动力相关蛋白Drp1在羊瘙痒因子139A感染小鼠脑组织中变化的研究

目的 研究动力相关蛋白1(Drp1)在羊瘙痒因子139A感染的小鼠脑组织中的变化。方法 采用Western Blot方法检测Drp1在羊瘙痒因子139A感染的脑组织匀浆中的含量变化及其亚硝基化水平。采用组织免疫荧光方法检测Drp1在脑组织中的分布。结果 在羊瘙痒因子139A感染终末期小鼠脑组织匀浆中,Drp1总量略有降低。对感染不同时间点的动态分析结果显示Drp1含量呈逐渐降低趋势,终末期稍有回升。感染终末期脑组织中亚硝基化水平明显增高。组织免疫荧光显示正常和羊瘙痒因子139A感染终末期小鼠脑组织中,Drp1与神经元细胞存在共定位现象。结论 Drp1在小鼠中枢神经系统中主要分布于神经元细胞,在羊瘙痒因子139A感染的小鼠脑组织中,Drp1总量略有降低,另外亚硝基化水平明显增高,提示在羊瘙痒病感染的小鼠脑组织中,线粒体动力相关蛋白的表达量及翻译后修饰水平出现了变化,在感染过程中起着重要作用。     

羊瘙痒因子139A感染引起小鼠脑组织中的1-ACT表达增加

目的 探究1-ACT在羊瘙痒因子139A感染小鼠脑组织中的变化情况。方法 利用蛋白免疫印迹、免疫组织化学、间接免疫荧光及荧光共聚焦方法分析羊瘙痒因子139A感染小鼠脑组织中1-ACT表达的变化和分布特点。结果 蛋白免疫印迹方法显示在羊瘙痒因子139A感染小鼠终末期脑组织中1-ACT的含量较正常对照小鼠明显上调且随着潜伏期的延长而逐渐增加;免疫组织化学方法发现1-ACT主要分布于羊瘙痒因子139A感染小鼠的皮层、丘脑和小脑区域;间接免疫荧光实验显示羊瘙痒因子139A感染终末期小鼠脑组织中补体成分C3含量明显增加,同时荧光共聚焦实验表明1-ACT与补体成分C3存在明显的共定位现象。结论 羊瘙痒因子139A感染终末期小鼠脑组织中1-ACT含量明显增加。     

CXCL1/CXCR2在羊瘙痒因子感染小鼠脑组织中的分布特征研究

目的 探究朊病毒感染小鼠脑组织CXC趋化因子配体1 (CXCL1)与CXC趋化因子受体2 (CXCR2)的分布特征。方法 通过免疫组织化学、免疫组织荧光双染实验明确羊瘙痒因子139A及ME7感染终末期小鼠脑组织中CXCL1/CXCR2的分布特征,确定CXCL1/CXCR2的靶细胞及与羊瘙痒因子样朊蛋白(PrPSc)沉积的关系。结果 通过全脑区免疫组化染色发现,CXCL1/CXCR2在羊瘙痒因子139A及ME7感染终末期小鼠脑组织中的含量明显升高,主要分布在海马、皮层、丘脑、小脑及延髓5个脑区。CXCL1与小胶质细胞和神经元细胞存在共定位,而CXCR2与神经元细胞存在共定位。在羊瘙痒因子139A及ME7感染终末期小鼠脑组织中CXCL1、CXCR2和PrP^Sc三者存在明显共定位。结论 CXCL1/CXCR2分布于朊病毒感染小鼠脑组织中朊病毒病理特征集中的脑区。     

寰枢椎周围软组织损伤对老龄小鼠脑衰老进程的影响

目的观察老龄小鼠寰枢椎肌肉、韧带、筋膜损伤对其脑衰老进程的影响。方法利用手术损伤小鼠寰枢椎周围软组织。利用跳台试验检测小鼠学习、记忆能力；组织切片HE染色观察脑海马神经元数量；免疫组织化学染色观察β-淀粉样多肽（Aβ）、caspase-3的表达；试剂盒检测血清NO及脑组织SOD、MDA的变化。结果与对照组相比，跳台试验：模型组小鼠反应期明显延长，潜伏期明显缩短，错误次数显著增加（P〈0．01）；HE染色可见模型组脑海马神经元数量明显减少，大脑皮层嗜神经现象明显；免疫组化：模型组脑海马区Aβ、caspase-3阳性细胞数均明显增加；生化指标检测结果：模型组小鼠脑组织MDA含量显著增加（P〈0．05），SOD活性明显下降（P〈0．01）；其血清NO含量也显著增加（P〈0．05）。结论寰枢椎软组织损伤促进老龄小鼠脑细胞凋亡，导致其学习、记忆功能明显减退，加速了脑衰老的进程。     

