人凝血酶原复合物在制备过程中凝血因子活性的检测和分析

目的对人凝血酶原复合物在制备工艺中凝血因子效价进行检测和分析。方法对PCC制备过程中的血浆、S/D病毒灭活、冻干工艺、干热病毒灭活前和后分别取样,检测分析凝血因子Ⅱ、Ⅶ、Ⅸ和Ⅹ效价,分别分析S/D病毒灭活、冻干工艺、干热病毒灭活前和后凝血因子效价的变化情况。分析PCC干热病毒灭活后的样品中四种凝血因子的效价比例。结果三批血浆中凝血因子Ⅱ、Ⅶ、Ⅸ和Ⅹ的活性在0.8~1.2 IU/m L之间。S/D病毒灭活前后PCC冻干工艺前后PCC中四种凝血因子活性无明显变化,而干热病毒灭活后PCC中四种凝血因子活性均有明显下降。三批PCC中四种凝血因子的比例基本一致,但是凝血因子Ⅶ效价较低,凝血因子Ⅱ效价偏高。结论通过PCC制备工艺中凝血因子效价的检测和分析可实现良好的质量控制。     

S/D灭活血浆内脂包膜病毒及病毒灭活血浆的研究

研究磷酸三丁酯(TNBP)／Triton X-100对血浆内脂包膜病毒的灭活效果。用VSV病毒和Sindbis病毒作指示病毒,加入血浆后再加磷酸三丁酯／Triton X-100,观察病毒的滴度变化及对血浆蛋白的影响。结果发现终浓度各为1％的磷酸三丁酯／Triton X-100在60min内可以灭活血浆内的两种指示病毒,而血浆蛋白的组成和功能变化很小。经层折、超滤后血浆内磷酸三丁酯和Triton X-100的残余量分别低于5μg/ml,表明S／D处理血浆的安全性和治疗作用都很好,其制剂冰冻血浆或冻干血浆可用于临床治疗凝血因子缺乏症,或用作血容量扩张剂。     

用β-丙内酯/紫外线照射灭活血浆和血浆衍生物中的肝炎病毒和HIV

<正>1956年,由LoGrippo和Hartmann介绍的人血浆冷灭菌法结合了化学试剂β-丙内酯/(β-PL)与紫外线照射的光化学灭菌效果。在不稳定血浆蛋白中所含病毒灭活的主要问题是破坏病毒和保留血浆蛋白的大部分活性。β-PL/UV灭菌过程导致活性的丢失对各蛋白是不同的。用该灭菌工艺对白蛋白和免疫球蛋白的活性不受影响。凝血因子的大部分活性降低。因为冷沉淀是生产FⅧ的原材料,对该物质用β-PL/VV的灭菌条件不同于对血浆的灭菌条件,Ⅷ浓缩物冷灭菌法的大规模生产至今还没有解决。Blotest静注免疫球蛋白制品用β-PL进行灭菌,未经UV照射。     

S／D处理血浆过程中的脂包膜病毒灭活试验观察

通过有机溶剂/去污剂对血浆中指示病毒VSV灭活的观察评估有机溶剂/去污剂对脂包膜病毒死活的效果。血浆样品与VSV病毒按9:1混合,然后用1％TNBP/1％ Triton X-100在30℃处理4h,测定开始样品中的病毒总量和S/D处理后不同时间取样内的病毒总量。实验中样品内加入1％TNBP/1％ Triton X-10015min后VSV病毒已全部灭活,灭活效果≥6.0log。按所述S/D处理方法可以完全灭活血浆内所有的脂包膜病毒而没有主要血浆蛋白的损失。     

S/D法灭活血液制剂中脂包膜病毒效果验证的研究

选用不同核酸的脂包膜病毒,其中RNA病毒为水疱性口炎病毒(VSV),DNA病毒为伪狂犬病毒(PRV),将两种指示病毒分别用于验证S/D法处理对纤维蛋白原、凝血酶原复合物、凝血因子Ⅷ、静注丙种球蛋白、免疫血浆等血液制剂的病毒灭活效果。结果该法对所有被处理的血液制剂中的PRV及VSV灭活能力分别为≥3.38~5.88和≥3.50~4.75logTCID50/0.1ml,表明S/D法对两种病毒核酸类型的脂包膜病毒有良好的灭活效果。     

