有望用于治疗朊病的新技术——RNA干扰

朊病也称传染性海绵状脑病,目前研究认为主要由3种原因引起:一是遗传性因素:由于编码PrPc的基因发生致病性突变所造成;二是后天获得性因素:由外源性的朊病毒感染引起;三是散发性因素:由不明原因造成.朊病是由朊病毒(即PrPSc)所引起,朊病毒能够自我复制,而且具有蛋白酶抗性,是目前惟一确定的病原因子.PrPSc的复制是通过正常细胞朊蛋白即PrPc转变而来.     

用于免疫治疗朊病毒病的抗体片段的研究进展

朊病毒是一种感染性蛋白质,能引起一系列的被称为传染性海绵状脑病(TSEs)或朊病毒病的致命性神经变性型疾病。在哺乳动物中,朊病毒通过召募普通的细胞蛋白(PrPc)并诱导其转化为具有致病性的PrPsc而实现自我复制。最近的研究结果表明,抗朊病毒的抗体能永久性地治愈被朊病毒感染的细胞。但是,全长的抗体和蛋白质不能通过血脑屏障(BBB),这妨碍了将其用于TSEs的体内治疗。采用另一种方式,即通过腺联病毒(AAV)转导将有朊病毒特异性的scFv传递到大脑内,尽管这不能完全保护其不受感染,但能延迟受感染小鼠的发病时间。通过直接向细胞培养物中加入羊痒病病毒,或使用慢病毒和腺联病毒双转导载体使其感染,作者研究了重组的抗PrP(D18)单链可变片段(scFv)抗朊病毒的效果。结果表明,在感染细胞中重组的抗PrP scFv能够减少蛋白酶K的含量。另外,证实了与AAV相比,慢病毒能更有效的使抗PrP scFv基因转染并减少感染的神经元细胞中PrPsc的含量。最后,作者使用生物信息学的方法构建了D18scFv-PrP复合物的结构模型。有趣的是,根据反馈的结果,在D18scFv将PrPc锚定到抗体凹槽过程中,ArgPrP151(朊蛋...     

用于免疫治疗朊病毒病的抗体片段的研究进展

朊病毒是一种感染性蛋白质,能引起一系列的被称为传染性海绵状脑病(TSEs)或朊病毒病的致命性神经变性型疾病.在哺乳动物中,朊病毒通过招募普通的细胞蛋白(PrPc)并诱导其转化为具有致病性的PrPsc而实现自我复制.最近的研究结果表明,抗朊病毒的抗体能永久性地治愈被朊病毒感染的细胞.但是,全长的抗体和蛋白质不能通过血脑屏障(BBB),这妨碍了将其用于TSEs的体内治疗.采用另一种方式,即通过腺联病毒(AAV)转导将有朊病毒特异性的scFv传递到大脑内,尽管这不能完全保护其不受感染,但能延迟受感染小鼠的发病时间.通过直接向细胞培养物中加入羊痒病病毒或使用慢病毒和腺联病毒双转导载体使其感染,作者研究了重组的抗PrP(D18)单链可变片段(scFv)抗朊病毒的效果.结果表明,在感染细胞中重组的抗PrP scFv能够减少蛋白酶K的含量.另外,证实了与AAV相比,慢病毒能更有效的使抗PrP scFv基因转染并减少感染的神经元细胞中PrPsc的含量.最后,作者使用生物信息学的方法构建了D18scFv-PrP复合物的结构模型.有趣的是,根据反馈的结果,在D18scFv将PrPc锚定到抗体凹槽过程中,ArgPrP151(朊蛋白的第151位氨基酸)是一个关键的氨基酸残基.以上结果表明,将靶向PrP的被动和主动的免疫方法相结合,可能是治疗干预朊病毒病的一个可行策略.     

