除去人血白蛋白中热原质方法的探讨（低温乙醇法）

根据热原质的性质,在热原质污染的人血白蛋白制品中加入新鲜冰冻血浆,用低温乙醇法工艺,经两步纯化可有效地去除制品中的热原质,制品回收率达90％以上,制品质量符合部颁标准。     

脑脊液微量白蛋白与血清白蛋白比值的临床意义

2007年3月-2008年1月我们对收治的85例神经系统疾病患者进行脑脊液(CSF)微量白蛋白(CAlb)检测,分析CAlb与血清白蛋白(SAlb)的比值,从而探讨CAlb与SAlb比值的临床意义,现将结果报告如下.     

赤丹退黄颗粒乙醇提取工艺正交试验研究

目的 为赤丹退黄颗粒提取工艺提供依据。方法 采用正交设计法，因素为溶剂用量、浓度、提取时间和次数，以芍药苷及出膏率指标综合分析。结果 最佳组合为A2B2C3D2，但考虑实际生产溶剂回收、降低成本、节约时间等因素，确定乙醇提取工艺为用5倍量的70％乙醇提取3次，每次2h，即A2B3C3D2。结论 提取时间对芍药苷和出膏率的影响最大，溶剂用量、浓度和提取次数三项水平间没有显性差异，特别是乙醇浓度在三水平间没有显性差异。     

延胡索醇回流法与超声法提取工艺比较研究

目的探讨延胡索药粉超声波提取的可行性,为工业化生产提供依据。方法以延胡索乙素为评价指标,首 先采用正交设计试验法筛选延胡索药粉超声波提取与醇回流提取各自的最优工艺条件,再用配对t检验法分析实验数据。结 果延胡索醇超声提取最优工艺参数为：延胡索药粉加人50%乙醇浸泡30 min,50%乙醇体积为药材量的6倍,超声时间1.0h。延胡索醇回流提取最优工艺参数为：延胡索药粉加入50%乙醇浸泡30 min,50%乙醇体积为药材量的10倍,水浴回流时间1.0 h。延胡索药粉醇回流与超声提取差别有统计意义,回流提取优于超声提取。结论延胡索采用回流提取为佳,按最 优工艺条件提取,延胡索乙素含量最高。     

猪血清白蛋白基因的克隆及其5′端调控序列功能分析

目的：克隆猪血清白蛋白全基因并对其5′端调控序列的转录功能进行分析。方法：以GenBank公布的猪血清白蛋白基因启动子序列和cDNA序列为依据，设计并合成D123／D61和D81／D84两对引物，同时从猪肝中提取猪基因组DNA，并以此为模板PCR扩增猪血清白蛋白部分基因片段作为噬菌斑原位杂交探针，对猪λ噬菌体文库进行筛选。经过4轮杂交和PCR交替筛选，从噬菌体文库中共获得4个基因片段，并对这4个片段进行测序、拼接。以绿色荧光蛋白作为报告基因，对猪血清白蛋白启动子在Hep(泛细胞中的表达进行研究。结果：共获得包括调控区在内的猪血清白蛋白全基因序列约35kb(GenBank登录号：AY663543)。将其与人的血清白蛋白基因相比对，发现基因的同源性为53.8％，其中编码区同源性为82．3％。荧光显微镜下可见EGFP在HepC2细胞中的表达。结论：获得猪血清白蛋白全基因。猪血清白蛋白基因启动子在HepC2有转录起始功能。此项工作一方面为利用基因打靶从猪体内制备生产人血清白蛋白的研究奠定了良好基础，另一方面为利用白蛋白启动子研究外源基因在肝组织的特异性表达及进行基因治疗提供了资料。     

评价改良的大鼠肝脏组织两步蛋白提取法

目的 探讨简单、易行的大鼠肝脏组织蛋白提取方法。方法 应用改良的大鼠肝脏组织两步蛋白提取法。结果 此方法提取蛋白方法简单，蛋白浓度高，易溶解，不易凝固、降解，电泳分离快。结论 该方法简单易行，费用低，质量高，可认为改良的两步法是提取大鼠肝脏组织蛋白的最佳方法。     

