人源隐孢子虫动物模型的建立与隐孢子虫种系发育关系分析

发布时间：2018-04-17 10:21

本文选题：隐孢子虫 + 线粒体　；参考：《河南农业大学》2006年硕士论文

【摘要】：隐孢子虫病是一种重要的人兽共患的原虫病，目前尚无用于治疗隐孢子虫病的特效药物。线粒体是真核生物中拥有蛋白和酶最多的细胞器，因而隐孢子虫线粒体也可能成为药物潜在的作用靶位。最近一些研究表明隐孢子虫存在线粒体诱生的细胞器，但和其他原生动物的线粒体存在着差异。隐孢子虫具有退化的线粒体，但缺少线粒体基因组，完全依赖核基因来编码所需的功能蛋白。 1．为了评价两种常用免疫抑制方法对小白鼠排卵囊规律的影响。作者将19日龄小白鼠随机分成两组，第一组通过饮水给予地塞米松，第二组采用导胃管灌服地塞米松。第3天，两组中每只小白鼠灌胃接种人源隐孢子虫，每只小白鼠感染量为1.5×10~6个卵囊。结果：两组均在感染的当天就有卵囊排出，感染后第6天粪便卵囊计数明显增加，随之出现3个高峰期，此后逐渐下降。免疫抑制组小白鼠在卵囊持续期不断有死亡但无腹泻症状。结论：用这两种免疫抑制方法，均能成功感染人源隐孢子虫，但通过灌服地塞米松可使小白鼠获得更高的OPG(每克粪便中的卵囊数)值和更长的排卵囊持续期。 2．为了近一步了解人源隐孢子虫在小白鼠体内的内生发育情况。作者采用19日龄昆明系小白鼠作为模型动物，随机分成8组，分3个感染剂量进行人源隐孢子虫分离株感染系列筛选试验，并确定随剂量和时间不同对人源隐孢子虫在小白鼠寄生部位的影响。感染后于4H、8H、12H、48H、72H破杀高剂量组。4D、6D、9D、12D，16D，20D破杀中剂量组。取每只小白鼠的消化道和其他脏器用福尔马林固定，作组织切片观察人源隐孢子虫在小白鼠体内的寄生部位和内生发育情况。结果表明隐孢子虫主要寄生在小白鼠十二指肠、空肠和回肠。在感染早期主要寄生在空肠和回肠，在感染中后期主要寄生在十二指肠和空肠。 3．为了从分子上了解本试验室所分离到隐孢子虫株所属的种或基因型，作者对所分离到隐孢子虫种株进行PCR扩增18SrRNA基因全序列和HSP70基因序列，并对PCR扩增片段进行测序，，然后利用Clustal X、DNAstar和Paup等软件对其进行序列分析比较、构建分子进化树，试图从分子上证明河南省不同地区不同种类隐孢子虫的遗传特征，以阐明隐孢子虫病的分子流行病学特点。通过18SrRNA基因全序列和HSP70序列分析，河南人源隐孢子虫分离株为小球隐孢子虫鼠基因型；河南鹿源隐孢子虫分离株不同与小球隐孢子虫鹿基因型；河南猪源隐孢子虫的两个分离株均为小球隐孢子虫猪基因型，即猪隐孢子虫；河南鹌鹑源的隐孢子虫两个分离株分别为贝氏隐孢子虫和火鸡隐孢子虫；河南乌鸡隐孢子虫和鸵鸟隐孢子虫分离株均为贝氏隐孢子虫；河南牛源隐孢子虫分离株为安氏隐孢子虫。 4．本试验以核编码的线粒体功能性蛋白基因Cpn60，AOX和PNO作为研究对象，对实验室分离到隐孢子虫分离株进行扩增测序，以确定在动物源隐孢子虫线粒体相关基因是否存在，对扩增出的序列进行比对分析构建进化树，以确定不同隐孢子虫株之间的进化关系，并与18SrRNA基因和HSP70基因构建的进化树作对照，进而评价这些基因是否更适合作为隐孢子虫分类和进化关系的基因座。结果通过CPN60、AOX和PNO基因编码序列构建进化树也同样适于作为隐孢子虫种系发育分析。
[Abstract]:Cryptosporidiosis is an important zoonotic protozoan disease, there is still no effective drug for the treatment of cryptosporidiosis. Mitochondria are organelles with most protein and enzyme in eukaryotes, so the mitochondrion of Cryptosporidium may be a potential target of drugs. The most recent studies show that Cryptosporidium insect mitochondria induced cell birth, but other protozoa and mitochondrial differences. Cryptosporidium has degenerated mitochondria, but the lack of mitochondrial genome, protein function is completely dependent on nuclear gene encoding is required.
1. in order to evaluate the effect of two kinds of commonly used methods of immunosuppressive mice pvax-cp15-p23-cp15 law. The 19 day old mice were randomly divided into two groups, the first group through drinking water treated with dexamethasone. The second groups by gavage. Dexamethasone guide tube for third days, two in each group mice were orally inoculated human Cryptosporidium, each mouse infection quantity is 1.5 * 10~6 oocysts. Results: the two groups were infected on the day of discharge oocysts, sixth days after infection fecal oocyst counts increased significantly, resulting in the 3 peak, then decreased gradually. The immunosuppressive mice in duration have been killed but oocysts without diarrhea conclusion: with the two kinds of immune suppression methods, can successfully infect human Cryptosporidium, but by gavage dexamathasone mice obtained higher OPG (the number of oocysts per gram feces) value and longer duration of oocyst.
