investigate the potential synergistic factors in embryo culture media to eventually define the porcine embryo culture media.     

恶性转化的成纤维细胞表皮生长因子受体的研究

本文用受体的放射性配基结合分析方法观察了C_3H小鼠胚胎成纤维细胞C_3H_(10)T1/2 CL8(简称NC_3H_(10))和~3H-TdR恶性转化的C_3H_(10)T1/2CL8(简称TC_3H_(10))的表皮生长因子受体(EGFR)。结果表明细胞恶性转化前后的EGFR都存在高亲和力和低亲和力两种结合位点,细胞恶性转化后能结合表皮生长因子的EGFR结合位点减少,Western blotting和受体的亲和交联分析表明EGFR的分子量为170kD,是单链多肽。     

单克隆人胰腺干细胞分离培养体系的建立

本研究探讨了建立单克隆人胰腺干细胞分离培养体系及单克隆人胰腺干细胞系．对一些影响干细胞增殖的因素进行了分析。无菌取人流产胎儿胰腺组织,切碎至1mm3,0．1％ Ⅳ型胶原酶消化,低糖DMEM、10％FBS、3．7g／LNaHCO3、0．08g／L青霉素及0．1g/L链霉素培养液贴壁培养细胞,2．5g／L胰蛋白酶＋0．4g／LEDTA消化传代。克隆环筛选单克隆干细胞,培养液中添加10ng／mLEGF．扩增单克隆干细胞。采用核型分析法检测干细胞染色体,MTT法测定干细胞生长曲线。胶原酶消化胰腺组织．获得单个细胞和细胞团。贴壁培养．原代上皮样胰腺干细胞克隆性生长。胰蛋白酶消化传代,上皮样胰腺干细胞逐渐被纯化。克隆环筛选,获得单克隆人胰腺干细胞。扩增培养,1例来源于4月龄男性流产胎儿胰腺组织干细胞建系,传50代。染色体核型分析．该干细胞系为正常的二倍体细胞。细胞生长曲线显示培养1—4d,干细胞生长缓慢,5-6d,进入倍增期。培养液中添加15％FBS,干细胞增殖较快。再添加15ng／mLEGF或10ng／mL IGF—Ⅱ．干细胞增殖更快。研究结果表明应用本实验建立的细胞分离培养体系获得了单克隆人胰腺干细胞系。     

Somatostatin 1-28 circulates in human plasma

The gel filtration profile of immunoreactive somatostatin in human plasma in the fasting state is not well established as a consequence of insufficient sensitivity of the combined chromatography and radioimmunoassay procedures usually employed. We here report the gel filtration profiles of plasma samples after somatostatin concentration by batchwise immunoaffinity chromatography. The results clearly and reliably document the presence of a circulating peptide in human plasma with a gel permeation chromatography profile identical to the one of synthetic somatostatin 1-28. Approximately 46% of the total somatostatin-like immunoreactivity in plasma is due to this component.