【作者】 蒋韦斌；

【导师】 林矫矫；

【作者基本信息】 上海师范大学 ， 动物学， 2009， 硕士

【摘要】 血吸虫病是由血吸虫感染引起的分布广泛、危害严重的人兽共患寄生虫病。本研究以血吸虫易感动物小鼠、非易感动物大鼠和对血吸虫具有天然抗病作用的东方田鼠作为动物模型,应用组织化学技术观察三种动物感染血吸虫后肺、肝组织病理学变化差异,应用芯片技术和生物信息学技术等比较分析三种动物感染血吸虫前后的基因差异表达,并重点对三种动物感染血吸虫后免疫相关分子、生长发育相关分子的表达差异进行深入分析,推测三种动物对血吸虫感染的可能应答机制,具体如下:1.对感染日本血吸虫的东方田鼠、大鼠和小鼠的肺、肝进行组织病理学观察,从组织水平了解三种动物感染日本血吸虫后肺肝组织的组织学变化差异及其引起的病理学变化差异。结果显示与适宜性宿主小鼠相比,在感染早期日本血吸虫在非适宜性宿主大鼠以及具有天然抗病作用的东方田鼠体内引起更强烈的免疫应答和病理损害,HE染色呈现免疫效应细胞以嗜酸性粒细胞为主,同时还有中性粒细胞和巨噬细胞等,这可能是东方田鼠具有先天抗日本血吸虫病的主要原因之一。2.利用大规模、高通量的全基因组寡核苷酸芯片技术,比较分析东方田鼠、WISTAR大鼠和BALB/c小鼠感染日本血吸虫前后肺和肝组织基因表达差异,应用SpotDataTM Pro V3.0软件和聚类软件对数据进行分析,采用实时定量PCR法进行验证。芯片杂交结果如下:（1）.攻虫感染十天后,东方田鼠、大鼠和小鼠与感染前相比肺上调基因数分别为2678、1980和364条;下调表达的基因数分别为1478、2306和16条;相应的肝上调表达基因基因数分别为330、1655和111条;下调表达基因数分别为190、540和179条。选择6个芯片杂交显示差异表达基因进行real time PCR验证,结果和芯片基本相符。（2）.分别筛选三种宿主肺中显著变化（Cy5/Cy3≥2.0和Cy5/Cy3≤0.5）的同源基因进行分层聚类分析,发现这类差异基因有1334条,为东方田鼠显著变化、大鼠相对变化而小鼠基本保持不变的基因。go的功能分析显示这些差异基因主要参与信号转导（signal transduction）、转录调节活性（transcription regulator activity）、细胞粘附（cell adhesion）、细胞凋亡（apoptosis）等;而肝中这类差异基因有265条,涉及细胞骨架构成（cytoskeleton construction）、催化活性（catalytic activity）、代谢作用（metabolism）、细胞分化（cell differentiation）、免疫应答（immune response）、生长发育（growth and development）等。（3）.通过聚类分析发现,肺肝组织共同差异表达基因有74条,其中东方田鼠显著上调基因46条,显著下调基因28条,这些基因主要参与了信号转导（Msr1、Scarb1、Pnrc1）、基因转录调控（Cnot2、Znrd1、Cited2、Xbp1、Gpbp1）、免疫应答（C1qa、C1ra、C1rb、Psmb8、Itgb2）等。（4）.感染日本血吸虫10d时东方田鼠肺、肝中一些与免疫相关的基因和一些细胞凋亡诱导基因表达上调,如肺中的补体成分1q（C1qa）、补体成分8a （C8a）、组织蛋白酶S（Ctss）、免疫球蛋白γFc受体1（cgr1）和免疫球蛋白γFc受体3 （cgr3）等;肝中有干扰素调节因子7（IRF-7）、组织蛋白酶S（Ctss）、CD74等;编程性细胞死亡6（Pdcd6）、半胱天冬酶7（Casp7）、酪氨酸蛋白激酶2（Jak2）等。而一些与生长发育相关的重要分子则在东方田鼠呈下调表达,如肺内甲状腺激素受体α（Thrα）、甲状腺激素应答基因（Thrsp）和类固醇11β脱氢酶1（Hsd11β1）基因,肝中胰岛素样生长因子1（IGF-1）基因。（5）.对基因表达谱进行深入分析还提示三种宿主肺、肝组织对血吸虫感染可能有不同的应答机制,东方田鼠通过Jak-STAT、VEGF、Notch以及FcεRⅠ等信号途径介导;大鼠通过补体级联途径介导;小鼠则通过多种细胞因子（主要是趋化因子和TNF）相互作用及Ca²⁺信号途径介导。本研究对深入探索东方田鼠抗日本血吸虫病的分子机理、发现东方田鼠抗血吸虫病的关键分子、阐明血吸虫与宿主的相互作用关系、开拓抗血吸虫疫苗和药物研究新途径都具有重要意义。更多还原