大肠腺癌中细胞增殖周期调控因子p16和cyclinD1的表达和意义

应用S－P免疫组织化学染色方法观察人大肠腺癌中细胞增殖周期调控因子p16和cyclinD1的表达和意义。结果表明，在良恶性细胞p16蛋白表达均位于细胞浆，CyclinD1蛋白表达主要位于细胞核。在大肠腺癌p16蛋白表达阳性率为49．2％，明显低于癌旁正常粘膜（789％），染色强度大部分为弱阳性或阳性（＋）。cyclinD1蛋白表达阳性率为67％，明显高于癌旁正常粘膜（28．9％），染色强度大部分为阳性（＋），强阳性（＋＋）。p16蛋白表达阳性率随大肠腺癌分化程度的降低而下降，与淋巴结转移无关。cyclinD1蛋白表达阳性率与大肠腺癌的分化程度、淋巴结转移均有关。本研究结果表明p16蛋白抑制肿瘤细胞增殖，而cyclinD1蛋白促进肿瘤细胞增殖这一细胞增殖周期调控机制，可能与大肠腺癌的发生发展有关。     

创伤小鼠脾细胞核因子—kB的表达变化

目的：探讨创伤后不同时间点脾细胞核因子-kappa B(NF-kB)的DNA结合蛋白和NF－kB p65蛋白亚型表达的动态变化。方法：采用小鼠双后肢闭合性砸伤＋骨折模型,于创伤后1、4、7日处死动物,分离、纯化脾细胞,提取细胞核蛋白。用电泳迁率改变试验（EMSA）检测NT－kB的DNA的结合活性,用免疫蛋白印迹法（Western blot)检测NF－kB p65蛋白亚型表达。结果：脾细胞NF－kB的DNA结合活性在创伤后1、4、7日表达明显增高。NF－kB p65蛋白亚型在创伤后1、4、7日表达明显增加。结论：创伤可明显增强脾细胞NF－kB的DNA结合活性和p65蛋白亚型的表达。在创伤后淋巴细胞活化及全身炎症反应综合征（SIRS）发生、发展过程中可能起重要作用。     

脑源性神经生长因子抗体对小鼠坐骨神经损伤后脊髓与背根节内Synaptophysin表达的影响

目的 探查小鼠坐骨神经压榨损伤后内源性脑源性神经营养因子(brain derived neuroliophic factor,BDNF)对Synaptophysin(SYN)在相应脊髓前角运动细胞与背根节神经元内表达的影响。方法 在小鼠一侧坐骨神经压榨损伤后腹腔注射BDNF抗体，中和内源性BDNF，然后用免疫组织化学方法观察SYN在与坐骨神经相连的脊髓前角运动细胞与背根节神经元的表达。结果 实验组SYN免疫反应阳性神经元的数目较对照组明显减少，阳性细胞的平均光密度也显著下降（P＜0.01)。结论 小鼠坐骨神经压榨损伤后内源性BDNF可能参与脊髓前角运动细胞与背根节神经元内SYN的表达。     

创伤小鼠脾细胞核因子κB的表达变化

目的：探讨创伤后不同时间点脾细胞核因子kappa B(NFκB)的DNA结合蛋白和NFκB p65蛋白亚型表达的动态变化。方法：采用小鼠双后肢闭合性砸伤+骨折模型,于创伤后1、4、7日处死动物,分离、纯化脾细胞,提取脾细胞核蛋白。用电泳迁移率改变试验(EMSA）检测NFκB的DNA结合活性;用免疫蛋白印迹法（Western blot）检测NFκB p65蛋白亚型表达。结果：脾细胞NFκB的DNA结合活性在创伤后1、4、7日表达明显增高。NFκB p65蛋白亚型在创伤后1、4、7日表达明显增加。结论：创伤可明显增强脾细胞NFκB的DNA结合活性和p65蛋白亚型的表达。在创伤后淋巴细胞活化及全身炎症反应综合征(SIRS)发生、发展过程中可能起重要作用。     

