【作者】 周永华；

【导师】 范红结； 高琪；

【作者基本信息】 南京农业大学 ， 兽医， 2010， 博士

【摘要】 弓形虫病是一种广泛流行于世界各地且严重危害健康的人兽共患病,病原体弓形虫具嗜神经细胞性特点,可侵蚀大脑神经细胞,导致宿主呈现一种慢性、隐匿或潜在的脑部感染。弓形虫感染宿主非常广泛,不同宿主对弓形虫的易感性不同,并且感染后的症状及转归也不相同。因此选择适宜的实验动物建立弓形虫感染的动物模型,直接关系到研究工作的成败,建立慢性感染状态的动物模型对人畜弓形虫病的防治研究也具有极大的实用价值。目前,缺乏良好的强毒RH株的慢性感染动物模型,已成为当前弓形虫病诊断及疫苗评价等相关研究的重要制约因素之一。大鼠与人相似,对弓形虫感染的敏感性低,感染后多呈隐性感染。因此大鼠感染弓形虫后的动物模型更易模拟人类感染弓形虫后的状况,对研究人类感染弓形虫的免疫学作用、母婴传播以及疫苗的设计和诊断试剂的研发具有更大的价值。目前国内外对大鼠弓形虫感染的研究已有报道,但大多采用弱毒虫株如Me49、Fukaya、Pru株等,很少应用弓形虫强毒(RH)株,并且感染后不易分离检查到弓形虫病原体而得到确切的感染依据。现有研究表明,弓形虫慢性感染有改变宿主行为的能力,特别是造成宿主的辨别力、学习记忆能力等的损害。海马是记忆形成的关键受损区,只有海马或海马周围脑区受损时,才出现空间定位的记忆障碍。目前关于弓形虫感染致中枢神经系统的损害作用机制了解较少,需要进行深入研究。本研究以清洁级实验动物大鼠为研究对象,建立慢性弓形虫感染的大鼠动物模型；并应用该动物模型通过物体识别试验和Morris’水迷宫观察弓形虫慢性感染对大鼠的学习认知能力的影响；并研究其海马组织的细胞因子、抗氧化水平、脑源性神经营养因子(BDNF)和N-甲基天冬氨酸受体(NMDA)、脑内单胺类神经递质去甲肾上腺素(NE)、多巴胺(DA)、5-羟色胺(5-HT)含量；脑组织神经丝(NF)mRNA表达、海马神经元细胞周期、细胞凋亡及其半胱氨酸蛋白酶-3 (caspase-3)和细胞色素C(Cytochrome C, Cyt c)表达及其海马组织的比较蛋白质组学等,探讨其可能的作用机制。其主要研究内容包括：1、弓形虫慢性感染RH株大鼠模型的建立通过经口或腹腔感染弓形虫RH株速殖子并辅以免疫抑制方法建立弓形虫慢性感染的大鼠模型进行了比较和研究。结果显示口服感染和腹腔感染弓形虫RH株90天后进行解剖,在其脑、肝组织印片、匀浆涂片中均未查见弓形虫速殖子抑或包囊,用B1基因PCR和B1与529bp基因双重PCR检测脑组织结果也为阴性,将部分大鼠脑组织匀浆后接种盲传小鼠,三代后均未出现临床症状,也无死亡。但在免疫抑制后感染弓形虫RH株组的大鼠脑组织中有2只通过小鼠接种回复分离成功RH速殖子。同时在感染弓形虫速殖子二月后再免疫抑制大鼠脑组织的匀浆涂片中4只发现查见少量弓形虫速殖子,并在双重PCR检测中有7只阳性。说明通过腹腔接种107弓形虫RH株速殖子可以建立大鼠的隐性感染模型。2、慢性弓形虫感染对大鼠的学习记忆能力及其海马细胞因子和抗氧化水平的影响通过被动回避实验和Morris水迷宫试验,观察弓形虫感染9周后对大鼠的学习记忆能力等行为学改变。应用放射免疫技术检测大鼠海马组织IL-1β、IL-6、TNF-α水平,同时以免疫组化检测NOS活性。应用黄嘌呤氧化酶法测定SOD活性、硫代巴比妥酸比色法测定MDA含量。结果显示慢性弓形虫感染对大鼠的记忆获得没有影响,但记忆消失要早于对照组；其Morris水迷宫测试中逃避潜伏期均明显延长,其距离百分比明显降低(P<0.05),感染组大鼠海马组织IL-1β、TNF-α水平明显高于对照组(P<0.05)；但IL-6水平与对照组相比差异无显著性(P>0.05)。感染组大鼠海马S0D活性较对照组降低(P<0.05)、海马MDA含量较对照组升高(P<0.05)。感染大鼠大脑皮层NOS阳性细胞数增加。提示慢性弓形虫感染对大鼠的学习记忆能力有影响,其作用机制可能与大鼠海马组织IL-1β、TNF-α细胞因子水平升高,自由基清除能力下降,氧化应激反应增强有关。3、慢性弓形虫感染对大鼠海马组织脑源性神经营养因子(BDNF)和N-甲基天冬氨酸受体(NMDA)亚单位NR2A、NR2B表达的影响将4 w大鼠感染弓形虫速殖子9 w后,应用免疫组化、原位杂交技术和图像处理方法分析海马BDNF,CA1、CA3和齿回状NR2A和NR2B的表达。结果显示：慢性弓形虫感染组大鼠免疫组化海马BDNF阳性染色颗粒显著高于对照组(P<0.05),原位杂交显示感染大鼠脑组织中BDNF mRNA表达均增加,海马阳性杂交信号高于对照组(P<0.05)。慢性弓形虫感染组大鼠在CA3区表达的免疫反应灰度值显著高于正常对照组(P<0.05),而在CA1区和齿状回表达的免疫反应灰度值与对照组相比较差异无显著性(P>0.05)。慢性弓形虫感染组大鼠的NR2B蛋白的表达在CA1和CA3区表达的免疫反应灰度值均显著高于对照组(P<0.05)；而在齿状回表达的免疫反应灰度值差异无显著性(P>0.05)。提示慢性弓形虫感染大鼠海马组织BDNF和BDNF mRNA表达增强,弓形虫慢性感染可影响其海马NMDA受体表达。4、慢性弓形虫感染对大鼠物体识别能力和脑内单胺类神经递质去甲肾上腺素(NE)、多巴胺(DA)、5-羟色胺(5-HT)含量的影响采用物体识别试验和高效液相色谱电化学法检测弓形虫感染9周后的大鼠物体识别能力和脑内单胺类神经递质NE、DA、5-HT含量。结果弓形虫感染大鼠的物体识别分辨指数(DI)显著低于对照组(P<0.01)。脑组织中NE、5-HT显著降低,而DA则显著升高,与对照组比较差异均有统计学意义(P<0.05)。提示慢性弓形虫感染可能通过影响脑内单胺类神经递质的合成从而降低物体识别能力和学习记忆能力。5、慢性弓形虫感染对大鼠脑组织神经丝mRNA表达和T细胞免疫水平的影响将4周龄雄性SD大鼠感染弓形虫9周后,应用RT-PCR法检测大鼠大脑组织中高相对分子质量NF(NF-H)、中相对分子质量NF(NF-M)和低相对分子质量NF(NF-L)mRNA表达水平；应用流式细胞术检测大鼠外周血CD3+、CD4+、CD8+ T淋巴细胞：酶联免疫吸附试验测定其血清中Y干扰素(IFN-y)、肿瘤坏死因子α(TNF-α)、白细胞介素4(IL-4)水平。结果显示弓形虫感染大鼠大脑组织NF-L mRNA下降为对照组的64.56%；NF-M下降为对照组的95.83%；NF-H下降为对照组的89.47%。弓形虫感染组大鼠外周血的CD4+T细胞和CD8+T细胞与对照组比较,差异均无统计学意义。而感染大鼠血清IFN-γ、TNF-α、IL-4水平升高。6、慢性弓形虫感染对大鼠海马神经元细胞周期、凋亡及其半胱氨酸蛋白酶-3(caspase-3)和细胞色素C (Cytochrome C, Cyt c)表达的影响应用流式细胞术检测慢性弓形虫感染大鼠海马神经元细胞周期和细胞凋亡,以罗丹明123染色检测其线粒体膜电位,同时以免疫组化法检测海马caspase-3和Cyt c蛋白表达。