超声处理对羊瘙痒病脑组织中PrPSc聚集状态的影响

目的 研究超声处理对感染羊瘙痒症仓鼠脑组织中PrP^Sc聚集状态的影响，寻找产生PrP^Sc低聚体的条件。方法 裂解液制备脑组织提取物，用各种超声条件处理不同阶段的脑组织提取物；以蛋白酶消化后的Western blot方法和图象分析系统观测PrP^Sc蛋白的分布和聚集状态。结果 适当的超声处理(15s共30次)可增加脑组织匀浆上清中PrP^Sc含量1．29～1．58倍；同样条件下超声处理可明显增加羊瘙痒因子263K感染仓鼠脑组织匀浆上清中PrP蛋白总量，而对正常对照仓鼠脑组织匀浆上清中PrP蛋白总量影响不大；对经常规高速离心获得的PrP^Sc的超声处理显示约90％的PrP^Sc存在于离心上清液中。结论 对感染动物脑组织进行超声处理可增加PrP^Sc的提取量，利于实验室检测。适当的超声处理可破碎大的相对分子质量的PrP^Sc聚集物，产生小的相对分子质量的PrP^Sc产物。     

不同剂量羊瘙痒因子263K颅内感染仓鼠临床终末期脑中GFAP表达的研究

目的 研究不同剂量羊瘙痒因子263 K经颅内注射感染仓鼠后,在发病的终末期星形胶质细胞增生程度是否与注射剂量及潜伏期长短有关.方法 以胶质纤维酸性蛋白(glial fibrill aryacidic protein,GFAP)作为星形胶质细胞增生的分子标志物,采用Western Blot和免疫组化方法检测感染仓鼠终末期脑匀浆和脑组织病理切片中的GFAP表达,经定量分析比较各感染剂量组间是否存在差异.结果与正常对照相比,不同剂量感染仓鼠发病终末期脑组织GFAP阳性细胞数量和总GFAP含量均明显升高,但各感染剂量组间无显著差异.结论 不同感染剂量羊瘙痒因子263K经颅内注射感染仓鼠在发病终末期脑中星形胶质细胞的增生程度相似,与感染剂量及潜伏期无关联性.     

一种朊蛋白错误折叠循环扩增方法的建立

目的建立一种类似于PCR的蛋白质扩增方法-蛋白错误折叠循环扩增技术(PMCA),用于朊病毒病脑组织中PrPSc的检测。方法将不同浓度的羊瘙痒因子263K毒株原液与正常仓鼠脑组织匀浆混合,经反复孵育/超声,共10~15个循环。WesternBlot检测扩增产物中蛋白酶K抗性PrPSc信号。结果在本研究试验体系下,263K毒株可以利用仓鼠脑组织为基质在体外迅速复制。所建立的PrPSc-PMCA技术可检测到10-5稀释的毒株原液中的PrPSc。与常规的脑组织免疫印记方法相比,敏感度提高了105~106倍。研究还显示PrPSc还可利用小脑和脑干为基质进行体外扩增复制。结论成功建立了PrPSc-PMCA技术,为朊病毒病的早期诊断和朊病毒生物学特性的研究提供了一种新的手段。     

上海脑库脑组织样本的质量控制分析

目的 通过对脑脊液pH值、脑组织RNA完整值(RIN)、转录组和蛋白质组检测以及形态学观察,评估上海脑库收集的脑组织用于后续生物学研究的可行性.方法 使用pH试纸检测新鲜脑脊液pH值;使用安捷伦RNA 6000 Nano芯片和2100生物分析仪对低温冻存的脑组织RNA进行完整性检测;使用BGIseq-500测序仪对低温...     

