| 题名 | 树鼩 (Tupaia belangeri）血栓模型与免疫细胞内毒素模型 |
| 作者 | 刘杰 |
| 学位类别 | 博士 |
| 答辩日期 | 2014-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 张云 |
| 关键词 | 树鼩 疾病动物模型 血栓 蛋白酶激活受体 Vorapaxar 内毒素 渗透压 |
| 其他题名 | Thrombosis and endotoxin model of tree shrew(Tupaia belangeri) |
| 学位专业 | 生物工程 |
| 中文摘要 | 近年来，小型哺乳动物树鼩受到越来越多的关注，因为和啮齿类相比，其与灵长类的亲缘关系更近。而且，树鼩动物模型在肝炎病毒感染、近视、抑郁症等疾病模型中有着独特的优势。用非人灵长类动物模型能够更好的模拟人的疾病状态，但非人灵长类动物资源非常匮乏，同时还有伦理以及昂贵的费用等问题。因此开发与灵长类亲缘关系近的小型动物模型是很有前景的。基于以上研究背景和实验室的研究方向，我们探索树鼩模型在血栓和细菌感染中的潜在优势。 血栓的形成是导致最常见疾病，如心肌梗死、中风发生的重要原因。血小板主要的功能是参与生理止血和病理的血栓形成。凝血酶是凝血系统中主要的蛋白酶，其介导的血小板聚集是通过活化蛋白酶激活受体（PARs）来完成的。有意思的是，人和小鼠血小板上表达的PARs并不相同：人表达PAR 1和4；小鼠表达PAR 3和4。作用于血小板PAR 1的治疗血栓的药物被认为出血风险小，治疗效果好。而基于人与常用的实验动物啮齿类血小板上PARs的表达差异，啮齿类动物不能用来评价作用于血小板PAR 1的药物。因此我们来探索树鼩模型在血栓疾病模型中是否具有独特的优势。 通过聚合酶链反应（PCR）、Western blot和流式细胞分析等技术，发现树鼩血小板上特异性地表达PAR1 和PAR4，与人的血小板上的表达模式相同。体外血小板实验表明，人血小板PAR1专一性抑制药物Vorapaxar能够抑制约40%树鼩血小板的聚集。建立的三氯化铁诱导的树鼩颈总动脉血栓模型表明，Vorapaxar在5mg/kg时能够延长三氯化铁引起的血流阻断时间约8分钟，但结果没有显著差异。本实验中初步建立的树鼩血栓模型还需要进一步优化。树鼩的血栓模型值得进一步深入研究。 内毒素（lipopolysaccharide，LPS）是革兰氏阴性菌的细胞壁成份，是其主要的致病因子。内毒素诱导的人与啮齿类动物基因表达差异很大。这表明人与啮齿类动物在免疫系统中存在大的差别。这解释了通过啮齿类动物模型筛选的针对免疫疾病类的药物效率很低的原因。因此我们建立树鼩免疫细胞内毒素模型，探讨树鼩模型在细菌感染疾病方面是否比啮齿类动物更优。 本实验中采用PCR和qPCR初步检测了树鼩免疫细胞（腹腔巨噬细胞和血液里的免疫细胞）对细菌内毒素LPS的免疫反应，发现树鼩免疫细胞诱导性的一氧化氮合成酶（iNOS）的表达与人相似而与小鼠不同。这提示树鼩的免疫系统可能与人更相似。后续需要系统性的研究内毒素诱导树鼩的基因表达，并与人和小鼠比较。 |
| 英文摘要 | Recently, the small animal tree shrew is becoming increasingly popular, in part because of its close evolutionary relationship with primates; in part because of its unique advantages in the study of hepatitis viruses infection, myopia, depression and so on. Although non-human primates’ models can best mimic the conditions of human pathology, they are terrible models when come to economic and ethical reasons. Thus, small animal models with close evolutionary relationship to primates are of interests. Based on the backgrounds of the tree shrew and the research interests in my laboratory, we evaluated the potential advantages of tree shrew models in thrombosis and bacterial infections. Using a combination of Polymerase Chain Reaction (PCR), Western blot and flow cytometer assays, we found that the tree shrew’s platelets (Tupaia belangeri) express protease activated receptor (PAR) 1 and 4. This result reveals that the expression of PARs on the tree shrew’s platelets is similar to that of the human counterparts, but different from that of mouse platelets with PAR3 and 4. Next, we established in vitro and in vivo ferric chloride induced thrombosis models to evaluate the effects of Vorapaxar, a human platelets’ PAR1 competitive inhibitor drug in its Phase III clinical trials, on the tree shrew’s platelets. In vitro study showed that Vorapaxar has a maximum inhibitory rate with 40% on tree shrew’s platelets aggregated by bovine thrombin. In vivo results revealed that a dose of 5mg/kg can prolong the occlussion time for about 8 mins (a result with no significant difference in statistics). Our models need to be optimized, and the tree shrew thrombosis models call for further investigations. The endotoxin response between human and mice, the most commonly used laboratory animal, differs dramatically. In our endotoxin response model of tree shrew immune cells, the expression of inducible nitric oxide synthase is significantly different. The endotoxin response of the tree shrew’ immune cells was similar to the condition of human, indicating a possibly suitable model of the tree shrew in bacterial infections. |
| 语种 | 中文 |
| 公开日期 | 2014-06-04 |
| 内容类型 | 学位论文 |
| 源URL | [http://159.226.149.42:8088/handle/152453/7881]  |
| 专题 | 昆明动物研究所_动物活性蛋白多肽组学 |
| 推荐引用方式 GB/T 7714 | 刘杰. 树鼩 (Tupaia belangeri）血栓模型与免疫细胞内毒素模型[D]. 北京. 中国科学院研究生院. 2014. |
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