头孢洛扎硫酸盐制备方法的改进

将起始原料ACLE·HCl首先与7-位侧链噻二唑化合物TATD反应，然后再与吡唑化合物UBT进行反应，并用三氟乙酸进行脱保护基反应。最后经过纯化析晶，合成头孢洛扎硫酸盐。该工艺得到头孢洛扎硫酸盐的纯度高，原料易得，纯化时不需要过柱，更适合工业化生产。     

Toxicity of palytoxin,purified ovatoxin-a,ovatoxin-d and extracts of Ostreopsis cf. ovata on the Caco-2 intestinal barrier model

Human intoxications in the Mediterranean Sea have been linked to blooms of the dinoflagellate Ostreopsis cf. ovata, producer of palytoxin (PlTX)-like toxins called ovatoxins (OVTXs). Exposure routes include only inhalation and contact, although PlTX-poisoning by seafood has been described in tropical regions. To address the impact of OVTXs on the intestinal barrier, dinoflagellate extracts, purified OVTX-a and -d and PlTX were tested on differentiated Caco-2 cells. Viability, inflammatory response and barrier integrity were recorded after 24 h treatment. OVTX-a and -d were not cytotoxic up to 20 ng/mL but increased IL-8 release, although to a lesser extent compared to PlTX. While PlTX and OVTX-a (at 0.5 and 5 ng/mL respectively) affected intestinal barrier integrity, OVTX-d up to 5 ng/mL did not. Overall, OVTX-d was shown to be less toxic than OVTX-a and PlTX. Therefore, oral exposure to OVTX-a and -d could provoked lower acute toxicity than PlTX.     

Epitope selection and their placement for increased virus neutralization in a novel vaccination strategy for porcine epidemic diarrhea virus utilizing the Hepatitis B virus core antigen

Porcine epidemic diarrhea virus (PEDV) is a member of the Alphacoronaviridae genus within the Coronaviridae family. It is the causative agent of porcine epidemic diarrhea, a disease that can have mortality rates as high as 100% in suckling piglets. PEDV causes severe economic loss, and has been in existence for decades. A panzootic starting in 2010 renewed interest in the development of a universal vaccine toward PEDV. This report details several design changes made to a Hepatitis B virus core antigen (HBcAg)-based recombinant vaccine strategy, and their effect in vivo. Initially, several multi-antigen vaccine candidates were able to elicit antibodies specific to three out of four B-cell epitopes inserted into the chimeric proteins. However, a lack of virus neutralization led to a redesign of the vaccines. The focus of the newly redesigned vaccines was to elicit a strong immune response to the YSNIGVCK amino acid motif from PEDV. Genetically modified new vaccine candidates were able to elicit a strong antibody (Ab) response to the YSNIGVCK epitope, which correlated with an increased ability to neutralize the CO strain of PEDV. Additionally, the location of the inserted PEDV epitopes within the vector protein was shown to affect the immune recognition toward the native HBcAg during vaccination.     

乳鼠面神经雪旺细胞的体外培养及其纯化

目的：体外培养雪旺细胞（SCs）时,在去除成纤维细胞（FCs）后,应用人参皂甙Rb1促进SCs的增殖以获得大量纯净的SCs,为周围神经组织工程学的发展奠定良好的基础.方法：本实验采用高浓度混合酶从乳鼠面神经中消化分离SCs,结合差速粘贴和阿糖胞苷处理进行纯化后,再用人参皂甙Rb1促进其分裂增殖.结果：S-100蛋白免疫荧光染色呈阳性反应的比例约占98%.结论：本方法快速、简便,能为SCs作为移植材料提供足够的来源,是纯化和促进SCs增殖较为理想的方法,具有潜在的临床应用前景.     

AB-8型大孔吸附树脂纯化左金丸有效部位的工艺优选

目的:优选左金丸的大孔树脂纯化工艺。方法:以总生物碱、盐酸小檗碱、吴茱萸碱的吸附-洗脱率为指标,采用单因素试验考察上样液质量浓度、径高比、洗脱剂等对左金丸大孔树脂纯化工艺的影响。结果:优选的纯化工艺为上样液质量浓度0.14g/mL,树脂柱径高比1︰10,上样量与树脂体积比1︰2.4(吸附3h),加2BV水洗除杂,用7BV70%乙醇洗脱,收集洗脱液。结论:该纯化工艺合理、稳定,可推广于大生产应用。     

黄蜀葵花中金丝桃苷的提取分离及纯度检测

目的 研究黄蜀葵花中金丝桃苷的提取、分离、纯化方法.方法 采用聚酰胺柱层析、梯度洗脱方法提取分离黄蜀葵花中的金丝桃苷,以氢谱和碳谱鉴定其结构,以薄层色谱法、HPLC法进行纯度检查.结果 制得的金丝桃苷经薄层色谱法检查无杂质斑点,经HPLC法检测其纯度98.5%以上.结论 所建立的分离、纯化方法简便,成本低.     