脓毒症小鼠不同组织炎症因子及T细胞亚群动态变化

目的 探讨CLP小鼠在脓毒症发病过程中不同脏器炎症浸润程度的变化以及不同免疫器官中T细胞亚群的相互关系。方法 对小鼠进行盲肠结扎穿孔术建立脓毒症模型，在术后0h、6h、12h、24h、48h及72h不同时间点检测小鼠血液、肺组织及回肠组织中IL-6、IL-10、IL-2、IL-4的含量变化，并以流式细胞技术检测小鼠脾脏中Treg及Th17比例变化、回肠黏膜固有层中CD4+T及CD8+T淋巴细胞比例变化。结果 肺组织及回肠组织中IL-6含量于术后6h达到峰值，血浆中IL-6含量于术后12h达到峰值，均显著高于同时间点假手术组（P＜0.01）。回肠组织中IL-10含量于术后6h达到峰值，明显高于假手术组（P＜0.01）。回肠组织中IL-2含量与术后出现持续上升，并在术后24h达到峰值，显著高于假手术组（P＜0.01）。血液、肺组织及回肠组织中IL-6、IL-10、IL-2、IL-4均于24h后大幅下降。CLP术后6h及24h的脾脏淋巴细胞Th17、Treg比例明显高于同时间点假手术组（P＜0.01）。回肠黏膜固有层中CD4+T细胞比例于术后12h达到最高，CD8+T细胞比例于术后24h达到最高，均显著高于假手术组（P＜0.01）。Th17、Treg、CD4+T及CD8+T细胞均于24h后出现下降。结论 脓毒症发病过程中，不同部位的炎症因子浸润程度不同，肠道黏膜中炎症反应出现最早且持续时间更长。脓毒症中后期CD8+T细胞的显著减少及Treg比例的相对性增加可能提示淋巴细胞增殖能力的下降。     

大鼠阿尔茨海默病模型中溶酶体组织蛋白酶D的表达及其意义

目的：观察阿尔茨海默病（AD）大鼠皮质神经元中溶酶体蛋白酶Cathepsin D的表达。方法采用β-淀粉样肽（Aβ）大鼠海马注射制作AD动物模型,用免疫组织化学染色和Western-blot印迹法检测大鼠颞底皮层Cathepsin D的表达。Y迷宫检测大鼠空间辨别性能和学习记忆能力。TUNEL法检测细胞凋亡情况。结果Aβ脑池内灌注造成Aβ沉积的AD模型在行为学和病理学改变上一定程度地模拟了AD。免疫组化和Werstern-bolt检测均显示实验组Cathepsin D阳性神经元数量较假手术组和正常组明显增加（P＜0.05）。TUNEL法检测显示AD大鼠颞底皮层神经元凋亡与正常组和假手术组相比显著升高（P＜0.05）。结论Cathepsin D在AD大鼠皮层脑组织中表达升高,可能参与了神经元凋亡等病理过程。     

Absence of autoantibodies against neurofilament proteins in the sera of scrapie infected mice

Autoantibodies against neurofilament proteins were not detected in any of the sera from the following scrapie infected mice: 19 mice infected with scrapie agent 139A in pre-clinical stage, 32 histologically confirmed scrapie mice and other 12 clinical scrapie mice infected with various strains. The test sera were assayed against acetone-fixed central neuron cultures from fetal mice by indirect immunofluorescence and immunoperoxidase techniques. The negative result suggests that autoantibodies against neurofilament proteins do not play a role in the pathogenesis of scrapie.     

Acceleration of scrapie disease in mice by an adenovirus

Coinfected mice were examined for a possible interaction between the scrapie agent and an adenovirus. A low titer (10(2) TCD50) of mouse adenovirus (MAdV) caused a significant acceleration of clinical signs of scrapie in mice infected 128 days previously with scrapie. In this experiment, the coinfected mice died 19 days earlier than mice infected with scrapie alone. When a higher titer of MAdV (10(4)-10(5) PFU) was used, a more drastic acceleration of scrapie disease was seen in mice infected 85 and 110 days previously with scrapie. At 85 days, coinfection caused mice to die 37 days earlier than mice infected with scrapie alone, whereas at 110 days, coinfection caused mice to die 52 days earlier than mice infected with scrapie alone. MAdV alone caused no clinical disease in normal mice. The brains of coinfected mice and mice that had been infected with scrapie alone showed a histopathology consistent with scrapie. A possible explanation for these findings is that the replication of the scrapie agent is accelerated by adenovirus. Defective parvoviruses are known to be helped by adenoviruses. Spleens from coinfected mice but not from mice infected with MAdV alone yielded, in cultures of BALB 3T3 cells, infectious MAdV and one or two smaller agents with the dimension and shape of a parvovirus.     

