实时荧光定量PCR检测PrP基因在金黄地鼠脑组织的动态表达

金黄地鼠是研究动物传染性海绵状脑病的理想模型动物之一,其脑组织内朊蛋白基因动态表达数据的测定对探讨该类疾病的发生、发展和分子致病机理具有重要意义。我们利用实时荧光定量RT-PCR技术,对不同年龄金黄地鼠大脑、小脑、丘脑和脑干PrP基因的表达进行了定量。结果发现,脑的四个检测部位都呈现高的表达量,但是同一年龄段不同组织每纳克总RNA中朊蛋白基因的表达量和每毫克组织中朊蛋白基因的表达量有显著的差别,不同组织在不同年龄出现表达高峰。本研究的结果对于探讨朊蛋白的基本功能和脑组织在传染性海绵状脑病病理发生中的作用,提供了基础数据。     

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.     

PrP106-126诱导的N2a细胞神经毒性中神经营养因子受体p75NTR的表达变化研究

神经元死亡是朊病毒病的主要病理学特征。朊蛋白多肽PrP106-126能够对神经细胞表现神经毒性,引起细胞凋亡。细胞表面蛋白神经营养因子受体p75^NTR的胞外区域可以与PrP106-126结合并产生促凋亡作用。作者以小鼠成神经瘤细胞N2a为细胞模型,应用荧光定量RT-PCR和Western Blot技术,以及DNA Ladder和AnnexinV-FITC/PI双重染色流式细胞凋亡检测技术对p75^NTR介导的PrP106-126神经毒性分子机制进行了研究。结果发现在PrP106-126诱导的N2a细胞凋亡过程中,p75^NTR的mRNA转录水平和蛋白表达水平均显著升高,以p75^NTR多克隆抗体sc-6189阻断PrP 106-126与p75^NTR的相互作用后,减弱了PrP106-126诱导的N2a细胞凋亡效果。该研究揭示了PrP106-126诱导的N2a细胞毒性中p75^NTR受体的表达变化,为解释朊病毒病的发病机制提供了重要数据。     

PrP蛋白与Shadoo蛋白研究进展

朊蛋白(PrP蛋白)在传染性海绵状脑病中具有重要作用,但其生物学功能至今没有明确。Shadoo蛋白是一种与朊蛋白在N端结构上极为相似的新蛋白,SPRN是Shadoo蛋白的结构基因。由于Shadoo蛋白与PrP蛋白具有相同的生物学性质,且它们在脑组织中重叠表达,因此也被认为是研究朊蛋白的关键因素。文章主要探讨PrP蛋白与Shadoo蛋白在基因结构、蛋白功能、朊病毒感染方面的关系。     

利用PRNP-/-羊制备朊蛋白多克隆抗体及其特性分析

<正>【目的】利用朊蛋白双基因敲除(PRNP-/-)羊制备朊蛋白多克隆抗体,并对其特性进行分析。【方法】构建山羊朊蛋白(PrP)原核表达载体,转入大肠杆菌并诱导表达,纯化获得羊PrP;将获得的PrP免疫PRNP-/-山羊,制备朊蛋白特异性的多克隆抗体;并对获得的朊蛋白多克隆抗体进行ELISA及Western-blot检测。【结果】获     

STC-1基因在大、小体型猪中表达与分布的差异分析

试验分别采集40日龄小体型猪（巴马猪）和大体型猪（大白猪）的心脏、肝脏、脾脏、肺脏、肾脏、头骨、骨骼肌组织,利用实时荧光定量PCR检测斯钙素-1（stanniocalcin 1,STC-1）基因mRNA在各个组织中的表达水平,并通过Western blotting检测STC-1蛋白在各个组织中的分布。实时荧光定量PCR检测结果表明,STC-1基因mRNA在巴马猪和大白猪肺脏、肾脏中相对表达水平较高,在骨骼肌中的表达水平最低;除心脏和骨骼肌外,巴马猪其余各组织中STC-1基因mRNA表达水平均显著高于大白猪（P < 0.05）。Western blotting检测结果表明,巴马猪肝脏中STC-1蛋白的表达量最高,而大白猪脾脏中STC-1蛋白表达量最高,两者差异显著（P < 0.05）;巴马猪肺脏、肝脏、骨骼肌及心脏组织中STC-1蛋白表达量均极显著高于大白猪（P < 0.01）;而巴马猪肾脏、脾脏中STC-1蛋白表达量极显著低于大白猪（P < 0.01）。本研究首次对大、小体型猪不同组织的STC-1基因mRNA表达水平及其STC-1蛋白分布进行检测,导致该基因表达与分布差异的原因可能与两种猪受外界环境应激及生长发育差异有关。     

