抗肿瘤因子内皮抑素基因的克隆及表达

为了克隆抗肿瘤因子内皮抑素基因并表达内皮抑素蛋白,从人胎盘组织中分离总ＲＮＡ,用ＲＴ－ＰＣＲ法扩增出５７０ｂｐ的ＤＮＡ片段,并成功地克隆到ｐＵＣ１８质粒载体中,经序列测定证实为内皮抑素基因,用Ｘｐｒｅｓｓ本外表达了内皮抑素蛋白并纯化成功。     

肿瘤新生血管抑制剂——血管抑素和内皮抑素

血管抑素和内皮抑素是两种内源性肿瘤新生血管抑制剂，主要通过抑制肿瘤内皮细胞的生长达到抑制肿瘤血管生成、诱导肿瘤细胞凋亡、防止肿瘤侵袭和转移的目的。作为两种最有前途的肿瘤新生血管抑制剂克服了肿瘤化疗过程中产生的耐药抗药性。文中对这两种抑制剂的结构与功能及应用研究最新进展作一综述。     

定点突变内皮抑素Zn2+的结合位点及突变基因的克隆表达

从人胚肝组织中提取总RNA, 以逆转录聚合酶链式反应（RT-PCR）法获得人内皮抑素编码序列, 采用定点突变技术将His2和His4双突变为Leu2和Val4. 将突变基因cDNA插入含有T7启动子的质粒pET-28b中构建表达质粒pMendo, 转化大肠杆菌BL21(DE3), 筛选表达菌株BL21-Mute, 表达菌株经IPTG诱导后以包涵体方式产生大量内皮抑素突变蛋白. SDS-PAGE分析表明, 表达的重组蛋白占菌株可溶性蛋白质的30%. 复性、 纯化的内皮抑素突变蛋白纯度达到98%, 失去抑制人脐静脉内皮细胞增殖的活性.     

内皮抑素与角膜新生血管相关研究进展

内皮抑素是目前已知最强的内源性血管生成抑制因子,它对以肿瘤血管为代表的活跃增殖的血管内皮细胞有强烈的选择性抑制作用,且无毒性、无耐药性,角膜新生血管形成与肿瘤新生血管类似,内皮抑素已成为治疗角膜新生血管性眼病的一种潜力药物.该文将结合内皮抑素的结构特点,围绕其抑制新生血管生成的作用机制,着重对其可能用于角膜新生血管发生及治疗方面的相关研究进展作一综述.     

人血管能抑素免疫兔抗血清制备及其特性测定

纯化的人血管能抑素包涵体进行SDS-PAGE分离，切胶回收目的蛋白，与弗氏不完全佐剂充分乳化后，进行多点皮下注射免疫新西兰白兔，制备兔抗人血管能抑素抗血清，抗血清去除交叉反应抗体后，进行Western Blotting分析抗血清与人血管能抑素及其N端和C端片段的抗原抗体反应特性。结果表明，经过4次加强免疫后，获得了高效价的兔抗人血管能抑素抗血清。抗血清去除交叉反应抗体后，Western Blotting分析表明，它能与人血管能抑素发生特异的抗原抗体反应，同时还能与人血管能抑素的N端片段和C端片段发生特异性较弱的抗原抗体反应。证明制备的兔抗人血管能抑素抗血清能应用于人血管能抑素及其N端和C端片段免疫学研究中。     

利用人内皮抑素基因构建植物表达载体及对烟草的遗传转化

将人内皮抑素(endostatin)基因重组于植物双元表达载体pGA 643,得到重组质粒pGE.用三亲融合法将其导入农杆菌LBA 4404中,采用叶盘法转化烟草,经诱导与筛选,获得了卡那霉素抗性植株.提取抗性植株总DNA,通过PCR扩增和Sou thern杂交检测,筛选出了整合有外源基因的转化植株.为进一步研究人内皮抑素在烟草中的表达及生物学功能奠定了基础.     

恩度对雌激素受体阳性的人乳腺癌细胞生长的抑制作用及其分子机制的研究

研究了重组人血管内皮抑素(恩度)对雌激素受体阳性的人乳腺癌MCF-7细胞株生长的抑制作用与其分子机制.采用MTT法、台盼蓝染色法和中性红染色法检测不同浓度恩度对MCF-7细胞生长的抑制作用,Hoechst33258荧光染色法和TUNEL分析法观察恩度诱导MCF-7细胞凋亡的形态变化,蛋白印迹法检测恩度作用的MCF-7细胞中与生长和凋亡相关蛋白的表达.结果表明,恩度显著性地抑制MCF-7细胞的生长并诱导细胞凋亡.此外,恩度显著性地减少MCF-7细胞VEGFR1,c-Met,ERα,Akt,NF-κB,Bcl-2和CyclinD1蛋白的表达,明显地上调Bax蛋白的表达,其作用的分子机制可能是通过抑制VEGFR1/c-Met/ERα-Akt-NF-κB信号传导通路,下调Bcl-2/Bax蛋白比率、降低CyclinD1蛋白的水平.     