超临界CO2法与乙醇回流法提取山茱萸中熊果酸含量的比较

目的考察超临界CO2法、乙醇回流法提取山茱萸中熊果酸的效果。方法分别采用超临界CO2法、乙醇回流法提取山茱萸中的熊果酸,以熊果酸作检测指标进行定量分析。结果2种方法所得提（萃）取物得率、熊果酸含量之间具有显著性差异。超临界CO2法所得萃取物得率（2．28％）较乙醇回流法（38．61％）低,其熊果酸含量（0．11％）也较乙醇回流法（0．25％）低。结论提取以熊果酸为主要成分的活性物质,乙醇回流法较好。     

正交设计优选白芷的渗漉法提取工艺

目的 优选白芷的渗漉法提取工艺.方法 以欧前胡素的含量为指标,应用正交试验设计筛选白芷的最佳提取工艺条件.结果 最佳提取工艺为A2B2C3D1,即用8倍量的70%乙醇浸泡24h,渗漉速度2m/mim.结论 优选得到的工艺稳定可行.     

猪血清白蛋白基因的克隆及其5''''端调控序列功能分析

目的克隆猪血清白蛋白全基因并对其5′端调控序列的转录功能进行分析.方法以GenBank公布的猪血清白蛋白基因启动子序列和cDNA序列为依据,设计并合成D123/D61和D81/D84 两对引物,同时从猪肝中提取猪基因组DNA,并以此为模板PCR扩增猪血清白蛋白部分基因片段作为噬菌斑原位杂交探针,对猪λ噬菌体文库进行筛选.经过4轮杂交和PCR交替筛选,从噬菌体文库中共获得4个基因片段,并对这4个片段进行测序、拼接.以绿色荧光蛋白作为报告基因,对猪血清白蛋白启动子在HepG2细胞中的表达进行研究.结果共获得包括调控区在内的猪血清白蛋白全基因序列约35 kb(GenBank登录号AY663543).将其与人的血清白蛋白基因相比对,发现基因的同源性为53.8%,其中编码区同源性为82.3%.荧光显微镜下可见EGFP在HepG2细胞中的表达.结论获得猪血清白蛋白全基因.猪血清白蛋白基因启动子在HepG2有转录起始功能.此项工作一方面为利用基因打靶从猪体内制备生产人血清白蛋白的研究奠定了良好基础,另一方面为利用白蛋白启动子研究外源基因在肝组织的特异性表达及进行基因治疗提供了资料.     

严重感染患者血清白蛋白动力学研究

目的了解感染状态下血清白蛋白的分解和分布动力学的变化,以进一步明确感染患者低白蛋白血症的发生机制。方法对照组10例,全部为男性健康志愿者;感染组10例,均为严重感染患者,APACHEⅡ评分8～22(平均13)。采用氯胺T法,用125I 标记人体白蛋白。所有受试者一次性从上臂静脉注射20μCi的125I白蛋白,分别在第0、1、2、4、8、12、24h,第2、3、4、5、6、7、9、11、13、15、18、22、25天抽血,测定γ射线量(dpm),拟合浓度时间曲线,计算标记白蛋白的半衰期(t1/2),分布容积(Vd),中央池向周边池的转运速率(K12)。结果感染组的125I标记白蛋白t1/2(d)明显短于对照组(8.2±1.4vs.12.5±1.7,P<0.01);感染组标记白蛋白从中央池向周边池的转运速率(K12)显著高于对照组[(4.4±1.9)×10-2/hvs.(2.4±0.6)×10-2/h,P<0.05];两组分布容积(Vd)则没有显著的差异(P>0.05)。结论在感染条件下,血清白蛋白从血管内到血管外的分布速率明显增加,白蛋白的分解速率也显著增加。     

人血白蛋白的功能及其在危重病治疗中的应用

人血白蛋白(HSA)是一种小分子量、非糖基化、带负电荷的血清蛋白,具有物质结合转运、协调维持血管内皮完整、抗氧化、抗炎、脏器功能保护、维持胶体渗透压等多种功能.综合分析现有的研究及Meta分析结果,HSA用于危重症治疗,可降低并发症发生率,对某些疾病如脓毒症、肝硬化合并自发性腹膜炎等,可显著改善患者预后,但不能确定其可降低危重病患者的病死率.     