NLRC5基因在SPF大白猪不同器官中的表达与分布

为了研究NLRC5基因在SPF大白猪不同组织中的表达和分布,试验以稀释的标准重组质粒为模板绘制标准曲线,建立实时荧光定量PCR方法,检测SPF大白猪肺脏、颌下淋巴结、腹股沟淋巴结、脾脏、空肠、胃、小肠、直肠、肾脏、心脏、回肠、肝脏、结肠、盲肠、扁桃体、肠系膜淋巴结等16种器官中NLRC5基因mRNA的表达差异和分布,并用Western-blot方法检测16种器官中NLRC5基因编码蛋白表达差异和分布。结果表明:NLRC5基因在SPF大白猪16种器官中均能表达,mRNA和蛋白表达水平大体一致,其中在淋巴结、脾脏和扁桃体等免疫器官中的表达量均较高,在胃和肠道等具有黏膜免疫功能的器官中表达量次之,在心脏和肝脏等非免疫器官中的表达量相对较低。说明试验获得了SPF大白猪不同器官中NLRC5基因的天然表达量,并且在免疫器官和具有黏膜器官中有较高的表达。     

PrP106-126对大脑皮质神经元和星形胶质细胞朊蛋白基因表达的影响

采用实时荧光定量RT-PCR的方法,对PrP106-126处理的大脑皮质神经元和星形胶质细胞模型进行了朊蛋白基因表达相对定量的研究。大脑皮质神经元经PrP106-126处理后,与SCR处理组和对照组相比,基因表达量明显下降。PrP106-126处理的星形胶质细胞与对照组和SCR处理组相比,朊蛋白基因的表达量显著升高。该试验结果为深入了解TSEs的分子致病机制和正常朊蛋白的功能提供了基础数据。     

朊蛋白生理功能的研究进展

朊蛋白（prion protein,PrPC）作为朊病的主要相关致病因子而受到广泛关注,虽然PrPC在疾病过程中的作用已有很多了解,但关于PrPC的正常生理功能还不是很清楚。就目前的研究而言,PrPC存在细胞核亚型和细胞质亚型两种形式,可与相应的配体及铜离子作用,并且通过细胞内吞或内陷方式被细胞膜获取;主要生理功能表现为保护神经系统,协助细胞的凋亡和分化、黏附,以及参与信号转导等。因此,文章对朊蛋白的生理功能作一总结,为朊病的治疗和朊蛋白的进一步研究提供了理论基础。     

朊蛋白在癌症发生中的作用

<正>常细胞型朊蛋白(PrPC)是一种高度保守、在所有哺乳动物体内广泛表达的细胞表面糖蛋白,人类PrPC在胚胎发育早期即已表达。研究表明,PrP在多种癌症中表达,包括胃癌、胰腺癌、大肠癌、乳腺癌、前列腺癌、肝癌(HCC)、口腔鳞状细胞癌(OSCC)等,影响这些癌症的发生与侵袭,并在多药耐性(MDR)获得中起重要作用。因此,朊蛋白有望成为治疗多种癌症的新靶标。论文就PrP的结构、功能及其在细胞功能和疾病发生发展中的作用,以及PrP在多种肿瘤发生发展、耐药性的作用等研究进行概述,并对PrP在癌症新型疗法未来发展中的潜在影响进行分析讨论,以期为PrP相关肿瘤病的防治方面做出贡献。     

Mapping PrPSc propagation in experimental and natural scrapie in sheep with different PrP genotypes

Twenty-one orally inoculated and seven naturally infected sheep with scrapie were examined for PrP(Sc) in peripheral tissues and in the central nervous system (CNS), using immunohistochemistry. In the inoculated group, VRQ (valine at codon 136, arginine at codon 154 and glutamine at codon 171)/VRQ sheep generally had a greater accumulation of the pathologic form of prion protein (PrP(Sc)) in peripheral tissues, as compared with VRQ/ARQ (alanine at codon 136, arginine at codon 154, and glutamine at codon 171) animals at corresponding time points after inoculation. PrP(Sc) was not detected in the ileal Peyer's patch, the spleen, the superficial cervical lymph node, and peripheral nervous tissues of several inoculated VRQ/ARQ animals. All inoculated VRQ/VRQ sheep, but only one of eight inoculated VRQ/ARQ animals, were PrP(Sc)-positive in the CNS. Thus, the propagation of PrP(Sc) seemed slower and more limited in VRQ/ARQ animals. Tissue and cellular localization of PrP(Sc) suggested that PrP(Sc) was disseminated through three different routes. PrP(Sc)-positive cells in lymph node sinuses and in lymphatics indicated spreading by lymph. The sequential appearance of PrP(Sc) in the peripheral nervous system and the CNS, with satellite cells as early targets, suggested the periaxonal transportation of PrP(Sc) through supportive cells. Focal areas of vascular amyloid-like PrP(Sc) in the brain of five sheep, suggested the hematogenous dissemination of PrP(Sc). There was a poor correlation between the amount of PrP(Sc) in the CNS and clinical signs. One subclinically affected sheep showed widespread PrP(Sc) accumulation in the CNS, whereas three sheep had early clinical signs without detectable PrP(Sc) in the CNS. A VV(136) (homozygous for valine at codon 136) sheep inoculated with ARQ/ARR (alanine at codon 136, arginine at codon 154, and arginine at codon 171) tissue succumbed to disease, demonstrating successful heterologous transmission. Less susceptible sheep receiving VRQ/VRQ or ARQ/ARR material were PrP(Sc)-negative by immunohistochemistry, enzyme-linked immunosorbent assay, and western blot.     