朊病毒的研究进展

朊病毒（Prion）是一种不含核酸的蛋白浸染因子,主要引起人和动物中枢神经疾病，即传染性海绵状脑病（TSE）。目前，由其引起的朊病毒病在世界多国发生，危害严重，经济损失巨大，并对人类健康构成很大威胁.本文就朊病毒的研究历史、朊病毒蛋白、朊病毒复制及其致病机理作了综述,并提出朊病毒研究中拟待解决的问题，以期为该类疾病的防治提供理论基础及依据。     

我国部分地区传染性羊痒病基因型的分布情况调查

引起羊痒病的病原朊蛋白是一种正常的唾液酸糖蛋白(PrPc)在三级结构发生改变后形成的异常蛋白(PrPsc)。该病的发生与绵羊朊蛋白编码基因PRNP遗传多样性密切相关,主要表现在PRNP第136、154和171位密码子组成的PRNP基因型与绵羊对痒病的抗病性的联系。根据已建立的一种利用荧光定量PCR扩增反应对羊痒病抗性基因进行筛选的方法,对我国部分地区的无角多赛特绵羊羊痒病基因分布情况进行调查,从而从品种选育水平上杜绝羊痒病的发生。     

实时荧光定量PCR构建绵羊PrP基因标准品质粒和标准曲线

羊痒病是一种传染性的致死性神经退行性疾病,常引起绵羊和山羊发病,该病在欧洲流行了大约250年,但是它的流行病学和传播机制还不是很清楚.正常的朊蛋白（PrPc）不能引起神经退行性病变,虽然目前已经对许多组织的PrP mRNA进行了检测,但是对它的生物学功能还知之甚少,对PrP基因表达的机制尚不清楚.研究显示,不同组织来源的细胞中朊蛋白的表达程度差异很大,主要出现于神经细胞中.PrPc在细胞中的高水平表达可促使PrPc向PrPsc转变,目前的研究中,对绵羊PrP mRNA的转录机制尚未阐释清楚.因此,对绵羊外周和中枢系统PrP mRNA的表达进行定量,有助于探讨各组织器官中的PrP在羊痒病的发生过程中的作用.     

朊病毒致病机理研究进展

朊病毒是一种无核酸但具有感染力的异常朊蛋白,可引起人和动物的神经退行性病变,该病结局是死亡。作者就朊病毒中枢神经系统感染机理,局部组织P rP sc聚集分布与P rP sc对神经组织损伤的关系、朊病中正常P rP c生理功能、细胞死亡可能机制、朊病中的神经炎—小胶质细胞的反应、补体激活作用;朊病毒外周神经系统感染机理,入侵门户、胞内转运机制、外周感染靶器官、胞内聚集与复制、神经入侵及朊病进程中已发现的几种受体细胞的重要作用等作一综述,为进一步研究该病提供理论依据。     

研究人员建立传染性朊病毒疾病小鼠模型

一个国际科学家小组建立了一种传染性朊病毒疾病小鼠模型。研究人员发现,通过改变两个核酸改变朊蛋白结构可以导致一种致命性的神经系统疾患。该研究的目的是研究可以引起慢性消耗性疾病即在鹿和麋鹿发现的传染性朊病毒疾病的朊蛋白变化。这种疾病与牛的牛海绵状脑病和人的Creutzfeldt-Jakob病类似。通过改变朊病毒基因中的两个核酸,     

痒病的防治措施与朊病毒

朊病毒是一种只有蛋白质而没有核酸的病毒,可以引起人类和动物中枢神经系统退行性病变。朊病毒病与艾滋病并列成为21世纪危害人类健康的顽疾。作者分别从病毒特性、感染途径和致病机理等方面,阐述了朊病毒的研究进展,并着重介绍了由朊病毒引起的代表性疾病绵羊痒病的流行病学特点、临床特征、病理变化、临床诊断要点及其综合防治措施,供广大兽医工作者和养殖户参考。     

痒病的防治措施与朊病毒

朊病毒是一种只有蛋白质而没有核酸的病毒,可以引起人类扣动物中枢神经系统退行性病变。朊病毒病与艾滋病并列成为21世纪危害人类健康的顽疾。作者分别从病毒特性、感染途径和致病机理等方面,阐述了朊病毒的研究进展,并着重介绍了由朊病毒引起的代表性疾病绵羊痒病的流行病学特点、临床特征、病理变化、临床诊断要点及其综合防治措施,供广大兽医工作者和养殖户参考。     