闵行区慢性病生物样本库的建立

随着社会人口老龄化的加剧,慢性病越来越成为重要的公共卫生问题和科学研究的重点。闵行区常住人口242.94万,纳入闵行区居民电子健康信息档案的糖尿病患者51379例,高血压患者184266例,肿瘤患者20000例。在慢性病的防治中,公共卫生研究人员对慢性病患者生物样本的需求量也大为上升,因此慢性病生物样本库的建立就成为摆在我们面前的一个紧急而重要的任务。     

启东肝癌高发区队列样本库的建立与应用

启东是我国的肝癌高发区,自1972年以来,启东现场采用流行病学、临床及实验室等方法,对肝癌的病因、肝癌的诊断和肝癌的治疗等进行了广泛的研究[1]。通过采集并分析大量血样、尿样及其他标本,为肝癌的实验研究提供了医学和生物学证据。20世纪80年代末尝试建立生物标本库,为启东肝癌研究的持续和深入发展增加了竞争力。现把我所生物样本库的建立与应用做一总结。     

杭州师范大学实验动物设施的建立

杭州师范大学新建实验动物中心设有标准化的实验动物生产设施与动物实验设施，总面积3090m^2，包括大小鼠生产屏障环境、实验屏障环境和大动物实验普通环境，各项指标已通过相关部门验收并投入使用。现为浙江省实验动物从业人员实习培训基地和大动物实验基地。本文介绍该动物中心的设施建设规模和设计工艺等情况及运行过程中的一些体会。     

新疆伊犁地区动物脑组织博尔纳病病毒p24基因片段的检测

目的 了解新疆伊犁地区动物脑博尔纳病病毒(Borna disease virus,BDV)感染现状,分析该病毒的种系来源,预防我国BDV大规模暴发流行.方法 采用巢式逆转录实时荧光定量聚合酶链反应(FQ-RT-PCR)的方法,对新疆地区200匹马、75头驴和100只牧羊犬脑组织标本进行BDVp24基因片段的检测.并对阳性标本进行BDV p40基因片段扩增电泳,同时排除GFP-p24和pMD-19质粒污染,最后克隆测序,进行基因同源性和氨基酸序列分析并分析BDV种系发生.结果 3例伊犁马、4例伊犁驴和9例牧羊犬的脑组织标本BDV p24检测阳性,阳性率分别为1.5%、5.3%、9.0%;BDV p40基因片段检测和质粒标准品GFP-p24、pMD-19检测均为阴性;BDV p24扩增产物序列与He/80株的同源性为100%.结论 新疆伊犁地区马、驴和狗可能存在BDV脑内的自然感染,该地区BDV流行株与He/80株存在同源性.     

皮肤角朊细胞与表皮干细胞双向电泳图谱的建立及差异分析

目的 建立皮肤角朊细胞与表皮干细胞的双向电泳图谱,并分析二者表达蛋白质的差异,为进一步研究体外调控表皮十细胞的增殖、分化提供线索.方法酶消化法获取单个表皮细胞悬液.Ⅳ型胶原快速贴壁法分选表皮干细胞.利用舣向电泳技术(2-DE)建立两种细胞的蛋白质表达图谱,并用Imagemaster 2D elite 5.0软件分析两种细胞的差异表达蛋一点.结果两种细胞的2-DE蛋白表达谱具有良好的重复性和可比性.皮肤角朊细胞与表皮干细胞的电泳图谱平均蛋白质点数分别为(982 ±18)个和(930 ±15)个,匹配点数为(850±13)个和(798±11)个,匹配率是86.56%和85.81%;两种细胞蛋白质间匹配点数是(886 ±8)个,匹配率是76.98%.找到差异蛋白点11个,其中只在表皮下细胞中表达或高表达的有8个;只在皮肤角朊细胞中表达或高表达的有3个.结论利用双向电泳法得到了分辨率较高儿重复性好的皮肤角朊细胞与表皮干细胞蛋白质组图谱,且二者存在有一定的差异.     