内皮抑素在癌性胸水诊断及治疗中的研究进展

恶性胸腔积液是晚期恶性肿瘤的严重并发症之一,积极的诊断及控制恶性胸水对患者的生活及生存质量具有重要意义。肿瘤浸润或转移至胸膜后血管内皮生长因子(VEGF)水平的升高、肿瘤新生血管的生成以及血管通透性的增高等是恶性胸腔积液形成的重要机制之一,而内皮抑素(ES)为内源性抗血管生成物质,其表达水平与多种恶性肿瘤的进展和预后有关,被认为是最有效的内源性血管生长抑制剂之一,近年来ES为癌性胸水诊断及治疗提供了新的途径,被许多学者广泛应用于临床。本文旨在介绍ES在癌性胸水诊断及治疗中的研究进展。     

真核生物多顺反子表达载体的构建及在Hela细胞中的表达

利用DNA重组技术将抗肿瘤血管生成的内皮抑素基因endostatin插入含有FHIT(抗肿瘤人脆性组氨酸三联体基因)和EGFP的pIRES2-FHIT-EGFP载体中构建了真核多顺反子表达载体pES-IRES-FHIT-IRES-EGFP,用脂质体法转染Hela细胞后,用G418筛选出稳定转染的细胞.经RT-PCR、RT-PCR southern blot、northern blot和免疫细胞化学分析,结果证明单顺反子和三顺反子在RNA水平都得到表达.在蛋白表达水平上,荧光观察和免疫细胞化学分析结果显示Endostatin、FHIT和EGFP在Hela细胞中均得到表达,为基因药物和肿瘤多基因治疗奠定了基础.     

人血管内皮抑制素基因在毕赤甲醇酵母中的表达

对发生突变的人血管内皮抑制素基因进行了PCR修正,并将其连接到pAO815载体上,然后克隆进大肠杆菌TOP10F＇中,提取质粒测序,证实序列正确.再用电激法转化毕赤甲醇酵母KM71,利用RDB平板和PCR技术筛选出阳性克隆菌落后,甲醇诱导表达,SDS-PAGE电泳检测显示重组的人血管内皮抑制素蛋自在毕赤甲醇酵母KM71中获得了有效表达.     

Progress in studies of angiostatin and its anti-tumor effects

Angiostatin is a 38 ku circulating angiogenesis inhibitor purified from the serum and urine of mice bearing a murine Lewis lung carcinoma. It is regarded as an internal fragment of plasminogen containing the first 4 Kringle structures. In vitro, angiostatin specifically inhibits endothelial cell proliferation. In vivo, it inhibits angiogenesis and suppresses the growth of such primary tumors as Lewis lung carcinoma and hemangioendotheliolma in mice.     

薄板坯连铸连轧过程中的静态软化行为

采用本钢Gleeble-2000热/力模拟试验机模拟了本钢薄板坯连铸连轧过程中奥氏体的静态软化行为.结果表明:与不含Nb钢相比,含Nb钢明显抑制了奥氏体的静态再结晶,软化率曲线出现了平台;与固溶Nb相比,析出Nb对静态再结晶的抑制作用更大;同传统流程相比,薄板坯连铸连轧生产线轧制低碳钢(包括含Nb钢和不含Nb钢)在道次间隔期间的静态再结晶激活能明显提高,更不容易发生静态软化行为.     

软骨血管生成抑制因子抑制血管生成及其抗肿瘤作用的研究

测定了小牛软骨血管生成抑制因子对血管内皮细胞细胞毒作用,对内皮细胞骨架系统及其运动迁移的抑制效应,对小鼠肿瘤生长的对抑制效应。     

1株羊蹄强拮抗内生芽孢杆菌H-g的分离鉴定

从羊蹄(Radix Rumicis Japonici)根中分离到1株对小麦赤霉(Fusarium graminearum)等多种植物病原真菌有强拮抗作用的内生芽孢杆菌H-g.通过形态特征和培养特征观察、生理生化实验及16S rDNA序列分析,初步鉴定该菌为枯草芽孢杆菌属,命名为bacillus subtilis subsp H-g.     

十六烷基三甲基溴化铵与氯化钠对不同浓度硫酸中冷轧钢的缓蚀协同作用

 用失重法和电化学方法研究了0.5-2.0mol/L硫酸中十六烷基三甲基溴化铵(CTAB)与氯化钠对冷轧钢的腐蚀影响.研究表明:CTAB与NaCl的混合物表现为阴极为主的混合型缓蚀剂,混合物的吸附符合Langmuir等温式.失重法和电化学方法都表明CTAB与NaCl对硫酸中钢的腐蚀存在缓蚀协同作用.     