结果显示大鼠弓形虫感染10周后,海马神经元细胞凋亡率显著增加(P<0.05),GO/G1期细胞百分率增高(P<0.05),即出现GO/G1期阻滞,S期细胞减少(P<0.05)；线粒体膜电位降低(P<0.05); caspase-3和Cyt c蛋白表达则均显著增高(P<0.05)。提示慢.性弓形虫感染可能导致大鼠海马神经元细胞的凋亡,进而引起一定的认知和学习记忆能力影响。7、慢性弓形虫感染大鼠与正常大鼠海马的比较蛋白质组学通过建立慢性弓形虫感染的大鼠模型,分离大鼠海马组织并提取海马蛋白质样品后,通过比较蛋白组学,分析其差异蛋白。通过基质辅助激光解吸/电离飞行时间质谱(MALDI-TOF-MS)对酶解后的肽段进行分析。经数据库(NCBI)检索,对差异蛋白质进行鉴定。结果发现有5个蛋白斑点在慢性弓形虫感染大鼠海马蛋白双向电泳图谱中消失,11个蛋白斑点在2组大鼠海马组织中含量发生了3倍以上的变化,其中弓形虫感染组上调4个,下调7个。质谱分析和数据库检索了16个差异表达蛋白,鉴定了9个蛋白质。分别是肌酸激酶、磷酸激酶1、ATP合酶、烯醇化酶、线粒体顺乌头酸酶、谷氨酰合成酶和肌动蛋白等,其表达的差异蛋白功能涉及能量代谢和信号传递等过程。蛋白差异点的发现为揭示弓形虫感染致海马损伤的机制以及研究弓形虫慢性感染致学习记忆能力影响提供了有益的线索。8、慢性弓形虫感染大鼠脊髓组织蛋白质的双向电泳分析和质谱鉴定采用双向凝胶电泳分离脊髓总蛋白,考染显色,Image Master图象分析系统分析,基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)分析获取肽质量指纹图谱(PMF),Mascot软件搜索MSDP、SWISS2PROT数据库鉴定蛋白质。结果获得重复性和分辨率较好的脊髓双向凝胶电泳图谱。斑点数分别检出268±13和301±15个蛋白点,对2张电泳图进行匹配后发现有7个蛋白斑点在2组大鼠脊髓组织中含量发生了3倍以上的变化,有差异的7个蛋白斑点经质谱鉴定的数据检索发现了3个差异表达蛋白质。分别为glyceraldehyde-3-phosphate dehydrogenase(甘油三磷酸脱氢酶)valosin-containing protein(含缬酪肽蛋白)和cytoplasmic 2(Gamma-actin)(类肌动蛋白)。弓形虫慢性感染对大鼠脊髓神经损伤机制可能与能量代谢受阻和细胞增殖有关。差异蛋白点有可能成为弓形虫慢性感染的治疗靶点。综上所述,慢性弓形虫感染对实验大鼠的学习记忆能力有一定程度的损害,其作用机制可能是弓形虫感染后影响了大鼠海马组织的细胞因子、单胺类神经递质、海马抗氧化水平以及蛋白差异表达等。其研究结果可为发现新的药物靶标奠定基础。更多还原

【Abstract】 Toxoplasmosis is a causative agent for zoonotic disease responsible for a wide variety of infections in animal and humans. Toxoplasma gondii has the ability of eroding neurons in brain, which lead to a chronic, hidden or latent infection in brain. Susceptivity is varied in different animal, as well as the symptom and sequel of the infection. A suitable animal model is a key point in researches. It has great value to established chronic infected animal model is important in research about diagnosis, vaccine and therapy of toxoplasmosis. Like human beings, rats is not susceptive to Toxoplasma gondii, it is generally asymptomatic after infected with Toxoplasma gondii. So, rat’s infection is more similar to human’s toxoplasmosis. It has more value in researching diagnosis, pathogenic biology, vaccine of toxoplasmosis.Recent studies have shown that chronic infection by Toxoplasma gondii can change host behavior and especially damage discretion and learning of host. Hippocampus is the damage zone crucial for memory formation. Only around the hippocampus or hippocampal brain regions damaged, there will be the spatial location of memory impairment. At present, mechanisms of central nervous system damage caused by Toxoplasma infection is poorly understood, and therefore need further research. The purpose of this study is to construct rat model which chronic infected by toxoplasma gondii. The contents of the study are as follows:1. Construction of rat model with chronic infection of Toxoplasma gondii RH strainBy mouth or abdominal cavity infected with Toxoplasma gondii RH strain, with the mothod of immune suppression, we constructed, compared, researched the rat model. The results showed that when dissected the rat infected with Toxoplasma gondii RH strain by mouth and abdominal cavity 90 days later, the tachyzoite or cyst could not be checked