苦参素白蛋白微球的制备

利用牛血清白蛋白为材料，制备了苦参素白蛋白微球。该微球直径小于２μｍ，含有苦参素２．１４％。体外实验证实，在ｐＨ７．４的０．１ｍｏｌ／Ｌ磷酸盐缓冲液中缓释作用明显，符合一级释放机理。     

米诺环素与牛血清白蛋白相互作用的研究

目的:研究人体生理条件下米诺环素与牛血清白蛋白(BSA)的相互作用机制.方法:利用荧光光谱法,并以Stern-Volmer方程确定药物与蛋白的作用类型.结果:根据不同温度下米诺环素对BSA的荧光猝灭作用,证明两者间为单一的动态猝灭过程,根据Stern-Volmer方程求出了米诺环素与牛血清白蛋白(BSA)的猝灭常数,并根据Fǎrster能量转移理论确定了生理条件下药物与蛋白的结合距离为3.03 nm.结论:在人体生理条件下米诺环素对牛血清白蛋白具有荧光猝灭作用且为动态猝灭过程.同步荧光技术确定米诺环素对BSA构象有一定的影响.     

血清-腹水白蛋白梯度与门脉高压性胃病的关系

 目的 测定门脉高压性胃病(portal hypertensive gastropathy,PHG)患者血清-腹水白蛋白梯度(serum ascites albumin gradient,SAAG),探讨PHG与SAAG之间的关系。方法 127例肝硬化并腹水病例分为PHG组与非PHG组,测量并比较两组SAAG;分析PHG组中SAAG与肝功能Child-Pugh分级、终末期肝病模型(MELD)评分、食管静脉曲张程度、门静脉直径及血小板计数/脾大小的关系;对PHG组中轻、重度情况下SAAG进行比较分析;绘制SAAG受试者工作特征(ROC)曲线,确定诊断PHG的SAAG最佳界限值。结果 PHG组与非PHG组的SAAG比较差异有统计学意义;PHG组的SAAG与Child-Pugh分级、MELD评分、食管静脉曲张程度呈正相关,不同水平SAAG的门静脉直径、血小板计数/脾大小比较差异有统计学意义;轻度PHG与重度PHG的SAAG比较差异有统计学意义;ROC曲线表明SAAG诊断PHG的最佳界值是18.57 g/L,敏感度和特异度分别为72.1%和93.2%。结论 SAAG反映了PHG病程中的门脉高压程度,对预测PHG的发生并判断其病情及预后有临床价值。     

Magnetization transfer in cross-linked bovine serum albumin solutions at 200 MHz: A model for tissue

We report results for proton 1/T₁, 1/T₂, and K, the rate of magnetization transfer from solvent to solute, for 5 and 10 wt. % solutions of bovine serum albumin, both native and chemically cross-linked, in undeuterated and ～50% deuterated water, at 4.7 T (200.1 MHz) and 19°C. At this field, although K > 1/T₁ for the cross-linked samples, magnetization transfer contributes little to 1/T₁ directly. Therefore K was measured using off-resonance irradiation of the protein protons. The data for all the samples can be fit using a theoretical model for magnetization transfer, with three parameters: the intrinsic longitudinal relaxation rates of solute and solvent protons, and K. The magnitude of Kis so large that the newly-identified, long-lived (～1 μs) hydration sites (S. H. Koenig, R. D. Brown III, and R. Ugolini, Magn. Reson. Med., 29, 77 (1993)) must be invoked to account for K, as is necessary to explain the differential effects of cross linking on the magnetic field dependence of 1/T₁ of protons and deuterons and the large 1/T₁ and 1/T₂ values below ～20 MHz in immobilized systems. Although these sites are few in number, their long resident lifetime becomes the correlation time for magnetization transfer when protein is immobilized, accounting for the large value of K. Recent data from several laboratories have shown that cross-linked protein, as used here, is a good model for 1/T₁ and 1/T₂ of tissue, as a function of temperature and magnetic field.     

Experimental biodistribution studies of 99mTc-recombinant human serum albumin (rHSA): a new generation of radiopharmaceutical

Recombinant human serum albumin (rHSA) produced by cultured fermentation has been prepared in the form of microcapsules nominally 3–5 m in diameter and radiolabelled with technetium-99m following reduction with stannous chloride. Radiochemical purity was assessed by chromatography on instant thin-layer chromatography and found to be greater than 90%. No evidence of aggregation was seen by microscopic examination. Imaging biodistribution studies in New Zealand white rabbits demonstrated targeting to the liver or lung, respectively, depending upon the size and surfactant properties of the microcapsules. This communication is the first to show scintigraphic studies using ^99m-Tc-labelled rHSA with the potential for lung, liver and cardiovascular imaging and demonstrates that recombinant DNA technology offers an important new source of materials suitable for use as radiopharmaceuticals without the need for pooled human blood products.     

