三种柔红霉素-白血病单抗免疫偶合物特性比较

用 3种方法将纯化的 Mc Ab1A1 与柔红霉素 (DNR)偶联 ,直接交联法制备的 Mc Ab1A1 - DNR偶合物克分子比为 1∶ 5 2 ,抗体活性有所下降 ,药物活性保留 84.6 % ;顺乌头酸酐法制备的 Mc Ab1A1 - CADNR偶合物克分子比为 1∶ 2 7,保持了较好的抗体活性 ,药物活性保留 5 0 % ;间接法制备的 Mc Ab1A1 - PAD- DNR偶合物克分子比为 1∶ 437,保持了较好的抗体活性 ,药物活性保留 91.6 %。 Mc Ab1A1 - PAD- DNR偶合物对 U937靶细胞的杀伤作用最强 ,Mc Ab1A1 - DNR次之。实验中观察到 ,L owry法不能用于含 DNR偶合物中抗体蛋白含量的测定 ,活细胞 EL ISA法不能用于含 DNR偶合物中抗体活性测定。     

急非淋M2型白血病单克隆抗体与高三尖杉酯碱偶合物的制备…

用羟基磷灰石，ＤＥＡＥ－Ｓｅｐｈａｃｅｌ和Ｓｅｐｈａｃｒｙ１ Ｓ－３００纯化了抗急非淋Ｍ２型白血病ＭｃＡｂ１Ｄ１，纯度为９９．９％，用直接交联方法将ＭｃＡｂ１Ｄ１与ＨＨＴ偶联，克分子比１：４０。该偶合物保持了抗体和药物活性，对急非淋Ｍ２型白血病靶细胞有选择性杀伤作用，而对急非淋Ｍ１型白血病细胞的Ｈｅｌａ细胞无明显杀伤作用。     

静注丙球激活补体活性测定方法的建立

目的　建立静注丙球 (IVIG)激活补体活性的测定方法 ,并对国内不同工艺生产的IVIG制品Fc段的生物学功能进行分析。方法　采用补体经典激活途径激活补体的方法。结果　应用该方法检测不同工艺生产的制品活性均有差别。该方法操作简便 ,重复性好。结论　将检测Fc段的生物学活性列为IVIG常规检测质控指标是可行的     

急非淋M_2型白血病单克隆抗体与高三尖杉酯碱偶合物的制备及体外对白血病细胞的杀伤作用

用羟基磷灰石、DEAE－Sephacel和SephacrylS－300纯化了抗急非淋M2型白血病MCAb1D1，纯度为99．9％。用直接交联方法将McAb1D1与HHT仍联，克分子比1：40。该偶合物保持了抗体和药物活性，对急非淋M2型白血病靶细胞有选择性杀伤作用，而对急非淋M1型白血病细胞和Hela细胞无明显杀伤作用。     

重组人肿瘤坏死因子可溶性受体Ⅱ(sTNFRⅡ)的纯化

目的 从大肠杆菌破碎液中纯化可溶性肿瘤坏死因子受体Ⅱ,获得能够中和TNF活性的受体蛋白。方法 菌体破碎液经热沉淀处理后,用金属鳌合层析柱纯化重组蛋白。并对蛋白的理化特性和生物学活性进行分析。结果 纯化的重组蛋白纯度大于95％,并且能中和TNF的生物学活性,抑制其对细胞的杀伤作用。结论该工艺能简便有效地纯化出TNF受体蛋白,为进一步深入研究奠定了基础。     

黄曲霉毒素G族人工抗原的合成与免疫效果研究

以黄曲霉毒素(Aflatoxin G1,AFG1)的半缩醛(Aflatoxin G2a,AFG2a)为中间物,通过还原烷基法,使黄曲霉毒素G1在8、9羟基上与牛血清白蛋白(BSA)实现偶联,得到偶联物。对偶联物进行紫外全波长扫描显示,其特征吸收峰与BSA和AFG1的特征吸收峰有明显差异。经完全透析后检测偶联物与BSA荧光强度差异,均表明BSA与黄曲霉毒素G1偶联,AFG1-BSA物质的量结合比为3.2∶1。以合成人工抗原免疫小鼠,用间接非竞争ELISA测得抗血清中抗体的效价可达1∶128 000,间接竞争ELISA测定抗体IC50为14.7 ng/mL。为下一步研制黄曲霉毒素G族单克隆抗体提供了关键试剂,为研究建立黄曲霉毒素G族免疫速测技术奠定了基础。     