荧光标记肿瘤转移体内模型的建立

目的 建立一种带荧光标记的肿瘤转移模型并探讨其应用价值.方法 人胃癌MGC-803细胞和小鼠宫颈癌U14细胞进行体外传代培养,转染pEGFP-N1质粒,24h检测转染效率,用无限稀释法筛选稳定表达绿色荧光蛋白(GFP)的单克隆细胞株MGC-803-GFP和U14-GFP.分别体内移植于BALB/c-nn裸鼠和C57BL/6J小鼠,观察成瘤潜伏期,绘制体内生长曲线,利用活体荧光成像系统活体连续观察GFP在体内的表达,观察肺部和淋巴结的转移情况,计算转移率.免疫组织化学检测与转移相关的分子CD44和E-cadherin在移植瘤中的表达.结果 MGC-803细胞和U14细胞pEGFP-N1转染24 h后的转染效率分别为30%和60%.获得了GFP(+)的单克隆MGC-803-GFP细胞株和U14-GFP细胞株.MGC-803-GFP在BALB/c-nu裸鼠和U14-GFP在C57BL/6J小鼠移植成瘤的潜伏期分别是3～5 d和2～4 d,成瘤率均为100%.利用活体荧光成像系统连续观察,可以清楚直观地观察表达GFP的MGC-803-GFP移植瘤在体内的生长,接种后第60天处死全部荷瘤鼠,解剖后仅有1只可见同侧腋窝下淋巴结的转移.接种U14-GFP后,分别在第28、37和52天观察了荷瘤小鼠肿瘤转移的发展过程.在U14-GFP原发瘤体积达到≥5 cm3时,肺部和淋巴结转移率分别为67%和100%.在MGC-803-GFP和U14-GFP的移植瘤中都可以检测到CD44的表达,而无E-cadherin的表达.结论 成功建立了表达绿色荧光蛋白单克隆细胞株MGC-803-GFP和U14-GFP,体内移植建立了荧光标记的肿瘤转移模型.该模型可用于可视化肿瘤体内的研究.     

Association of prion protein genotype and scrapie prion protein type with cellular prion protein charge isoform profiles in cerebrospinal fluid of humans with sporadic or familial prion diseases

The present study investigates whether posttranslational modifications of cellular prion protein (PrP^C) in the cerebrospinal fluid (CSF) of humans with prion diseases are associated with methionine (M) and/or valine (V) polymorphism at codon 129 of the prion protein gene (PRNP), scrapie prion protein (PrP^Sc) type in sporadic Creutzfeldt-Jakob disease (sCJD), or PRNP mutations in familial Creutzfeldt-Jakob disease (fCJD/E200K), and fatal familial insomnia (FFI). We performed comparative 2-dimensional immunoblotting of PrP^C charge isoforms in CSF samples from cohorts of diseased and control donors. Mean levels of total PrP^C were significantly lower in the CSF from fCJD patients than from those with sCJD or FFI. Of the 12 most abundant PrP^C isoforms in the examined CSF, one (IF12) was relatively decreased in (1) sCJD with VV (vs. MM or MV) at PRNP codon 129; (2) in sCJD with PrP^Sc type 2 (vs. PrP^Sc type 1); and (3) in FFI versus sCJD or fCJD. Furthermore, truncated PrP^C species were detected in sCJD and control samples without discernible differences. Finally, serine 43 of PrP^C in the CSF and brain tissue from CJD patients showed more pronounced phosphorylation than in control donors.     

Morphological characteristics of the brain lesions in mice infected with a homogenate of L cells latently infected with the scrapie agent

Degeneration of neurons of the Ammon horn lower branch both in the early and terminal stages of the disease of mice infected with the homogenate of L cells latently infected with the scrapie agent (the L-S system) was frequently detected alongside with brain lesions typical of slow infections (vacuolation). Examinations of chromosomes in metaphase plates of L-S cells carried out by several methods including the TAC system for texture analysis of the image (Leutz, BRD) revealed three marker chromosomes new for continuous L cells, the appearance of true chromatid translocations as well as significant changes in chromosome numbers. Besides, ultrastructural features of L-S cells at later stages of cultivation were revealed. It is assumed that the active effect of the scrapie agent on L cells infected with it resulted in the emergency of a new antigen capable to induce selective affection of the neurons of the Ammon horn lower branch in susceptible mice.     