Variable patterns of distribution of PrP(CWD) in the obex and cranial lymphoid tissues of Rocky Mountain elk (Cervus elaphus nelsoni) with subclinical chronic wasting disease

Sections of medulla oblongata, taken at the level of the obex, palatine tonsil and medial retropharyngeal lymph node from 10,269 captive Rocky Mountain elk (Cervus elaphus nelsoni), were examined by immunohistochemical staining with monoclonal antibody for the prion protein associated with the transmissible spongiform encephalopathy of cervids, chronic wasting disease (PrP(CWD)). The protein was detected in 226 of them. On the basis of the anatomical location of the deposits in the brainstem of 183 elk, four distinct patterns of distribution of PrP(CWD) within the parasympathetic region of the dorsal motor nucleus of the vagus nerve and the adjacent nuclei were observed. Mild gross lesions of chronic wasting disease (serous atrophy of fat) were observed in only three elk, all with spongiform degeneration; the other elk were considered to be in the preclinical stage of the disease. In contrast with the relatively predictable distribution of prion protein (PrP) in the brain and cranial nodes of sheep and mule deer, the distribution of PrP(CWD) in the brain and nodes of the elk was more variable and unrelated to their PrP genotype. One hundred and fifty-five of the 226 positive elk had deposits of PrP(CWD) in the brainstem and lymphoid tissues, 43 had deposits only in the lymphoid tissue and 28 had deposits only in the brainstem.     

Pre-clinical and clinical diagnosis of scrapie by detection of PrP protein in tissues of sheep.

The usefulness of detecting the scrapie-associated fibrillar protein (PrP) in the lymphoreticular organs of sheep as a diagnostic tool was investigated. The PrP was detected by means of a rabbit-anti-sheep PrP polyclonal antibody by Western blot analysis. PrP was detected in samples from the central nervous system (CNS) of five of six sheep showing clinical signs of natural scrapie infection, in spleen samples from four of the six sheep and in lymph node samples taken from three of the sheep. PrP was detected in the spleen and lymph node samples, but not in the CNS samples from one of the six sheep that was clinically and histopathologically abnormal. This animal appeared to be in the early clinical stage of the disease. A total of 47 clinically normal sheep were examined for the presence of PrP. It was detected in spleen samples from three of the 47 sheep and in lymph node samples from three of the 39 sheep tested. Similarly, PrP was detected in a sample of lymph node obtained surgically from one of three experimentally infected sheep 14 months after inoculation. The PrP-positive sheep and one of the remaining PrP-negative sheep showed clinical signs of scrapie six and five months later respectively. One sheep euthanased 18 months after experimental infection was positive for PrP in the CNS, spleen and lymph node, but five other sheep which were killed or died two, eight, 16, 18 and 21 months after infection were negative or doubtful for the detection of PrP.     

PrP基因在金黄地鼠淋巴和外周组织中表达水平的动态检测

利用实时荧光定量RT—PCR技术，构建了标准重组质粒，制备了标准曲线，对不同年龄金黄地鼠腹股沟浅淋巴结、脾、心、肝、肺和肾提取总RNA，反转录后进行PrP基因的表达定量。结果发现，淋巴组织呈现高的表达量，外周组织的表达量比较低；不同组织在不同年龄出现表达高峰。     

Comparison of brain PrPd distribution in ovine BSE and scrapie

Scrapie and bovine spongiform encephalopathy (BSE) are both prion diseases affecting ruminants, and these diseases do not share the same public health concerns. Surveillance of the BSE agent in small ruminants has been a great challenge, and the recent identification of diverse prion diseases in ruminants has led to the development of new methods for strain typing. In our study, using immunohistochemistry (IHC), we assessed the distribution of PrP(d) in the brains of 2 experimentally BSE-infected sheep with the ARQ/ARQ genotype. Distribution of PrP(d) in the brain, from the spinal cord to the frontal cortex, was remarkably similar in the 2 sheep despite different inoculation routes and incubation periods. Comparatively, overall PrP(d) brain distribution, evaluated by IHC, in 19 scrapie cases with the ARQ/ARQ, ARQ/VRQ, and VRQ/VRQ genotypes, in some cases showed similarities to the experimentally BSE-infected sheep. There was no exclusive neuroanatomical site with a characteristic and specific PrP(d) type of accumulation induced by the BSE agent. However, a detailed analysis of the topography, types, and intensity of PrP(d) deposits in the frontal cortex, striatum, piriform cortex, hippocampus, mesencephalon, and cerebellum allowed the BSE-affected sheep group to be distinguished from the 19 scrapie cases analyzed in our study. These results strengthen and emphasize the potential interest of PrP(d) brain mapping to help in identifying prion strains in small ruminants.     