Effect of prior treatment with thioglycolate on the incubation period of intraperitoneally injected scrapie

Injection of mice with thioglycolate 5 days prior to intraperitoneal injection of scrapie brain homogenate led to a statistically significant increase in scrapie incubation periods. This was seen with two different scrapie strains (ME7 and 139A), in different mouse strains (C57BL/6J and Compton White), and at several dilutions of the scrapie inoculum.     

Scrapie incubation periods and end-point titers in mouse strains differing at the H-2D locus

In extending findings on the influence of the mouse H-2D locus on the scrapie incubation period, we showed that with the intracerebral (i.c.) route of injection, SJL and NZW mice (s and z alleles, respectively) had shorter incubation periods than C57BL mice (b allele) at several concentrations of two scrapie strains, ME7 and 139A. The three mouse strains have the same Sinc genotype, s7s7. Incubation period data among the three mouse strains after intraperitoneal (i.p.) injection revealed a different rank order of incubation periods from that seen after i.c. injection. End-point titers in the three mouse strains were similar after i.c. injection, but both scrapie strains yielded very low titers in NZW mice after i.p. injection. There were marked differences between 139A and ME7 incubation periods after both i.c. and i.p. injections in the three mouse genotypes, providing an additional parameter that distinguishes these two scrapie strains.     

A miRNA Signature of Prion Induced Neurodegeneration

MicroRNAs (miRNAs) are small, non-coding RNA molecules which are emerging as key regulators of numerous cellular processes. Compelling evidence links miRNAs to the control of neuronal development and differentiation, however, little is known about their role in neurodegeneration. We used microarrays and RT-PCR to profile miRNA expression changes in the brains of mice infected with mouse-adapted scrapie. We determined 15 miRNAs were de-regulated during the disease processes; miR-342-3p, miR-320, let-7b, miR-328, miR-128, miR-139-5p and miR-146a were over 2.5 fold up-regulated and miR-338-3p and miR-337-3p over 2.5 fold down-regulated. Only one of these miRNAs, miR-128, has previously been shown to be de-regulated in neurodegenerative disease. De-regulation of a unique subset of miRNAs suggests a conserved, disease-specific pattern of differentially expressed miRNAs is associated with prion–induced neurodegeneration. Computational analysis predicted numerous potential gene targets of these miRNAs, including 119 genes previously determined to be also de-regulated in mouse scrapie. We used a co-ordinated approach to integrate miRNA and mRNA profiling, bioinformatic predictions and biochemical validation to determine miRNA regulated processes and genes potentially involved in disease progression. In particular, a correlation between miRNA expression and putative gene targets involved in intracellular protein-degradation pathways and signaling pathways related to cell death, synapse function and neurogenesis was identified.     

Tubulovesicular structures in experimental Creutzfeldt-Jakob disease and scrapie

Tubulovesicular structures, measuring 20-50 nm in diameter, were found in dilated neuronal processes in brains from mice infected with the Fujisaki strain of Creutzfeldt-Jakob disease virus. These particles were similar to those observed in brains from hamsters infected with scrapie. These structures are consistently present in naturally occurring and experimentally induced spongiform encephalopathies, irrespective of the host species or virus strain. Their role in pathogenesis is undetermined.     

Evidence of ssDNA in tubulofilamentous particles: their relationship to scrapie-associated fibrils.

Abnormal tubulofilamentous particles were identified by electron microscopy using a simple touch negative staining technique from brains of mice infected with four strains of the scrapie agent. Treatment by three proteolytic enzymes and subsequent treatment with DNase and mung bean nuclease of grids prepared from the infected animals confirmed previous observations that the tubulofilamentous particles observed in scrapie-effected brains are complex structures. The core of the tubulofilamentous particle scrapie-associated fibrils was revealed by treatment with SDS. Treatment with proteolytic enzymes and subsequent treatment with DNase or mung bean nuclease or S1 nuclease also revealed typical and transitional stages of scrapie-associated fibrils. However, treatment with RNase A had no effect. The data suggest that nucleic acid is a single-stranded DNA protected by a protein coat.