【Abstract】 Schistosomiasis caused by schistosome, is a wide spread parasitic zoonosis that causes serious healthy problem to both human and animals. The research useed mouse, a susceptible host of schistosome, rat, a unsusceptible host, and Microtus fortis, a resistant host, as animal model. After challenge infection, the histopathological change in lungs and livers of the three kinds of animal was observated with histochemistry technique. Gene difference expression was analysed and compared with microarray and bioinformatics technique. The dissertation focused on the different expression of immune-associated genes and development-associated genes and infered the possible response mechanisms of three kinds of animal infected with schistosome. The results were as follows:The histopathological change in lungs and livers of Microtus fortis, rat and mouse were observed by hematoxylin-eosin stain and Olympus light microscope.The Schistosoma japonicum caused more intensive immune response and pathological lesion in the body of Microtus fortis and rat than mouse, the susceptible host. Hematoxylin-eosin stain showed that the immune effector cells nearly were eosinophile granulocyte, supplemented by heterophil granulocyte, macrophage, et al, which may be one of the main reasons why Microtus fortis can naturally resist to Schistosoma japonicum.Gene difference expression of the lung and the liver tissues between Microtus fortis,rats and mice infected with Schistosoma japonicum for 10d were analyzed by oligonucleotide chip.Data analysis was performed on the basis of a significant two-fold change in signal using SpotDataTM Pro V3.0 and cluster3.0 software in order to find out gene function cluster associated with schistosomiasis resistence.Real-time PCR were used for validation. The chief results are showed as fo11ows:Ten days after challenge infection, in Microtus fortis,2678 lung and 330 liver genes with Gene Ontology annotation were up-regulated and 1748 lung and 190 liver genes were down-regulated in comparison with controls;in rats,1980 lung and 1655 liver genes were up-regulated and 2306 lung and 540 liver genes were down-regulated;in mice,364 lung and 111 liver genes were up-regulated and 16 lung and 179 liver genes were down-regulated. The differential expression was validated in 6 randomly selected genes using quantitative RT-PCR.Homologous genes of Microtus fortis,rats and mice are highlighted on the basis of a significant（Cy5/Cy3≥2.0或≤0.5）. Hierarchy cluster analysis showed that 1334 lung genes were identified as differentially regulated, which were significant different in Microtus fortis, relatively different in rats and nearly basically fixed in mouse. These genes could be functionally categorized into signal transduction,transcription regulator activity,signal,cell adhesion and apoptosis. These kind of genes in liver could be functionally categorized into cytoskeleton construction,catalytic activity,metabolism,cell differentiation, immune response, growth and development. Cluster analysis showed that 74genes,including Msr1,Cnot2,C1qa,Psmb8,Itgb2,and so on,were identified as differentially regulated between the two groups.Some immune-associated genes （such as C1qa, C8a, Ctss, cgr1, cgr3, IRF-7 and CD74） in Microtus fortis infected with Schistosoma japonicum for 10 days were up-regulated. Some development-associated genes （such as Thrα, Thrsp, Hsd11β1 and IGF-1） were down-regulated while some apoptosis induction genes （such as Pdcd6, Casp7, Jak2） were up-regulated.The profound analysis on gene expression pattern suggests that the lung tissues and liver tissues of three hosts infected with Schistosoma japonicum have different response mechanisms. Microtus fortis induces immune response via Jak-STAT, VEGF, Notch and FcεR. Rat induces immune response via complement cascade pathway. Mouse induces immune response via Ca²⁺ signal pathway and the interreaction between chemotatic factor and TNF.The results reported here were meaningful for exploring the molecular mechanism of schistosoma-resistance of M.fortis , finding out the key molecule of resisting to Schistosoma japonicum, elucidate the interaction between Schistosoma japonicum and its host, bringing out a new way to develop anti-schistosomula vaccines and drugs.更多还原