Accumulation of pathogenic prion protein (PrPSc) in nervous and lymphoid tissues of sheep with subclinical scrapie

All sheep older than 1 year of age from a flock of the Rygja breed in which clinical scrapie was detected for the first time in two animals (4%) were examined for accumulation of pathogenic prion protein (PrPSc) by immunohistochemistry in the obex, the cerebellum, and the medial retrophayngeal lymph node. In addition, six lambs, 2-3 months old, all offspring of PrPSc-positive dams, were examined for PrPSc in the ileal Peyers' patch (IPP), the distal jejunal lymph node, the spleen, and the medial retropharyngeal lymph node (RPLN). In this flock, 35% (17/48) of the adult sheep showed accumulation of PrPSc, an eightfold increase compared with clinical disease. All positives carried susceptible PrP genotypes. Three sheep had deposits of PrPSc in the RPLN and not in the brain, suggesting that this organ, easily accessible at slaughter, is suitable for screening purposes. Two 7-year-old clinically healthy homozygous V136Q171 ewes showed sparse immunostaining in the central nervous system and may have been infected as adults. Further, two littermates, 86-days-old, showed PrPSc in the IPP. Interestingly, one of these lambs had the intermediate susceptible PrP genotype, VA136QR171. In addition to early immunolabeling in the dorsal motor nucleus of the vagal nerve, a few of the sheep had early involvement of the cerebellum. In fact, a 2-year-old sheep had sparse deposits of PrPSc in the cerebellum only. Because experimental bovine spongiform encephalopathy (BSE) in sheep seems to behave in a similar manner as natural scrapie, these results, particularly regarding spread of infectivity, may have implications for the handling of BSE should it be diagnosed in sheep.     

Co-existence of classical scrapie and Nor98 in a sheep from an Italian outbreak

Nor98 is an atypical scrapie strain characterized by a molecular pattern and brain distribution of the pathological prion protein (PrP^Sc) different from classical scrapie. In Italy, 69 atypical cases have been identified so far and all were characterized as Nor98 strain. In this paper we report an unusual case in a sheep which showed immunohistochemical and molecular features of PrP^Sc different from the other atypical cases. The sheep was from an outbreak where the index and the other four cases were affected by classical scrapie. Histopathological, immunohistochemical and Western blot analyses on the brain of the unusual case revealed the simultaneous presence of pathological features characteristic of Nor98 and classical scrapie. Interestingly, the prevalent disease phenotype in the brainstem was classical scrapie-like, while in the cerebral cortex and cerebellum the Nor98 phenotype was dominant. The sub-mandibular lymph node was positive and showed a PrP^Sc molecular pattern referable to classical scrapie. The PrP genotype was AL₁₄₁RQ/AF₁₄₁RQ. Taken together, the occurrence of classical scrapie in the outbreak, the PrP genotype, the involvement of different cellular targets in the brain and the pathological and molecular PrP^Sc features observed suggest that this unusual case may result from the co-existence of Nor98 and classical scrapie.     

Experimental transmission of US scrapie agent by nasal, peritoneal, and conjunctival routes to genetically susceptible sheep

Scrapie is a naturally occurring fatal neurodegenerative disease of sheep and goats. This study documents incubation periods, pathologic findings, and distribution of abnormal prion proteins (PrP(Sc)) by immunohistochemistry in tissues of genetically susceptible sheep inoculated with US sheep scrapie agent. Four-month-old Suffolk lambs (QQ at codon 171) were inoculated by 1 of 3 different routes (nasal, peritoneal, and conjunctival) with an inoculum (No. 13-7) consisting of a pool of scrapie-affected sheep brains. Except for 3 sheep, all inoculated animals were euthanized when advanced clinical signs of scrapie were observed between 19 and 46 months postinoculation (MPI). Spongiform lesions in the brains and labeling of PrP(Sc) in central nervous system and lymphoid tissues were present in these sheep. One intranasally inoculated sheep euthanized at 12 MPI had presence of PrP(Sc) that was confined to the pharyngeal tonsil. These results indicate that the upper respiratory tract, specifically the pharyngeal tonsil, may serve as a portal of entry for prion protein in scrapie-infected environments.