Experimental transmission of abnormal prion protein (PrPsc) in the small intestinal epithelial cells of neonatal mice

Using an immunohistochemical method, we attempted to detect the transmission of abnormal prion protein (PrPsc) to the enterocytes of the small intestine of neonatal mice by oral exposure with sheep brain affected by scrapie. Five 1-day-old neonatal mice were exposed by oral inoculation to the homogenized brain of a scrapie-affected sheep. In the small intestine of all mice 1 hour after inoculation, immunoreactivity with antinormal prion protein (PrPc) antibody was seen in the cytoplasm of villus enterocytes. This finding suggests transmission of abnormal PrPsc into the cytoplasm of enterocytes. In control mice treated with normal sheep brain, no PrPc signal was seen in enterocytes of the small intestine. Immunopositivity for neurofilament protein and glial fibrillary acidic protein was seen in the cytoplasm of enterocytes of mice inoculated with scrapie and normal sheep brain. This suggests that the enterocytes of neonatal mice can absorb PrPsc and other macromolecular proteins of the sheep brain affected by scrapie and may be more important than previously thought as a pathway for PrPsc transmission in neonatal animals.     

朊蛋白构象改变和种属突破机制的研究进展

异常朊蛋白(P rP sc)是一种可引起人和动物传染性脑退化病的不含核酸的蛋白因子。P rP c是一个正常的蛋白,有正常的生理功能,主要分布于神经元表面,属于肌醇磷脂锚蛋白类。由P rP c向P rP sc转变即产生疾病。作者简单介绍了正常和异常朊蛋白结构、功能、构象改变及朊病毒种属屏障的研究进展,并提出了目前尚待研究的问题。     

Glycosylation of prion strains in transmissible spongiform encephalopathies

This is a review of prion replication in the context of the cell biology of membrane proteins especially folding quality control in the endoplasmic reticulum (ER). Transmissible spongiform encephalopathies, such as scrapie and BSE, are infectious lethal diseases of mammalian neurons characterised by conversion of the normal membrane protein PrPC to the disease-associated conformational isomer called PrPSc. PrPSc, apparently responsible for infectivity, forms a number of different conformations and specific N-glycosylation site occupancies that correlate with TSE strain differences. Dimerisation and specific binding of PrPc and PrPSc seems critical in PrPSc biosynthesis and is influenced by N-glycosylation and disulfide bond formation. PrPsc can be amplified in vitro but new glycosylation cannot occur in cell free environments without the special conditions of microsome mediated in vitro translation, thus strain specific glycosylation of PrPSc formed in vitro in the absence of these conditions must take place by imprintation of PrPc from existing glycosylation site-occupancies. PrPSc formed in cell free homogenates is not infectious pointing to events necessary for infectivity that only occur in intact cells. Such events may include glycosylation site occupancy and ER folding chaperone activity. In the biosynthetic pathway of PrPSc, early acquisition of sensitivity of the GPI anchor to phospholipase C can be distinguished from the later acquisition of protease resistance and detergent insolubility. By analogy to the co-translational formation of the MHC I loading complex, it is postulated that PrPSc or its specific peptides could imprint nascent PrPc chains thereby ensuring its own folds and the observed glycosylation site occupancy ratios of strains.     

CNA42 monoclonal antibody identifies FDC as PrPsc accumulating cells in the spleen of scrapie affected sheep

Natural scrapie, new variant Creutzfeldt-Jakob disease and murine experimental transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative disorders. The agent responsible for these diseases is closely related to PrPsc, an abnormal isoform of the cellular prion protein. Before reaching the brain, it invades and replicates in lymphoid organs such as spleen, tonsils and lymph nodes. Follicular dendritic cells (FDC) may support the prion replication in lymphoid tissues of sheep as shown in murine models infected with scrapie. In sheep, specific antibodies recognising FDC are lacking. The CNA42 mAb, directed against human FDC was used to identify these cells in sheep spleen. As well as showing that the pre-treatments needed for immunohistochemical detection of PrPsc did not prevent labelling by the CNA42 mAb, accumulation of PrPsc in FDC of spleens of scrapie affected sheep was demonstrated using a double immunolabelling strategy. Thus, the CNA42 antibody represents a suitable tool to identify FDC and investigate their role in natural sheep scrapie.     