Technetium-99m labelled human serum albumin for ventriculography: a comparative evaluation of six labelling kits

In this study we have compared the characteristics of six labelling kits for the preparation of technetium-99m labelled human serum albumin (^99mTc-HSA) and evaluated the usefulness of the various ^99mTc-HSA preparations as blood pool tracer agents. The amount of the principal ingredients, i.e. HSA and stannous ions, varies largely between the studied kits and this is probably a reason for the observed differences in the labelling rate. Analysis of the reaction mixtures after labelling of the respective kits with ^99mTc showed in each preparation the presence of four to five radioactive components in variable relative amounts. The retention time of the main component on size-exclusion high-performance liquid chromatography (SEC-HPLC) was identical for all preparations. Biodistribution of the HPLC-isolated fractions was studied in mice. The components with the shortest and longest retention times on HPLC show poor retention in the plasma. The three intermediate fractions, including the principal peak, are initially retained relatively well in the blood (60%–70% of the injected dose after 10 min), but clearly to a lower degree than iodine-125 labelled HSA. Moreover, they diffuse out of the vascular compartment at a much higher rate than ¹²⁵I-HSA. The biological behaviour of the main component of the various preparations was clearly different, despite the identical retention time on SEC-HPLC. Study of the total preparations in mice and a rabbit showed that two of them are cleared rapidly from the blood and cannot be considered valuable blood pool tracers. Diffusion of the other preparations out of the blood is slower but also considerable and compromises their use for ventriculography.K.A. Verbeke is a Research Assistant for the Belgian National Fund for Scientific Research     

Comparison of the stability of Y-90-, Lu-177- and Ga-68- labeled human serum albumin microspheres (DOTA-HSAM)

IntroductionMicroparticles derived from denatured human serum albumin (DOTA-derivatized human serum albumin microspheres, or DOTA-HSAM) are attractive carriers of radionuclides for both therapeutic and diagnostic purposes. In this article, we describe a labeling procedure for diagnostic (Ga-68) and therapeutic (Y-90, Lu-177) radionuclides and report on the results of stability studies of these products.MethodsDOTA-HSAM was labeled in 0.5 M ammonium acetate buffer, pH 5.0, containing 0.02 mg/ml detergent. After adding the radionuclide, the mixture was shaken for 15 min at 90°C. Labeling yields and in vitro stability were determined by thin-layer chromatography. For determination of the in vivo stability of Ga-68 and Y-90 DOTA-HSAM, the particles were injected intravenously in Wistar rats.ResultsLabeling yields up to 95% in the case of Ga-68 and Lu-177 were achieved. Ga-68-labeled DOTA-HSAM showed high in vitro and in vivo stability. The amount of particle-bound radioactivity of Lu-177 DOTA-HSAM declines slowly in a linear manner to approximately 72% after 13 days. For Y-90, the labeling yield decreased with increasing radioactivity level. We presume radiolysis as the reason for these findings.ConclusionThe labeling of DOTA-HSAM with different radionuclides is easy to perform. The radiation-induced cleavage of the labeled chelator together with the rather short half-life of radioactivity fixation in vivo (3.7 days) is, in our opinion, opposed to therapeutic applications of DOTA-HSAM. On the other hand, the high stability of Ga-68 DOTA-HSAM makes them an attractive candidate for the measurement of regional perfusion by PET.     

重组人血清白蛋白在酵母中的表达及分析鉴定

目的：实现重组人白蛋白（rHSA)在巴斯德毕赤酵母(Pichia pastoris)中的高效表达,方法：将本室构建的rHSA表达载体转化毕赤酵母,用G418筛选抗性克隆,SDS－PAGE与Western印迹检测并鉴定表达产物;采用生化法、电泳扫描及Brodford法、竞争ELISA3种方法测定摇瓶培养条件下rHSA的表达量。结果：Western印迹分析发现转移至膜上的酵母表达产物能与抗人血清白蛋白单克隆抗体结合,相对分子质量与人血白蛋白一致,证明在酵母中成功地表达了rHSA。在甲醇诱导下摇瓶培养,rHSA表达量随诱导表达时间的延长而增加,第8天产量约为3．5g/L。结论：人血清白蛋白表达载体与毕赤酵母基因组整合,获得酵母表达株,摇瓶培养,该株表达量为3．5g/L。