in the smear of brain, liver or homogenate, with the result of PCR using gene B1 or double PCR using gene B1 and 529bp, showed negative, when innoculated with the homogenate of rat’s brain, the mouse appeared no clinical symptom or death. But when immune suppression firstly, then infected with Toxoplasma gondii RH strain, we can separate the tachyzoite. And then, after infected the rat with tachyzoite, with the result of immune suppression, a few tachyzoites could be checked in 4 rats, meanwhile,7 positive rats were checked by the method of double PCR. All of these showed that by infected with Toxoplasma gondii RH strain using intraperitoneal injection, the infected model can be constructed.2.The effects of chronic Toxoplasma gondii infection on capacity of learning and memory and cytokinese of hippocampus in ratsAfter 9 weeks of infection, passive avoidance tests and Morris water maze tests were carried out to observe behavior changes such as learning and memory ability. The level of IL-1β、IL-6 and TNF-αof hippocampus in rats were examined by radioimmunoassay, the activity of SOD with xanthine oxidase, the content of MDA with thiobarbituric acid. The results showed that there were no difference in memory acquirement in all rats, but memory retention of infected rats was earlier than control group’s. The escape latency of the rats in Toxoplasma gondii infection groups was significantly greater than the control group’s and the percent of distance between the quadrant was previously decreased (P< 0.05). Compared with control groups, the level of IL-1βand TNF-αin hippocampus increased significantly in the Toxoplasma gondii infection group (P< 0.05), but the level of IL-6 was opposite (P> 0.05). SOD activity in hippocampus in Toxoplasma gondii infection rats mgprot is lower than in the control group (P<0.05). MDA content in hippocampus of the infected group increased, compared with the control group (P<0.05). Toxoplasma gondii infection could lead to obvious effects in learning and memory capacity of rats. Regulating the level of IL-1β、TNF-αcytokines and decrease hippocampal scavenging and increase oxidative stress in rats hippocampus by chronic Toxoplasma gondii infection maybe one of the mechanisms for affecting learning and memory ability of Toxoplasma gondii infection rats.3. Expression effects of subunits NR2A, NR2B of neurotrophic factor (BDNF) and N-methyl-aspartate receptor (NMDA) derived from rat hippocampus which were infected by Toxoplasma gondii Four-week old rats were infected by Toxoplasma gondii tachyzoite. After 9 weeks, use the application of immunohistochemistry, insitu hybridization and image processing to analysis the expression of hippocampal BDNF, CA1, CA3 and the dentate gyrus NR2A and NR2B. The results showed that the positive immunohistochemical staining granules of Toxoplasma gondii infection in rats’hippocampal BDNF were significantly higher than control groups, situ hybridization showed that rats infected with Toxoplasma gondii brain