静注人免疫球蛋白(pH4)Fc段生物学活性检测条件的优化

目的优化静注人免疫球蛋白(intravenous immunoglobulins,IVIG)Fc段生物学活性检测条件,并验证该方法的重复性。方法基于《中国药典》三部(2010版)人免疫球蛋白Fc段激活补体的试验方法,优化致敏红细胞的抗原(白喉类毒素)效价(25、50、100、150、200和250 Lf/ml)、IVIG浓度(5、10、20、30、40和50 mg/ml)和补体效价(15、50、75、100和150 CH50/ml)3个试验条件;采用优化的试验条件,对1批IVIG制品重复测定10次,计算变异系数(CV),验证该方法的重复性。结果在致敏红细胞的抗原(白喉类毒素)效价100～200 Lf/ml、IVIG浓度50 mg/ml和补体效价≥75 CH50/ml的条件下,补体介导的溶血反应动力学曲线呈典型的"S"型,可准确计算出IVIG Fc段激活补体的指数(IFc)。采用优化的试验条件,对1批IVIG制品重复测定10次,溶血反应动力学曲线几乎重合为一条,10次测定的IFc值的CV=8.91%。结论优化了IVIG Fc段生物学活性检测条件,优化后的检测方法重复性较好。     

激活素A对巨噬细胞系RAW264.7细胞吞饮活性及TLR4表达的双向调节作用

目的探讨激活素A(ActivinA)对巨噬细胞系RAW264.7细胞活性的调节作用及其可能的机制。方法取对数生长期的小鼠巨噬细胞系RAW264.7细胞,加入1μg/ml脂多糖(LPS),继续培养8h,采用ELISA法检测细胞分泌ActivinA水平;分别加入ActivinA、LPS和ActivinA+LPS,中性红染料法检测细胞吞饮活性;流式细胞术分析细胞表面分子MHCⅠ、MHCⅡ及Toll样受体4(TLR4)的表达水平。结果LPS呈时间依赖性刺激RAW264.7细胞分泌ActivinA;ActivinA可明显促进静息RAW264.7细胞的吞饮活性,而对MHCⅠ、MHCⅡ及TLR4的表达水平无明显影响;ActivinA和LPS共同作用,ActivinA明显抑制了LPS活化的RAW264.7细胞的吞饮活性,并下调TLR4的表达。结论ActivinA可能以自分泌/旁分泌形式参与巨噬细胞活性调节,其抑制LPS作用与TLR4表达有关。     

新型静注人免疫球蛋白的制备及鉴定

目的制备新型静注人免疫球蛋白(IVIG),并对其质量进行鉴定。方法采用超滤浓缩法制备了8批10%IVIG制品,以甘氨酸代替糖类作为稳定剂的主要配方,并优化其浓度(12~18 g/L)。检测10%IVIG制品的总体质量、Ig G亚类分布、Fc活性及稳定性。结果最佳甘氨酸浓度为15 g/L。10%IVIG制品的总体质量均合格,Ig G亚类分布及Fc活性均与5%IVIG制品相似,且该制品在25℃存放6个月和2~8℃存放6年后的总体质量均合格。结论制备的10%IVIG质量优良,且具有良好的稳定性。     

星形丁苯热塑性弹性体的合成

以正丁基锂为引发剂、环忆烷为溶剂、四氢呋喃为活性添加剂和结构调节剂,研究了分别以本二甲酸二甲酯（ＤＭＰ）和邻苯二甲酸二七酯（ＤＰＰ）为偶联剂合成（ＳＢ）。Ｒ的工结果表明,用ＤＰＰ作偶联剂,礤偶联效率（ＣＥ）可达９３％以上,偶联反应在４５内完成;偶联反应温度对偶联反应无明显影响;当四氢呋喃／正丁基锂（ＴＨＦ／Ｌｉ,摩尔比）为０时,ＣＥ与偶联剂／正丁基锂无关;当ＴＨＦ／Ｌｉ大于０,ＤＰＰ／Ｌｉ大于０．     

