黄鳝血清转铁蛋白的提取及表征

对从黄鳝血液中提取的蛋白层析纯化后进行生物质谱测试,确定为血清转铁蛋白. 利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、原子力显微镜、圆二色谱法对蛋白的分子量、表面形貌、二级结构进行了表征. 结果表明,该转铁蛋白分子量为74 kDa,含量为0.750 mg/mL,含a-螺旋29.8%,b-折叠6.6%.     

转铁蛋白的研究进展

转铁蛋白（Transferrin,TRF,TF,又称为铁传递蛋白、血清运铁蛋白、运铁蛋白、嗜铁蛋白、铁糖蛋白）是一种重要的β-球蛋白,是脊椎动物体内铁的运输者。1945年,Holmberg和Laurell首次在人血清中发现了这种非血红素结合铁的转铁蛋白,此后人们又在哺乳动物以及鱼类、两栖类及爬行类的血清中分别发现了转铁蛋白的存在。由于在抗菌、杀菌以及在肿瘤和癌症防治等方面的突出作用,转铁蛋白在医药界已越来越受到人们的重视并开展了转铁蛋白作为药物载体的大量研究。     

转铁蛋白修饰聚乳酸纳米微粒表面的99mTC标记及体内分布

以转铁蛋白溶液为外水相,聚乳酸丙酮溶液为油相,纳米沉淀法制备了表面结合转铁蛋白的聚乳酸纳米微粒,以二氯亚锡为还原剂,直接法和CDPTA螯合法对纳米微粒进行99mTc放射性标记,以C6胶质瘤细胞实验考察了标记对纳米微粒表面转铁蛋白活性的影响,结果表明直接法标记率较高,大于8011%,对转铁蛋白活性有影响。CDPTA螯合法标记法较低(7213%),对转铁蛋白活性影响较小。以脑部荷胶质瘤大鼠为动物模型,鼠尾静脉注射放射性标记纳米微粒,SPECT示踪和γ计数器检测显示:以转铁蛋白表面修饰的聚乳酸纳米微粒经静脉注射后主要分布于肝、脾,与正常鼠相比,荷胶质瘤大鼠对纳米微粒的摄取率有所提高。     

乳铁蛋白纯化和检测方法研究进展

乳铁蛋白是一种具有抗微生物活性的铁离子结合糖蛋白,属于转铁蛋白家族的一员,具有调节炎症反应和激活免疫系统等功能,因此乳铁蛋白被广泛应用于医学和食品领域。本综述汇集了乳铁蛋白纯化和检测方法的研究进展,并比较了各方法之间的优缺点。     

Vero细胞无血清培养基的优化

目的通过试验设计(design of experiments,DOE)方法和统计学分析快速优化Vero细胞无血清培养基。方法以DMEM/F12为基础培养基,通过Plackett-Burman试验、最陡爬坡试验和Box-Behnken试验考察7种添加物胰岛素、人转铁蛋白、透明质酸、牛血清白蛋白、亚硒酸钠、表皮生长因子和高密度脂蛋白对Vero细胞生长的影响,以CCK-8测定的吸光度值(A490)为响应值,优化Vero细胞无血清培养基。用最优化的因子浓度配制的Vero细胞无血清培养基培养Vero细胞,对优化结果进行验证。结果通过Plackett-Burman试验筛选出胰岛素、牛血清白蛋白和人转铁蛋白对Vero细胞生长影响较大;采用最陡爬坡试验和Box-Behnken试验进一步优化,Design-Expert 8.06软件进行回归分析,得到胰岛素、人转铁蛋白和牛血清白蛋白的最佳浓度分别为5.625μg/ml、14.034μg/ml和3.92 mg/ml,在此优化条件下的A490值为2.3,较基础培养基提高了3.41倍。用最优的胰岛素、人转铁蛋白和牛血清白蛋白浓度配制的Vero细胞无血清培养基培养Vero细胞的A490值为2.148,为预测值的93.4%,符合度较高。结论应用DOE方法快速高效地优化了Vero细胞无血清培养基,为无血清培养基的研制奠定了基础。     

肝癌细胞转铁蛋白受体靶向多肽类似物BP9a的合成

以2-CTC树脂为载体、Fmoc-氨基酸为原料,采用Fmoc/t Bu固相多肽合成法得到肝癌细胞转铁蛋白受体靶向多肽BP9N末端连接半胱氨酸的类似物BP9a,实现其作为靶向载体与抗肿瘤药物分子进行化学偶联的功能。BP9a粗品经反相半制备高效液相色谱仪(RP-HPLC)纯化后纯度达到98.42%,总收率为39.38%。     