Prion biology relevant to bovine spongiform encephalopathy

Bovine spongiform encephalopathy (BSE) and chronic wasting disease (CWD) of deer and elk are a threat to agriculture and natural resources, as well as a human health concern. Both diseases are transmissible spongiform encephalopathies (TSE), or prion diseases, caused by autocatalytic conversion of endogenously encoded prion protein (PrP) to an abnormal, neurotoxic conformation designated PrPsc. Most mammalian species are susceptible to TSE, which, despite a range of species-linked names, is caused by a single highly conserved protein, with no apparent normal function. In the simplest sense, TSE transmission can occur because PrPsc is resistant to both endogenous and environmental proteinases, although many details remain unclear. Questions about the transmission of TSE are central to practical issues such as livestock testing, access to international livestock markets, and wildlife management strategies, as well as intangible issues such as consumer confidence in the safety of the meat supply. The majority of BSE cases seem to have been transmitted by feed containing meat and bone meal from infected animals. In the United Kingdom, there was a dramatic decrease in BSE cases after neural tissue and, later, all ruminant tissues were banned from ruminant feed. However, probably because of heightened awareness and widespread testing, there is growing evidence that new variants of BSE are arising "spontaneously," suggesting ongoing surveillance will continue to find infected animals. Interspecies transmission is inefficient and depends on exposure, sequence homology, TSE donor strain, genetic polymorphism of the host, and architecture of the visceral nerves if exposure is by an oral route. Considering the low probability of interspecies transmission, the low efficiency of oral transmission, and the low prion levels in nonnervous tissues, consumption of conventional animal products represents minimal risk. However, detection of rare events is challenging, and TSE literature is characterized by subsequently unsupported claims of species barriers or absolute tissue safety. This review presents an overview of TSE and summarizes recent research on pathogenesis and transmission.     

Expression of alpha-hemoglobin stabilizing protein and cellular prion protein in a subclone of murine erythroleukemia cell line MEL

Alpha-Hemoglobin stabilizing protein (AHSP) functions as the erythroid-specific molecular chaperon for alpha-globin. AHSP gene expression has been reported to be downregulated in hematopoietic tissues of animals suffering from prion diseases though the mechanism remains to be clarified. Herein, we demonstrate that MELhipod8 cells, a subclone of murine erythroleukemia (MEL) cells, have prion protein (PrPc) on the cell surface and have highly inducible expression of the AHSP and alpha- and beta-globin genes, resembling the expression pattern of the PrP and AHSP genes in bipotential erythroid- and megakaryocyte-lineage cells followed by erythroid differentiation in normal erythropoiesis. Moreover, MELhipod8 cells exhibit greater effective erythroid differentiation with a population of hemoglobinized normoblast-like cells than that observed for the parental MEL cells. These findings suggest that MELhipod8 cells could provide a mechanism for downregulation of the AHSP gene in prion diseases.     

Biology of PrPsc accumulation in two natural scrapie-infected sheep flocks.