An Fc–Small Molecule Conjugate for Targeted Inhibition of the Adenosine 2A Receptor

The adenosine A_2A receptor (A_2AR) is expressed in immune cells, as well as brain and heart tissue, and has been intensively studied as a therapeutic target for multiple disease indications. Inhibitors of the A_2AR have the potential for stimulating immune response, which could be valuable for cancer immune surveillance and mounting a response against pathogens. One well‐established potent and selective small molecule A_2AR antagonist, ZM‐241385 (ZM), has a short pharmacokinetic half‐life and the potential for systemic toxicity due to A_2AR effects in the brain and the heart. In this study, we designed an analogue of ZM and tethered it to the Fc domain of the immunoglobulin IgG3 by using expressed protein ligation. The resulting protein–small molecule conjugate, Fc–ZM, retained high affinity for two Fc receptors: FcγRI and the neonatal Fc receptor, FcRn. In addition, Fc–ZM was a potent A_2AR antagonist, as measured by a cell‐based cAMP assay. Cell‐based assays also revealed that Fc–ZM could stimulate interferon γ production in splenocytes in a fashion that was dependent on the presence of A_2AR. We found that Fc–ZM, compared with the small molecule ZM, was a superior A_2AR antagonist in mice, consistent with the possibility that Fc attachment can improve pharmacokinetic and/or pharmacodynamic properties of the small molecule.     

A “Clickable” MTX Reagent as a Practical Tool for Profiling Small‐Molecule–Intracellular Target Interactions via MASPIT

We present a scalable synthesis of a versatile MTX reagent with an azide ligation handle that allows rapid γ‐selective conjugation to yield MTX fusion compounds (MFCs) appropriate for MASPIT, a three‐hybrid system that enables the identification of mammalian cytosolic proteins that interact with a small molecule of interest. We selected three structurally diverse pharmacologically active compounds (tamoxifen, reversine, and FK506) as model baits. After acetylene functionalization of these baits, MFCs were synthesized via a CuAAC reaction, demonstrating the general applicability of the MTX reagent. In analytical mode, MASPIT was able to give concentration‐dependent reporter signals for the established target proteins. Furthermore, we demonstrate that the sensitivity obtained with the new MTX reagent was significantly stronger than that of a previously used non‐regiomeric conjugate mixture. Finally, the FK506 MFC was explored in a cellular array screen for targets of FK506. Out of a pilot collection of nearly 2000 full‐length human ORF preys, FKBP12, the established target of FK506, emerged as the prey protein that gave the highest increase in luciferase activity. This indicates that our newly developed synthetic strategy for the straightforward generation of MFCs is a promising asset to uncover new intracellular targets using MASPIT cellular array screening.     

抗体阻断表面黏附分子DNAM-1和LFA-1对人自然杀伤细胞肿瘤杀伤活性的抑制作用

目的探讨抗体阻断表面黏附分子DNAM-1和LFA-1对体外扩增的人自然杀伤(Natural killer,NK)细胞的肿瘤杀伤活性的影响。方法体外活化人外周血单个核细胞来源的NK细胞,并检测其表面黏附分子DNAM-1和LFA-1的表达水平;通过Trans-well阻隔试验,阻断NK细胞与靶细胞接触,考察细胞-细胞直接接触对NK细胞杀伤活性的影响;通过NK细胞毒性试验,引入抗DNAM-1单克隆抗体和抗LFA-1单克隆抗体,阻断相应信号途径,考察相关黏附分子在NK细胞肿瘤杀伤过程中的作用。结果体外活化的NK细胞高表达表面黏附分子DNAM-1和LFA-1。阻断NK细胞与靶细胞接触,NK细胞的肿瘤杀伤效应大大降低。应用单克隆抗体阻断DNAM-1或LFA-1信号途径,可显著降低NK细胞肿瘤的杀伤效应;联合阻断DNAM-1和LFA-1信号途径,可进一步降低NK的细胞杀伤效应。结论细胞-细胞间直接接触是NK细胞发挥肿瘤杀伤效应的重要机制,表面黏附分子DNAM-1和LFA-1介导的信号途径对维持NK细胞肿瘤杀伤活性具有重要意义。     