光谱法研究黄腐酸与转铁蛋白的结合

正人血清转铁蛋白(Tf)在抗癌药物向主要的增生性癌细胞递送中显示出了过度表达转铁蛋白受体的潜能。黄腐酸因具有广泛的生物和药理活性而引起人们的普遍关注。黄腐酸与Tf的相互作用对深入了解黄腐酸的抗癌活性具有重要意义。本研究在生理条件下,利用荧光猝灭、热力学、同步荧光和圆二色谱(CD)法研究了黄腐酸与Tf的相互作用机制。黄腐酸与Tf结合,形成了新的复合物,并计算得出在288、298、310 K时,荧光猝灭表观缔合常数     

老年脑梗死合并营养不良患者早期肠内营养支持治疗效果评价

目的探讨老年脑梗死合并营养不良患者应用肠内营养支持治疗效果。方法将40例老年脑梗死合并营养不良患者随机分为营养干预组和常规治疗组各20人。在抗血小板、稳定斑块、活血化瘀、改善微循环等治疗原则基础上,给予营养干预组进行个体化营养支持治疗。比较治疗前后血红蛋白、总蛋白、白蛋白、转铁蛋白、MNA-SF评分和BADL评分。结果营养干预组血红蛋白、总蛋白、白蛋白、转铁蛋白水平较治疗前明显升高(P0.05);常规治疗组总蛋白、白蛋白、转铁蛋白水平较治疗前明显降低(P0.05);两组MNA-SF及BADL评分均有改善,但营养支持组较对照组改善更为明显(P0.05)。结论应用肠内营养支持治疗明显改善老年脑梗死合并营养不良患者营养状态,改善日常生活活动能力,改善临床预后。     

人血浆转铁蛋白的分离与纯制

介绍了用硫酸铵盐析和 DEAE-Sephadex A-50层析技术由 Cohn 氏组分Ⅳ制备纯人血浆转铁蛋白的过程。聚丙烯酰胺凝胶电泳、免疫电泳、吸收光谱和铁结合活性等检定的结果与文献报道的一致。     

人血清转铁蛋白标准物质纯化

建立了一种从Cohn血浆分离副产物组分IV中纯化高纯度转铁蛋白(Tf)标准物质的技术,对Cohn组分IV进行复溶、离心和过滤,采用Capto DEAE阴离子交换色谱进行初分离,再通过穿透式Octyl-Sepharose 4 Fast Flow疏水色谱进一步精制;对纯化产品进行了结构、纯度、活性等表征,采用十二烷基磺酸钠?聚丙烯酰胺凝胶电泳、基质辅助激光解吸电离飞行时间质谱、高效液相色谱及酶联免疫吸附法对Tf标准物质进行了分析. 结果表明,Tf产品纯度大于99%,收率为79%;圆二色光谱分析显示Tf的二级结构未发生改变,铁离子结合能力实验表明Tf可结合2个铁离子,保持活性.     

Multivalent Display and Receptor‐Mediated Endocytosis of Transferrin on Virus‐Like Particles

The structurally regular and stable self‐assembled capsids derived from viruses can be used as scaffolds for the display of multiple copies of cell‐ and tissue‐targeting molecules and therapeutic agents in a convenient and well‐defined manner. The human iron‐transfer protein transferrin, a high affinity ligand for receptors upregulated in a variety of cancers, has been arrayed on the exterior surface of the protein capsid of bacteriophage Qβ. Selective oxidation of the sialic acid residues on the glycan chains of transferrin was followed by introduction of a terminal alkyne functionality through an oxime linkage. Attachment of the protein to azide‐functionalized Qβ capsid particles in an orientation allowing access to the receptor binding site was accomplished by the Cu^I‐catalyzed azide–alkyne cycloaddition (CuAAC) click reaction. Transferrin conjugation to Qβ particles allowed specific recognition by transferrin receptors and cellular internalization through clathrin‐mediated endocytosis, as determined by fluorescence microscopy on cells expressing GFP‐labeled clathrin light chains. By testing Qβ particles bearing different numbers of transferrin molecules, it was demonstrated that cellular uptake was proportional to ligand density, but that internalization was inhibited by equivalent concentrations of free transferrin. These results suggest that cell targeting with transferrin can be improved by local concentration (avidity) effects.     