Adaptation of the agent of scrapie to hamsters

The agent of scrapie (Compton strain) can be transmitted from mice to hamsters; the incubation period of the disease is 5–6 months. Passage of the agent of scrapie through suspensions of brain tissue was repeated 10 times. The scrapie agent was found in the spinal cord and spleen but it could not be found in the liver, kidneys, adrenals, and lungs of the infected animals in the last stage of the disease.Division of General Epidemiology, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR O. V. Baroyan.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 2, pp. 199–201, February, 1976.     

Evidence that the transmission of one source of scrapie agent to hamsters involves separation of agent strains from a mixture.

A previous paper (Kimberlin & Walker, 1977) described an experimental model of scrapie in hamsters in which the incubation period decreased progressively over the first 4 passages before becoming stable at the 5th and subsequent passes. Studies have been made of some of the agent strains present in brains taken from the 2nd, 3rd, 4th and 6th hamster passes. The results indicate the presence of at least two strains of agent at the 3rd passage level. One of these (431K) is highly pathogenic for mice and the other (263K) has an extremely low pathogenicity for mice. However only one of these strains (263K) is present in hamster brain after the 6th serial passage. It is suggested that the 'adaptation' of scrapie to hamsters may involve the selection, from a mixture, of a single strain which is highly pathogenic for hamsters. The possibility of modification of the properties of agent strains on passage discussed.     

Immunisation with a synthetic prion protein-derived peptide prolongs survival times of mice orally exposed to the scrapie agent

Several lines of evidence suggest that immunisations may be helpful in the prophylaxis and treatment of neurodegenerative amyloidoses like Alzheimer's disease and prion infections. We used a synthetic prion protein-derived peptide (PrP105–125) and a recombinant PrP fragment (PrP90–230) as antigens for the active immunisation of mice, which were subsequently infected by dietary exposure to the scrapie agent. Immunisation with PrP105–125 prolonged the survival times significantly. In contrast, immunisation with PrP90–230 or adjuvants alone had no effect on the disease development. An epitope mapping of the antibodies raised against PrP90–230 revealed that reactivities against previously defined protective epitopes were either underrepresented or absent. These results point towards the possibility to prevent prion spread via the food chain by vaccinating humans or other species at risk to contract prion diseases.     

Characteristics of prion protein (PrP(Sc)) in the brains of hamsters inoculated serially with a mouse-passaged scrapie strain

Syrian hamsters were inoculated intracerebrally with a mouse-passaged scrapie strain. After serial passage, the incubation period decreased, but the vacuolar lesion profiles remained unchanged. In immunoblot analysis, accumulated prion protein (PrP) showed hamster PrP characteristics from the first passage. However, immunohistochemical examination revealed a changing pattern of accumulated PrP with each passage. In particular, there were many PrP plaques in the subpial and subependymal region at the third passage, no such plaques having been observed in the same region at the first passage. These results suggest that the species barrier influences not only the incubation period but also the pattern of accumulation of PrP in affected brains.     

Borreliacidal activity of sera from hamsters infected with the Lyme disease spirochete.

An in vitro borreliacidal assay that accurately reflects the levels of protective antibody determined by passive transfer of immunity studies was developed. Borreliacidal antibody in sera obtained from normal hamsters infected with Borrelia burgdorferi was readily detected. When immune serum containing complement was incubated with B. burgdorferi organisms, spirochetes were killed within 2 h. Treating immune serum with anti-hamster immunoglobulin G abrogated the borreliacidal activity. Killing of B. burgdorferi in serum was detected 1 week after infection; it peaked at week 3 and gradually declined. Relatively high levels of borreliacidal antibody were found, especially in week 3 immune serum, which could be diluted 1,280-fold. The decrease in borreliacidal antibody after infection may account for occurrences of reinfection and the remitting course of Lyme disease.