Sheep scrapie is a prion disease that requires interaction of exogenous prions with host prion protein (PrP) supporting prion formation. Disease is associated with deposition of a host-generated conformational variant of PrP, PrPsc, in a variety of tissues, including brain, resulting in fatal spongiform encephalopathy. Efficiency of PrPsc formation is determined by polymorphisms in the PrP-coding sequence. This article adds to previous data of natural sheep scrapie, concentrating on the effect of host genotype and age on PrPsc accumulation patterns during preclinical and clinical disease. Two entire scrapie-infected, predominantly Suffolk-cross, sheep flocks euthanized for regulatory purposes were genotyped and analyzed for PrPsc deposition in various tissues using single- and dual-label immunohistochemistry. Scrapie, as defined by PrPsc deposition, occurred in 13/80 sheep. Preclinical disease was evident in nearly 70% of infected sheep, ranging in age from 14 months to 7 years. PrPsc accumulated systemically in the nervous tissue, various lymphoid tissues, both alimentary tract related and non-alimentary tract related, and the placenta. Clinical neurological illness was always associated with spongiform encephalopathy and PrPsc deposition in the brain. Only 6 of 9 sheep with preclinical scrapie had PrPsc deposition in the brain but widespread PrPsc deposition in peripheral lymphoid tissue, supporting previous data showing peripheral PrPsc accumulation preceding deposition in the brain. PrPsc colocalized with a marker for follicular dendritic cells throughout the lymphoid system. PrPsc also accumulated in the peripheral nervous system, particularly the nervous supply of the gastrointestinal tract. Abundant PrPsc was evident in trophoblast cells of placentomes but not in the endometrium, myometrium, or associated nervous plexus. PrPsc deposits were not observed in the mammary parenchyma or bone marrow. Scrapie susceptibility was defined genetically by PrP codon 171: PrPsc deposition was restricted to PrP genotype AA136RR154QQ171 in 12/13 cases or AV136RR154QQ171 in 1/13 cases. The earliest accumulation was observed in the single VRQ/ARQ heterozygous animal, consistent with the reported high scrapie susceptibility and brief incubation period observed in breeds with predominance of the V136R154Q171 allele. Disease occurred within, as well as independent of, mother-daughter lines, suggesting both maternal and nonmaternal transmission in the flocks.     

Investigation of the effects of experimental autolysis on the detection of abnormal prion protein in lymphoid and central nervous system tissues from elk and sheep using the Western blotting method

Tissues unsuitable for standard immunohistochemical and histopathological examinations for chronic wasting disease (CWD) in cervids and for scrapie in sheep are frequently submitted for testing. This study investigated the effects of experimental autolysis on the detection of abnormal prion protein (PrPsc) in lymphoid and central nervous system (CNS) tissues from elk and sheep. The PrPsc was detected using a Western blotting (WB) test following PrPsc enrichment using sodium phosphotungstic acid (PTA) precipitation (PTA-WB). A commercial enzyme-linked immunosorbent assay (ELISA) was used as a reference test for quantitative measurement. This study showed that the amount of PrPsc in lymphoid and CNS tssues from elk and sheep decreased gradually as a result of autolysis, but PrPsc was still detectable after 5 and 15 d incubation at 37°C by PTA-WB for all lymphoid and CNS samples. The results of the ELISA supported those of PTA-WB, particularly for CNS tissues. In conclusion, autolysis at 37°C for 15 d would not significantly affect the detection of PrPsc in lymphoid and CNS tissues by WB and ELISA and, particularly, PTA-WB is a valuable and alternative confirmatory test to detect PrPsc in autolyzed lymphoid and CNS samples.     

Prion protein self-interaction in prion disease therapy approaches

Transmissible spongiform encephalopathies (TSEs) or prion diseases are unique disorders that are not caused by infectious micro-organisms (bacteria or fungi), viruses or parasites, but rather seem to be the result of an infectious protein. TSEs are comprised of fatal neurodegenerative disorders affecting both human and animals. Prion diseases cause sponge-like degeneration of neuronal tissue and include (among others) Creutzfeldt-Jacob disease in humans, bovine spongiform encephalopathy (BSE) in cattle and scrapie in sheep. TSEs are characterized by the formation and accumulation of transmissible (infectious) disease-associated protease-resistant prion protein (PrP(Sc)), mainly in tissues of the central nervous system. The exact molecular processes behind the conversion of PrP(C) into PrP(Sc) are not clearly understood. Correlations between prion protein polymorphisms and disease have been found, however in what way these polymorphisms influence the conversion processes remains an enigma; is stabilization or destabilization of the prion protein the basis for a higher conversion propensity? Apart from the disease-associated polymorphisms of the prion protein, the molecular processes underlying conversion are not understood. There are some notions as to which regions of the prion protein are involved in refolding of PrP(C) into PrP(Sc) and where the most drastic structural changes take place. Direct interactions between PrP(C) molecules and/or PrP(Sc) are likely at the basis of conversion, however which specific amino acid domains are involved and to what extent these domains contribute to conversion resistance/sensitivity of the prion protein or the species barrier is still unknown.