腺病毒载体介导的HSV-tk基因杀伤肿瘤细胞的研究

目的 探讨ＨＳＶ－ｔｋ基因对各种肿瘤细胞的杀伤作用及人、鼠瘤细胞对其敏感性及瘤细胞表面的αｖ 整合素对腺病毒载体转染的影响。方法用同源重组法构建 ＡｄＣＭＶ ｔｋ基因;蚀斑形成法对重组腺病毒进行扩增、 滴定;Ｘ－ｇａｌ染色法观察腺病毒转染率,结晶紫染色法检测 ＡｄＣＭＶｔｋ／ＧＣＶ的杀伤作用。结果 ＡｄＣＭＶｔｋ对肿瘤细胞 的杀伤强度在一定范围内与ＡｄＣＭＶｔｋ的剂量呈正相关;对人类的Ｈｅｌａ、ＧＲＣ、Ｈｅｐ－２瘤细胞达１００％转染所需的腺病 毒感染复数量（ｍｕｌｔｉｐｌｉｃｉｔｙ　ｏｆ　ｉｎｆｅｃｔｉｏｎ,ｍ．ｏ．ｉ）较鼠Ｌｅｗｉｓ、ＢＴＴ７３９瘤细胞的ＭＯＩ量低;在相同ＭＯＩ病毒量时,有αｖ整合 素的瘤细胞转染率高于无αｖ整合素的瘤细胞。结论ＡｄＣＭＶｔｋ／ＧＣＶ具有较明显的杀伤肿瘤细胞的作用,且有种属 差异,对人瘤细胞作用大于鼠瘤细胞,瘤细胞表面的αｖ整合素有利于腺病毒的转染。     

The selective delivery of anticancer agents using target-sensitive liposomes

Target-sensitive (TG-S) liposomes having modified antibodies on their surface were employed to study the release of calcein and the selective delivery of the anticancer agents, doxorubicin (DOX) and methotrexate (MTX). The release of calcein from TG-S liposome occurred when the various target cells were contacted with liposomes and it was proportionally increased with the increase of antibody affinity to the target cells. Increasing the concentration of antigen molecules (major histocompatibility, MHC) on the surface of RMA-S, the release of calcein and drugs from TG-S liposomes contacting with RMA-S also rised. The destabilization of TG-S liposomes was only induced above a threshold density of surface antigen on the target cell membrane. The growth inhibition of specific target cells by the liposomal drugs was always stronger than that of the non-specific ones. For specific target cells, the IC₅₀ of liposomal DOX was about 2 times greater than that of free DOX, on the while, for non-specific target cells, more than 5 times. This indicates that the liposomal drugs were transferred preferentially to the specific target cells than the non-specific ones. Based on this phenomenon, the TG-S liposomal MTX were also applied for the selective elimination of the specific target cells in the mixed culture of specific and non-specific target cells.     

整合素αvβ_3拮抗剂-苯丁酸氮芥偶联毒素的合成及其体外抗肿瘤活性

目的合成整合素(Integrin)αvβ3拮抗剂-苯丁酸氮芥偶联毒素,并检测其体外抗肿瘤活性。方法化学合成In-tegrinαvβ3拮抗剂,并与苯丁酸氮芥通过酰胺键偶联,MTT法检测偶联毒素对人脐静脉内皮细胞ECV304和肝癌细胞HepG2的抑制作用。结果合成的偶联毒素经核磁共振和质谱分析鉴定,表明结构正确,纯度达90%以上,对HepG2细胞的抑制效果弱于苯丁酸氮芥,但对ECV304细胞的抑制特异性较好。结论Integrinαvβ3拮抗剂-苯丁酸氮芥偶联毒素可抑制HepG2和ECV304细胞的增殖,为癌症治疗提供了一个新途径。     