BDNF mRNA expression were increased, positive hybridization signals higher than the hippocampus. The expression in the CA3 area gray value of immune response was significantly higher than the control groups, while the expression of immune response in CA1 and dentate gyrus gray value had no significant differences compared with the control groups. The expression of NR2B protein expression in the CA1 and CA3 areas of the immune response gray were significantly higher than control groups(P<0.05); while the expression in the dentate gyrus gray value of immune response was no significant difference(P>0.05), which suggested that chronic infection of Toxoplasma gondii prompted hippocampus BDNF and BDNF mRNA expression. Toxoplasma chronic infection can affect the expression of hippocampal NMDA receptors.4. Effects of Toxoplasma gondii chronic infection in rats’on object recognition and brain monoamine nerrotransmitters norepinephrine, dopamine (DA), 5-hydroxytryptamine(5-HT) contentThe object recognition test and morris water maze tests were carried out to observe such behavior change as the learning and memory ability. Meanwhile, the monoamine neurotransmitter NE, DA and 5-HT contents in Toxoplasma gondii infection rats brain were detected with high performance liquid chromatography-electrochemical detection (HPLCECD). For ORT, infection groups of three doses showed significantly less exploration time on new object (P<0.05); the discrimination index (DI) of infection groups showed very significant lower DI (P<0.01), compared with control group. The contents of NE,5-HT in infected rats significantly reduced, while the DA was significantly increased. Compared with the control groups, there were significant differences (P<0.05). Suggest that Toxoplasma chronic infection may influence the monoamine neurotransmitter synthesis and therefore reduce the learning and memory ability.5. Effect of Toxoplasma gondii infection on levels of neurofilameat mRNA and cellular immunity in ratsFour-week old male SD rats were injected with Toxoplasma. After 9 weeks, mRNA levels of light molecular NF (NF-L), medium molecular NF (NF-M) and high molecular NF (NF-H) in cerebrum was determined by RT-PCR. The percentages of CD4+,CD8+ T lymphocytes were examined by means of flow cytometery, and the peripheral blood serum levels of IFN-y, TNF-a, IL-4 were analyzed by ELISA. The results showed that the mRNA levels of NF-L in rat cerebrum decreased significantly to 64.56%, the NF-M decreased to 95.83%, and NF-H declined to 89.47% of the control. Compared with the control group, no significant changes were observed on the levels of CD4+ T lymphocytes and CD8+ T lymphocytes. The levels of IFN-y, TNF-a, IL-4 in experimental rats’ sera raised.6. Changes of apoptosis and its related protein expressions of hippocampal neurons in rats with Toxoplasma gondii chronic infectionDetect Cell cycle and the percentage of the hippocampal neuron apoptosis in the rats suffered from Toxoplasma infected rats was