Aspirin causes short-lived inhibition of bradykinin-stimulated prostacyclin production in man

Acetylsalicylic acid (aspirin) inhibits prostanoid synthesis by irreversible acetylation of fatty acid cyclooxygenase (EC 1.14.99.1). It thereby inhibits synthesis of pro-aggregatory thromboxane A2 (TXA2) by platelets and is widely used in the treatment and prophylaxis of vascular disease. Its efficacy, however, may be reduced since it also inhibits formation of prostacyclin (PGI2) which is a vasodilator and anti-aggregatory agent. There is uncertainty over the optimum dose regimen for aspirin since although it inhibits platelet thromboxane production for many days, the magnitude and duration of its effect on PGI2 production by vascular endothelium in vivo is unknown. Resting plasma concentrations of PGI2 (measured as the stable hydrolysis product 6-oxo-PGF1 alpha) are at or below the limit of sensitivity of the most sensitive assays and cannot therefore be used to demonstrate a reduction in production. Bradykinin stimulates PGI2 synthesis by cultured human vascular endothelial cells and we have shown that it stimulates PGI2 production by man in vivo. We report here that an oral dose of aspirin (600 mg) causes rapid and substantial inhibition of bradykinin-stimulated PGI2 production, but recovery occurs within 6 hours; this implies that endothelial PGI2 synthesis would be spared most of the time during dosing once daily with even this relatively large dose of aspirin.     

磁场对碳酸钙结晶过程及晶型影响的研究

通过测定电导率变化研究了磁场对碳酸钙结晶过程的影响,利用扫描电子显微镜(SEM)和X射线衍射仪(XRD)对碳酸钙晶型进行了表征.结果表明,当Ca2+浓度低于2.5 mmol/L时,磁处理抑制碳酸钙的结晶速度;当Ca2+浓度高于2.5 mmo1/L时,磁处理促进了碳酸钙的结晶速度且抑制了晶粒的长大.随着磁场强度的增大,磁...     

The gene product Murr1 restricts HIV-1 replication in resting CD4+ lymphocytes

Although human immunodeficiency virus-1 (HIV-1) infects quiescent and proliferating CD4+ lymphocytes, the virus replicates poorly in resting T cells. Factors that block viral replication in these cells might help to prolong the asymptomatic phase of HIV infection; however, the molecular mechanisms that control this process are not fully understood. Here we show that Murr1, a gene product known previously for its involvement in copper regulation, inhibits HIV-1 growth in unstimulated CD4+ T cells. This inhibition was mediated in part through its ability to inhibit basal and cytokine-stimulated nuclear factor (NF)-kappaB activity. Knockdown of Murr1 increased NF-kappaB activity and decreased IkappaB-alpha concentrations by facilitating phospho-IkappaB-alpha degradation by the proteasome. Murr1 was detected in CD4+ T cells, and RNA-mediated interference of Murr1 in primary resting CD4+ lymphocytes increased HIV-1 replication. Through its effects on the proteasome, Murr1 acts as a genetic restriction factor that inhibits HIV-1 replication in lymphocytes, which could contribute to the regulation of asymptomatic HIV infection and the progression of AIDS.     

A synthetic peptide that is a bombesin receptor antagonist

The tetradecapeptide bombesin was originally isolated from frog skin. Bombesin-like peptides have since been detected in mammalian gastrointestinal tract, brain and lung. These peptides have potent pharmacological effects on the central nervous system; they cause contraction of intestinal, uterine and urinary tract smooth muscle; and stimulate the release of other peptides including gastrin, cholecystokinin, motilin, pancreatic polypeptide, neurotensin, insulin, enteroglucagon, prolactin and growth hormone. Specific plasma membrane receptors for bombesin have been demonstrated on pancreatic acinar cells, brain membranes and pituitary cells. Studies defining the physiological importance of bombesin have been impeded by the lack of a bombesin receptor antagonist. Here we describe experiments which demonstrate that a peptide originally described as a substance P receptor antagonist, [D-Arg, D-Pro, D-Trp, Leu ]substance P, is also a bombesin receptor antagonist. This peptide competitively inhibits the ability of bombesin to stimulate enzyme secretion from dispersed pancreatic acini, and also inhibits the action of other peptides that interact with the bombesin receptor.     

气相色谱——电子捕获检测器法研究亚硒酸根对汞的生物甲基化影响

用气相色谱－电子捕获检测器法研究了底泥中汞的甲基化作用，结果为汞的甲基化是微生物过程，甲基化的量只占总汞的很少一部分。Ｓｅ（Ⅳ）抑制汞的甲基化。