亮氨酸拉链提高小鼠血浆中剪接的凝血因子Ⅷ凝血活性

培养细胞实验表明,亮氨酸拉链通过改善内含肽(intein)的蛋白质剪接效率,提高双载体转B区缺失型凝血因子Ⅷ(BDD-FⅧ)基因细胞剪接FⅧ蛋白的分泌量和活性.本文从C57BL/6小鼠门静脉注射含亮氨酸拉链和Ssp DnaB内含肽融合的BDD-FⅧ的重链和轻链基因双表达载体,48 h后,检测到血浆的重链分泌量和FⅧ活性分别为(298±67)μg/L和(1.15±0.29)U/mL,明显高于不含亮氨酸拉链的双载体转BDD-FⅧ基因对照小鼠((179±59)μg/L和(0.58±0.19)U/mL).结果表明,亮氨酸拉链通过改善蛋白质反式剪接,提高基于蛋白质剪接的双载体转BDD-FⅧ基因小鼠血浆的凝血活性,为进一步双腺相关病毒(AAV)载体转BDD-FⅧ基因的甲型血友病基因治疗研究提供了依据.     

人凝血因子Ⅷ工艺研究进展

人凝血因子Ⅷ(Human coagulation factorⅧ,FⅧ)是人血浆中重要的蛋白质,是人体内源性凝血途径的主要成分,血液中FⅧ过低会导致不同程度的凝血功能障碍。目前国内FⅧ大多以血浆冷沉淀为起始原料,采用离子交换层析纯化获得"#$。在此工艺中,血浆的采集、血浆融化、冷沉淀离心、冷沉淀的溶解、层析前样品的预纯化、离子交换层析条件、病毒灭活、冻干工艺均对FⅧ的质量影响较大,因此主要从这几方面进行了综述。     

有机溶剂／表面活性剂处理人血浆的生产和体外鉴定

作者发展了用最终浓度1％( W/W) TNBP和Triton-100于30℃保温4小时的改良有机溶剂/表面活性剂(S/D)处理法灭活人血浆病毒。本法灭活≥10~6黑猩猩感染剂量(CID_(50))的HBV,≥10~5GID_(50)的HCV和≥10~(6·2)组织培养感染剂量(TCID_(50))的HIV。病毒灭活后的11批血浆被冻干,12批被冰冻直到使用。上述血浆经过了广泛的实验室试验,包括凝血因子1-XIII, Von willebrand因子,血浆蛋白溶酶原,血凝抑制剂,纤维蛋白溶酶和其他临床上重要的血浆蛋白质的鉴定。在S/D处     

亚甲蓝光化学法灭活新鲜冰冻血浆病毒及其效果评价

目的:探讨以亚甲蓝光化学法(MB-P)灭活新鲜冰冻血浆病毒及其效果。方法:选取2013年3月-2016年11月来青海省血液中心无偿献血者捐献的400 mL全血共1500份,分离新鲜冰冻血浆200 mL后,再分为两份即对照组和观察组各100 mL,各1500份。对照组不经灭活,实验组经MB-P病毒灭活。比较两组的纤维蛋白原(FIB)、总蛋白(TP)、凝血因子VⅢ(FVⅢ)及白细胞(WBC)的含量。再随机抽取500例接受了上述病毒灭活血浆输注的患者,观察其输血反应的发生情况。结果:观察组的FIB和TP含量及FVⅢⅠ活性比对照组有所降低,但差异不具有统计学意义(P0.05);观察组的WBC含量较对照组明显降低,且具有统计学差异(P0.05);FIB、TP、FVⅢⅠ的回收率的分别为85.43%、91.08%和80.49%,WBC的残留率为0.21%;500例接受了上述病毒灭活血浆输注的患者中,有1例出现发热症状。结论:经MB-P灭活病毒血浆的有效成分含量较高,符合国家相关标准,并且WBC含量和输血不良反应发生率均较低,满足临床安全输血的要求,但仍需严格要求血液收集和血浆病毒灭活的操作过程并严格掌握输血适应症,避免人为造成的输血风险。     

洋葱伯克霍尔德菌外膜蛋白双向电泳的建立与优化

目的:洋葱伯克霍尔德菌外膜蛋门的分离及双向电泳图谱的建立和优化.方法:用月桂酰基氯酸钠法提取外膜蛋白,以同相pH梯度为第一向和SDS-PAGE为第二向进行双向电泳,对裂解液成分,IPG胶条的pH和凝胶染色方法等进行优化.结果:获得外膜蛋白浓度为2.87μg/μl;最佳裂解液成分为:7mol/L尿素,2mol/L硫脲,4%Chaps,2%pharmalyte,65 mmol/L DTT,0.5%Triton X-100,10mmol/L Tris.结论:提取的外膜蛋白满足双向电泳条件;获得理想的外膜蛋白双向电泳图谱用于后续实验中.     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.