measured with flow cytometry (FCM) and their mitochondrial membrane potential was measured with FCM under the Rhodamine 123 dying. The protein expressions of Cyt c and caspase-3 were detected with immunohistochemical methods. The results show that apoptosis in hippocampal neurons was significantly increased after 10 weeks of toxoplasma gondii infection (P<0.05). The cell percentage of G0/G1 phase increased significantly (P<0.05), while the cell percentage of S phase decreased significantly (P<0.05); the mitochondrial membrane potential decreased significantly (P<0.05), and the expressions of caspase-3 and Cyt c proteins increased significantly (P<0.05). It shows that Toxoplasma gondii chronic infection can lead to the apoptosis of the hippocampal neuron, and then lead to a certain learning and memory capabilities.7. Comparative proteome analysis of rat hippocampus from Toxoplasma gondii chronic infected and healthy ratsEstablish animal model of the Toxoplasma inection rat. Isolated hippocampus and extract hippocampal protein sample, and then total protein were analyzed by 2-DE. We obtained proteins of the satisfactory 2-DE patterns of the spinal cord. Totally 268±13 and 301±15 protein spots were obtained in the Toxoplasma gondii infection and control rats spinal cord maps respectively, which 7 spots increased or decreased in quantity.3 protein spots were identified by MALDL-TOF-MS,and similar to glyceraldehyde-3-phosphate dehydrogenase、valosin-containing protein and Gamma-actin. Proteomics method of Toxoplasma chronic infection was established in spinal cord. The differentially displayed proteins in the spinal cord may provide further insight into molecular mechanisms and useful clues for developing new drugs for its treatment.8. Proteome research of Toxoplasma gondii infection spinal cord by two -dimensional gel electrophoresis analysis and mass spectrometry identificationTo establish animal model of the Toxoplasma infection rat, and spinal total protein were analysed and extracted by 2-DE, blue staining and analyzed with PDQuest 1.0 software. We obtained proteins of the satisfactory 2-DE patterns of the spinal cord. Totally 268±13 and 301±15 protein spots were obtained in the Toxoplasma gondii infection and control rats spinal cord maps respectively,of which 7 spots increased or decreased in quantity.3 protein spots were identified by MALDL-TOF-MS,and similar to glyceraldehyde-3-phosphate dehydrogenase、valosin-containing protein and Gamma-actin. The differentially displayed proteins in the spinal cord may provide further insight into molecular mechanisms and useful clues for developing new drugs for its treatment.In summary, Toxoplasma gondii chronic infection damage learning and memory abilities of rats. The mechanism of Toxoplasma gondii infection may be related to hippocampus of cytokines, monoamine neurotransmitter, hippocampal antioxidant levels and protein differential expression and so on. The results provided basis for discovering new drug targets.更多还原