人髓核间充质干细胞的分离提纯方式及生物学活性鉴定

目的 :探索人髓核间充质干细胞(nucleus pulposus mesenchymal stem cells,NPMSCs)的提纯方法并鉴定其生物学活性。方法:收集3例腰椎间盘突出症患者的退变髓核组织(Pfirrmann分级均为Ⅳ级),利用酶消化法分离细胞。采用两种方法分离提纯NPMSCs,一组细胞采用贴壁法培养(贴壁组),另一组通过流式细胞分选技术利用NPMSCs表面阳性标志物CD73、CD90、CD105获得NPMSCs(流式组)。将两种方法获得的NPMSCs进行体外培养扩增,分别进行形态学观察,细胞计数试剂盒(Cell Counting Kit,CCK-8)检测增殖能力。贴壁组NPMSCs采用流式细胞分选仪在进行分选之前检测免疫表型,流式组NPMSCs在生长达80%~90%融合时进行免疫表型的检测。向成骨、成脂、成软骨诱导分化,诱导28d后分别进行茜素红染色观察其成骨能力、油红O染色观察其成脂能力、甲苯胺蓝染色观察其成软骨能力,利用Imag J软件计算染色区域所占的面积百分比。比较两组NPMSCs在形态学、免疫表型及增殖和分化能力的差异。结果:形态学观察发现,两组NPMSCs均呈漩涡状生长,贴壁组NPMSCs可见散在的单个细胞生长;流式组NPMSCs长梭形形态更长,排列更加紧密,少见散在的单个贴壁生长细胞。流式细胞分选后所得的NPMSCs占细胞总数的(89.67±2.52)%,可以进行体外培养扩增,细胞为典型的长梭形特征,漩涡状生长,在接种后12~15d达80%~90%融合,增殖能力在接种后5~13d明显高于贴壁组NPMSCs(P0.05)。流式组NPMSCs的CD73、CD90、CD105的表达率明显高于贴壁组NPMSCs(P0.05),并且低表达CD34、CD45及HLA-DR。两种方法获得的NPMSCs均能完成三系诱导分化,流式组成骨、成脂、成软骨染色区域百分比均明显高于贴壁组(P0.05)。结论:利用流式细胞分选技术从人退变髓核组织中可获得较高纯度的NPMSCs,并能进行后续培养扩增。与贴壁法获得的NPMSCs相比,流式细胞分选的NPMSCs具有更强的增殖与分化能力。     

星点设计-效应面法优化刺五加总黄酮大孔树脂纯化工艺

目的:应用星点设计-效应面法优化刺五加总黄酮大孔树脂纯化工艺。方法:以上样质量浓度、乙醇浓度、乙醇洗脱用量为自变量,总黄酮洗脱率为因变量,对自变量中各水平多元线性回归及二项式拟合,用星点设计-效应面法优化出最佳纯化工艺。结果:单因素试验基础上,选择AB-8型大孔树脂,结合实际确定最佳纯化工艺为上样质量浓度为0.2g/m L,乙醇浓度为80%,乙醇洗脱用量为6.0BV,在此最佳条件下,总黄酮洗脱率的最大值达92.5%,与预测值(91%)相符。结论:采用星点设计-效应面法优化的大孔树脂纯化工艺方法简便,稳定可行,预测性好。     

Antigenicity of a recombinant NS3 protein representative of ATPase/helicase domain from hepatitis C virus

It has been shown that the Hepatitis C virus nonstructural NS3 protein possesses at least two enzymatic domains: a serine-protease domain and an adenosine triphosphatase (ATPase)/helicase domain. In this report, a truncated fragment of NS3 (26 kDa), representing main epitopes from the (ATPase)/helicase domain, has been expressed in Escherichia coli. The recombinant protein was purified by Ion Metal Affinity Chromatography (IMAC) with more than 90% purity. The recognition of B-cell linear epitopes in the NS3 protein was evaluated by immunoblot. The recombinant NS3 protein was reduced and carboxymethylated, and the recognition of either conformational and/or linear B-cell determinants was evaluated by ELISA. The inclusion of the recombinant NS3 protein in a third-generation diagnostic system UltraMicroELISA (UMELISA) allowed an increase in the sensitivity, due to the detection of a new variety of false-negative sera in blood donor test samples.     

Turnover of 125I-labelled tissue kallikrein following intraduodenal or intravenous administration

Tissue kallikrein is released in the body both physiologically and in many inflammatory disorders. Little is, however, known about the turnover of released tissue kallikrein in humans. Approximately 1 mg of tissue kallikrein (mol wt 43 000 Da) was purified from 85 L human urine by: (1) ultracentrifugation, (2) filtration through an aprotinin-coupled Sepharose 4B column, followed by (3) gel filtration over a Sephadex G-75 column. The elimination, after intraduodenal or intravenous administration of purified tissue kallikrein radiolabelled with ¹²⁵I, was followed by collecting serial samples of plasma, urine and faeces from three volunteers. Within 72 h, about 96% of the intraduodenally administered radioactivity had been excreted in urine, and approximately 5.4% in faeces, mainly as ¹²⁵I. No intact ¹²⁵I-tissue kallikrein was found in plasma, urine or faeces after the intraduodenal instillation of the protein. The plasma half-life of ¹²⁵I-tissue kallikrein up to 3 h after intravenous injection was 9 min and, thereafter, 20 h. The ¹²⁵I-tissue kallikrein was quickly bound to a plasma protein with a mol wt of about 67 kDa, but some of the radioiodinated tissue kallikrein was still unbound 15 min after injection, judged by gel filtration on Sephadex G-200 columns. Most of the radioactivity was excreted in the urine as ¹²⁵I, but about 4- 6% was recovered as free ¹²⁵I-tissue kallikrein. Conclusion: The use of tissue kallikrein as an oral drug appears, therefore, to be useless. Tissue kallikrein released into plasma seems to be quickly bound to a protein with a mol wt of 67 kDa, probably kallistatin or Protein C inhibitor, but some tissue kallikrein seems to be unbound and may have some physiological or pathophysiological action. The unbound tissue kallikrein is, at least partly, cleared from the circulation by the kidneys, and tissue kallikrein in the urine may partly be derived from plasma.