Irreversible changes in protein conformation due to interaction with superparamagnetic iron oxide nanoparticles

The understanding of the interactions between nanomaterials and proteins is of extreme importance in medicine. In a biological fluid, proteins can adsorb and associate with nanoparticles, which can have significant impact on the biological behavior of the proteins and the nanoparticles. We report here on the interactions of iron saturated human transferrin protein with both bare and polyvinyl alcohol coated superparamagnetic iron oxide nanoparticles (SPIONs). The exposure of human transferrin to SPIONs results in the release of iron, which changes the main function of the protein, which is the transport of iron among cells. After removal of the magnetic nanoparticles, the original protein conformation is not recovered, indicating irreversible changes in transferrin conformation: from a compact to an open structure.     

Transferrin: from inorganic biochemistry to medicine

Transferrin is one of the key proteins of iron metabolism in mammalians. It is responsible for the transfer of the essential iron(III) ions through the biological fluids from absorption to storage and utilization sites. Moreover, transferrin is involved in the metabolism of other metal ions that are either trace or toxic elements. In recent years the crystal structure of transferrin has been solved at high resolution. This has allowed an extensive reinterpretation of the many spectroscopic studies carried out on this protein in the last decade as well as the elucidation of some interesting structure-function relationships. We review here recent progresses in transferrin biochemistry, particular focus being given to those aspects that are relevant from a medical point of view.     

Transferrin Receptors in Erythropoiesis

Erythropoiesis is a highly dynamic process giving rise to red blood cells from hematopoietic stem cells present in the bone marrow. Red blood cells transport oxygen to tissues thanks to the hemoglobin comprised of α- and β-globin chains and of iron-containing hemes. Erythropoiesis is the most iron-consuming process to support hemoglobin production. Iron delivery is mediated via transferrin internalization by the endocytosis of transferrin receptor type 1 (TFR1), one of the most abundant membrane proteins of erythroblasts. A second transferrin receptor—TFR2—associates with the erythropoietin receptor and has been implicated in the regulation of erythropoiesis. In erythroblasts, both transferrin receptors adopt peculiarities such as an erythroid-specific regulation of TFR1 and a trafficking pathway reliant on TFR2 for iron. This review reports both trafficking and signaling functions of these receptors and reassesses the debated role of TFR2 in erythropoiesis in the light of recent findings. Potential therapeutic uses targeting the transferrin-TFR1 axis or TFR2 in hematological disorders are also discussed.     

A DNA Aptameric Ligand of Human Transferrin Receptor Generated by Cell-SELEX

General cancer-targeted ligands that can deliver drugs to cells have been given considerable attention. In this paper, a high-affinity DNA aptamer (HG1) generally binding to human tumor cells was evolved by cell-SELEX, and was further optimized to have 35 deoxynucleotides (HG1-9). Aptamer HG1-9 could be taken up by live cells, and its target protein on a cell was identified to be human transferrin receptor (TfR). As a man-made ligand of TfR, aptamer HG1-9 was demonstrated to bind at the same site of human TfR as transferrin with comparable binding affinity, and was proved to cross the epithelium barrier through transferrin receptor-mediated transcytosis. These results suggest that aptamer HG1-9 holds potential as a promising ligand to develop general cancer-targeted diagnostics and therapeutics.     

Human Serum Transferrin Fibrils: Nanomineralisation in Bacteria and Destruction of Red Blood Cells

Fibrils formed by human serum transferrin [(1–3 μM ) apo‐Tf, partially iron‐saturated (Fe_0.6‐Tf) and holo‐Tf (Fe₂‐Tf) forms], from dilute bicarbonate solutions, were deposited on formvar surfaces and studied by electron microscopy. We observed that possible bacterial contamination appears to give rise to long, pea‐pod‐like (PPL) structures for Fe₂‐Tf, attributable to the formation of polyhydroxybutyrate (PHB) storage granules, under the nutrient‐limiting conditions used. These PPL structures contained periodic nanomineralisation sites susceptible to uranyl stain. Extended incubation of transferrin solutions (about four days) gave rise to extensive transferrin fibril structures. Optical microscopy and AFM studies showed that red blood cells (RBCs) readily adhere to these fibrils. Moreover, the fibrils appear to penetrate RBC membranes and to induce rapid cell destruction (within about 5 h). It is speculated that in situations in vivo where transferrin fibrils can form, such interactions might have adverse physiological consequences, and further studies could aid the understanding of related pathological events.     