2. in order to further our understanding of human Cryptosporidium in mice of endogenous development. The author adopts 19 day old Kunming mice as animal model, randomly divided into 8 groups, 3 of infective dose of Cryptosporidium infection series screening test, and determined with dose and time different effects on mice in parts of the parasitic human Cryptosporidium infection. After 4H, 8H, 12H, 48H, 72H break to kill high dose group.4D, 6D, 9D, 12D, 16D, 20D in the break to kill dose. Take each mouse digestive tract and other organs were fixed in formalin as the organization section observation of human Cryptosporidium growth in mice in vivo and parasitic site. Results show that Cryptosporidium parasites mainly in mouse duodenum, jejunum and ileum. In early infection mainly parasitic in jejunum and ileum, late in infection mainly parasitic on twelve duodenum and jejunum.
3. in order to understand the test chamber from the molecule isolated species or genotypes of Cryptosporidium isolates belong to the author the isolated Cryptosporidium isolates were amplified by PCR 18SrRNA gene sequence and HSP70 gene sequence, and amplified PCR fragments were sequenced, and then use Clustal X, DNAstar and Paup software analysis and comparison of the sequences, the phylogenetic trees, trying to prove from the molecular genetic characteristics of Cryptosporidium in different areas of Henan Province, to clarify the molecular epidemiology of cryptosporidiosis. Through the complete sequence of 18SrRNA gene and HSP70 sequence analysis, strain of Cryptosporidium parvum genotype in Henan source separation Cryptosporidium; and different strains of Cryptosporidium parvum genotype isolated by Henan deer deer sporozoans; Henan porcine Cryptosporidium isolates were two for Cryptosporidium parvum swine genotype, i.e. Cryptosporidium suis Henan; quail source of Cryptosporidium isolates were two Cryptosporidium and Cryptosporidium meleagridis; Henan chicken and ostrich Cryptosporidium Cryptosporidium isolates were Cryptosporidium baileyi; Henan Cryptosporidium from bovines by isolates of Cryptosporidium andersoni.
In this experiment, 4. mitochondrial functional nuclear protein gene Cpn60 encoding, AOX and PNO as the research object, the laboratory isolates of Cryptosporidium isolates was amplified and sequenced to determine the source of Cryptosporidium in animal mitochondrial related genes exist on the amplified sequence comparison analysis of the phylogenetic tree, to determine the different hidden the evolutionary relationship between spore strains, and phylogenetic tree was constructed with 18SrRNA gene and HSP70 gene as controls, and to evaluate whether these genes are more suitable as the locus of Cryptosporidium taxonomy and evolutionary relationships. The results by CPN60, AOX and PNO gene encoding sequence phylogenetic tree is also suitable for Cryptosporidium species development analysis.

【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R-332;R383

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