3株新城疫病毒体外对人喉癌Hep-2细胞杀伤效应的比较

目的比较新城疫病毒(NDV)强毒株D817、弱毒株7793和La Sota疫苗株对人喉癌Hep-2细胞的杀伤效应。方法3株病毒经扩增、噬斑纯化后,分别感染Hep-2细胞和喉正常组织细胞,一定时间后,显微镜下观察细胞形态变化,MTT比色法检测病毒对细胞的杀伤效应。结果3株病毒均能在Hep-2细胞中增殖并杀伤细胞,杀伤效应以D817株最高,La Sota株其次,7793株最低,且差异均无统计学意义。杀伤效应与病毒剂量和作用时间呈正相关,显微镜下可见明显的细胞病变。而对喉正常组织细胞的杀伤效应不明显。结论NDV D817株和7793株对喉癌细胞的杀伤效应与La Sota疫苗株相似,而对喉正常组织细胞的杀伤效应不明显,有望成为喉癌生物学治疗新的候选病毒株。     

Enhanced Anti-Tumoral Activity of Methotrexate-Human Serum Albumin Conjugated Nanoparticles by Targeting with Luteinizing Hormone-Releasing Hormone (LHRH) Peptide

Active targeting could increase the efficacy of anticancer drugs. Methotrexate-human serum albumin (MTX-HSA) conjugates, functionalized by luteinizing hormone-releasing hormone (LHRH) as targeting moieties, with the aim of specifically targeting the cancer cells, were prepared. Owing to the high expression of LHRH receptors in many cancer cells as compared to normal cells, LHRH was used as the targeting ligand in this study. LHRH was conjugated to MTX-HSA nanoparticles via a cross-linker. Three types of LHRH targeted nanoparticles with a mean particle size between 120-138 nm were prepared. The cytotoxicity of LHRH targeted and non-targeted nanoparticles were determined on the LHRH positive and negative cell lines. The internalization of the targeted and non-targeted nanoparticles in LHRH receptor positive and negative cells was investigated using flow cytometry analysis and fluorescence microscopy. The cytotoxicity of the LHRH targeted nanoparticles on the LHRH receptor positive cells were significantly more than non-targeted nanoparticles. LHRH targeted nanoparticles were also internalized by LHRH receptor positive cells significantly more than non-targeted nanoparticles. There were no significant differences between the uptake of targeted and non-targeted nanoparticles to the LHRH receptor negative cells. The active targeting procedure using LHRH targeted MTX-HSA nanoparticles could increase the anti-tumoral activity of MTX.     

Methotrexate Ameliorates Systemic Inflammation and Septic Associated-Lung Damage in a Cecal Ligation and Puncture Septic Rat Model

Background: Sepsis is a serious, heterogeneous clinical entity produced by a severe and systemic host inflammatory response to infection. Methotrexate (MTX) is a folate-antagonist that induces the generation of adenosine and also inhibits JAK/STAT pathway; MTX it is widely used as an anti-inflammatory drug to control the immune system. Objective: The aim of this study was to assess the beneficial effects of a single and low dose of MTX in the systemic response and acute lung injury (ALI) induced by sepsis. As in the clinics, we treated our animals with antibiotics and fluids and performed the source control to mimic the current clinic treatment. Methods and main results: Sepsis was induced in rats by a cecal ligation puncture (CLP) procedure. Six hours after induction of sepsis, we proceeded to the source control; fluids and antibiotics were administered at 6 h and 24 h after CLP. MTX (2.5 mg/Kg) was administered 6 h after the first surgery in one CLP experimental group and to one Sham group. A protective effect of MTX was observed through a significant reduction of pro-inflammatory cytokines and a decrease infiltration of inflammatory cells in the lung. In addition, we found a regulation in adenosine receptor A2aR and the metalloproteinases by MTX. Conclusion: A single, low dose of MTX attenuates sepsis lung-associated damage by decreasing pro-inflammatory response, infiltration of pro-inflammatory cells and avoiding defective tissue lung remodeling.