Genetically engineered brain drug delivery vectors: cloning, expression and in vivo application of an anti-transferrin receptor single chain antibodystreptavidin fusion gene and protein

A single chain Fv antibody–streptavidin fusion proteinwas expressed and purified from bacterial inclusion bodies followingcloning of the genes encoding the variable region of the heavychain and light chain of the murine OX26 monoclonal antibodyto the rat transferrin receptor. The latter undergoes receptormediated transcytosis through the brain capillary endothelialwall in vivo, which makes up the blood–brain barrier (BBB);therefore, the OX26 monoclonal antibody and its single chainFv analog may act as brain drug delivery vectors in vivo. Attachmentof biotinylated drugs to the antibody vector is facilitatedby production of the streptavidin fusion protein. The bi-functionalityof the OX26 single chain Fv antibody–streptavidin fusionprotein was retained, as the product both bound biotin and therat transferrin receptor in vitro and in vivo, based on pharmacokineticand brain uptake analyses in anesthetized rats. The attachmentof biotin–polyethyleneglycol–fluorescein to theOX26 single chain Fv antibody–streptavidin fusion proteinresulted in illumination of isolated rat brain capillaries inconfocal fluorescent microscopy. In conclusion, these studiesdemonstrate that genetically engineered single chain Fv antibody–streptavidinfusion proteins may be used for non-invasive neurotherapeuticdelivery to the brain using endogenous BBB transport systemssuch as the transferrin receptor.     

Theranostic Polyaminocarboxylate–Cyanine–Transferrin Conjugate for Anticancer Therapy and Near‐Infrared Optical Imaging

Iron chelation therapy has been recognized as a promising antitumor therapeutic strategy. Herein we report a novel theranostic agent for targeted iron chelation therapy and near‐infrared (NIR) optical imaging of cancers. The theranostic agent was prepared by incorporation of a polyaminocarboxylate‐based cytotoxic chelating agent (N‐NE3TA; 7‐[2‐[(carboxymethyl)amino]ethyl]‐1,4,7‐triazacyclononane‐1,4‐diacetic acid) and a NIR fluorescent cyanine dye (Cy5.5) onto a tumor‐targeting transferrin (Tf). The N‐NE3TA–Tf conjugate (without Cy5.5) was characterized and evaluated for antiproliferative activity in HeLa, HT29, and PC3 cancer cells, which have elevated expression levels of the transferrin receptor (TfR). The N‐NE3TA–Tf conjugate displayed significant inhibitory activity against all three cancer cell lines. The NIR dye Cy5.5 was then incorporated into N‐NE3TA–Tf, and the resulting cytotoxic and fluorescent transferrin conjugate N‐NE3TA–Tf–Cy5.5 was shown by microscopy to enter TfR‐overexpressing cancer cells. This theranostic conjugate has potential application for dual use in targeted iron chelation cancer therapy and NIR fluorescence imaging.     

Aberrant Transferrin and Ferritin Upregulation Elicits Iron Accumulation and Oxidative Inflammaging Causing Ferroptosis and Undermines Estradiol Biosynthesis in Aging Rat Ovaries by Upregulating NF-Κb-Activated Inducible Nitric Oxide Synthase: First Demonstration of an Intricate Mechanism

We report herein a novel mechanism, unraveled by proteomics and validated by in vitro and in vivo studies, of the aberrant aging-associated upregulation of ovarian transferrin and ferritin in rat ovaries. The ovarian mass and serum estradiol titer plummeted while the ovarian labile ferrous iron and total iron levels escalated with age in rats. Oxidative stress markers, such as nitrite/nitrate, 3-nitrotyrosine, and 4-hydroxy-2-nonenal, accumulated in the aging ovaries due to an aberrant upregulation of the ovarian transferrin, ferritin light/heavy chains, and iron regulatory protein 2(IRP2)-mediated transferrin receptor 1 (TfR1). Ferritin inhibited estradiol biosynthesis in ovarian granulosa cells in vitro via the upregulation of a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and p65/p50-induced oxidative and inflammatory factor inducible nitric oxide synthase (iNOS). An in vivo study demonstrated how the age-associated activation of NF-κB induced the upregulation of iNOS and the tumor necrosis factor α (TNFα). The downregulation of the keap1-mediated nuclear factor erythroid 2-related factor 2 (Nrf2), that induced a decrease in glutathione peroxidase 4 (GPX4), was observed. The aberrant transferrin and ferritin upregulation triggered an iron accumulation via the upregulation of an IRP2-induced TfR1. This culminates in NF-κB-iNOS-mediated ovarian oxi-inflamm-aging and serum estradiol decrement in naturally aging rats. The iron accumulation and the effect on ferroptosis-related proteins including the GPX4, TfR1, Nrf2, Keap1, and ferritin heavy chain, as in testicular ferroptosis, indicated the triggering of ferroptosis. In young rats, an intraovarian injection of an adenovirus, which expressed iron regulatory proteins, upregulated the ovarian NF-κB/iNOS and downregulated the GPX4. These novel findings have contributed to a prompt translational research on the ovarian aging-associated iron metabolism and aging-associated ovarian diseases.