伯氏疟原虫重要内质网塑形蛋白的初步研究

发布时间：2018-01-15 03:24

本文关键词：伯氏疟原虫重要内质网塑形蛋白的初步研究　出处：《天津医科大学》2017年博士论文　论文类型：学位论文

更多相关文章： 脑型疟疾 疟原虫 内质网 YOP1 SEY1

【摘要】：重度疟疾目前仍是撒哈拉以南非洲主要的致死疾病之一,其中脑型疟疾最为凶险,因为其发病初期的临床表现和其他脑病很难区分,而一旦发病又会很快致死。脑型疟疾的发生机制尚不清楚,也缺乏有效的治疗。伯氏疟原虫(Plasmodium berghei ANKA,Pb)感染C57BL/6小鼠可以表现出类似人类脑型疟疾的症状,但是发病率并不稳定。前期研究显示小鼠脑型疟疾的发病率受到感染剂量的影响,因此我们通过分别注射103和106疟原虫感染的红细胞来接种C57BL/6小鼠,并比较脑型疟疾的发病率。结果显示,低剂量感染组小鼠全部发生血脑屏障的破坏,即脑型疟疾,而高剂量感染组只有约30%的小鼠出现该现象。低剂量感染组小鼠的虫血率不超过10%,并于四天后出现活动减少、木僵、偏瘫和抽搐等症状;而高剂量感染组虫血率明显高于低剂量组,且只有小部分小鼠出现脑型疟疾症状。组织病理学检查发现低剂量感染组小鼠出现脑组织的出血和水肿,与其临床症状一致。这些数据证明通过低剂量含虫红细胞感染小鼠可以有效建立脑型疟疾的实验模型。在此基础上,我们又进行了血常规和生理代谢方面的检测,结果显示低剂量感染组小鼠的白细胞和血小板数量比高剂量组和未感染组有明显下降,但是单核细胞比例上升、血糖下降且血脂升高。对免疫相关细胞的流式检测显示,虽然高、低剂量感染组之间并没有统计学差异,但和未感染组相比,树突状细胞的数量和比例降低,单核细胞的数量和比例上升,T淋巴细胞的活化增加。单细胞寄生虫通常利用分泌蛋白来致病。引发疟疾的疟原虫在进入哺乳动物血液后,会大幅重构自身表面,并改造被感染的红细胞表面的蛋白组分。因此,扰乱疟原虫蛋白分泌的手段将成为研究疟原虫致病性和免疫特性的关键。内质网是蛋白分泌的关键细胞器。我们的前期研究揭示内质网蛋白YOP1介导管状结构的形成,而SEY1介导形成网络所需的膜融合,这些内质网塑形蛋白的突变导致细胞不同程度的生长和分泌缺陷。我们通过基因的同源序列比对,确定了伯氏疟原虫存在YOP1和SEY1的同源蛋白,体外脂质体重组实验和蔗糖密度梯度实验等验证了PbYOP1具有诱导膜高曲度形成的功能和寡聚化趋势。酵母和哺乳动物细胞内质网形态回补实验进一步确认了PbYOP1和PbSEY1具有维持内质网管状网络形态的功能。为了深入研究PbYOP1和PbSEY1在疟原虫发育和侵染过程中的生理功能,我们通过疟原虫转染实验构建了PbYOP1或PbSEY1缺失的重组疟原虫。我们的研究结果确立了小鼠脑型疟疾的模型,为区分脑型疟疾和非脑型疟疾奠定了重要的基础,并找到了一些可用于早期诊断的线索。而伯氏疟原虫内质网塑形蛋白重组子的构建,为后期利用蛋白组学分析诱发脑型疟疾的关键分泌蛋白提供了重要工具,最终将用于阐明内质网形态与功能在疟原虫生长发育、侵染、和免疫隔离等过程中的作用机制。
[Abstract]:Severe malaria in sub Saharan Africa is still one of the leading killers, including cerebral malaria is the most dangerous, because the clinical manifestations of early onset of encephalopathy and other difficult to distinguish, but once the disease will soon die. The mechanism of cerebral malaria is not clear, the lack of effective treatment of Plasmodium berghei. (Plasmodium berghei ANKA, Pb) infected C57BL/6 mice can exhibit similar to human cerebral malaria symptoms, but the incidence is not stable. Previous studies have shown that the pathogenesis of mouse cerebral malaria infection rate is affected by the dose, so we were injected through 103 and 106 infection of Plasmodium red blood cells inoculated into C57BL/6 mice and compared the brain the incidence of malaria infection. The results showed that low dose group mice all occurred in the BBB, namely cerebral malaria, and high dose infection group, only about 30% of the mice appeared the phenomenon The low dose group of mice infected. Parasitemia does not exceed 10%, and reduced activity in four days, stupor, hemiplegia and convulsions and other symptoms; while high dose infection group parasitemia was significantly higher than the low dose group, and only a small part of mouse cerebral malaria symptoms. Histopathological examination found low dose infection mice had cerebral hemorrhage and edema, consistent with the clinical symptoms. These data proved that the experimental model by low dose containing insect can effectively establish cerebral malaria infected red blood cells in mice. On this basis, we also examined the blood and physiological metabolism, the results showed that low doses of white blood cells and infected mice the number of platelets in high dose group and non infection group decreased significantly, but the proportion of monocytes increased, elevated blood glucose and blood lipid. Decreased flow cytometry on immune cells, although high, low dose of infection There was no statistically significant difference between groups, but compared with the uninfected group, the number and proportion of dendritic cells decreased, the number and proportion of mononuclear cells increased, increased activation of T lymphocytes. Single cell secretory protein to pathogenic parasites usually use. Malaria causing Plasmodium in mammals into the blood, a reconstruction of the surface. Protein group and surface modification of infected red blood cells. Therefore, disrupting the Plasmodium protein secretion will become a key means of malaria parasite pathogenic and immunological characteristics. The endoplasmic reticulum is a critical organelle protein secretion. Our previous study reveals endoplasmic reticulum protein YOP1 mediates the formation of tubular structure, and SEY1 mediated formation the network required for membrane fusion, the endoplasmic reticulum shaping protein mutations lead to different degrees of cell growth and secretion by homologous sequence alignment. We determined the gene. The homologous protein of Plasmodium berghei YOP1 and SEY1 in vitro, liposome reconstitution experiments and sucrose density gradient experiments verified that the PbYOP1 has the function of inducing membrane and high curvature of the poly trend. Yeast and mammalian cells morphology of endoplasmic reticulum covering experiments further confirmed that PbYOP1 and PbSEY1 can maintain the tubular network morphology of endoplasmic reticulum function. In order to study the physiological function of PbYOP1 and PbSEY1 in the process of development of parasites and infection, we constructed recombinant Plasmodium PbYOP1 or PbSEY1 deletion by Plasmodium transfection experiments. The results of our study established a mouse model of cerebral malaria, laid an important foundation for the distinction between cerebral malaria and cerebral malaria, and found some can be used for early diagnosis of clues. And the construction of Plasmodium berghei recombinant protein of the endoplasmic reticulum remodeling, for further use of proteomic analysis by The key secretory proteins of cerebral malaria provide an important tool, which will eventually be used to elucidate the mechanism of endoplasmic reticulum morphology and function in the process of malaria growth, development, infection and immune isolation.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R382

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