少棘蜈蚣Kv1.3离子通道抑制剂的结构与功能及其对转基因小鼠银屑病模型的作用
文献类型：学位论文
| 作者 | 康迪 |
| 学位类别 | 硕士 |
| 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 | 赖仞 |
| 关键词 | 少棘蜈蚣 毒素 银屑病 Kv1.3 |
| 其他题名 | Structure-function of a Kv1.3 inhibitor from centipede （Scolopendra subspinipes mutilans ）venom and its effect in a transgenic mouse model of psoriasis |
| 学位专业 | 生物化学与分子生物化学 |
| 中文摘要 | 银屑病是一种由环境和遗传因素长期相互作用而引发，以表皮角朊细胞增生、炎症细胞浸润为主要特征的慢性自身免疫性疾病。很多研究都证实其发病机制和Kv1.3过度表达或激活相关。特异性的Kv1.3抑制剂有可能成为治疗银屑病的一个新途径。 本论文从少棘蜈蚣毒液中分离纯化得到一种特异性的Kv1.3离子通道的抑制剂，对其结构和功能进行了研究并检测了它对银屑病动物模型K14-VEGF转基因小鼠的治疗作用。 收集的蜈蚣毒液通过凝胶过滤层析、反向高压液相色谱从蜈蚣毒液中分离得到一种对Kv1.3通道有选择性抑制作用的蜈蚣毒素，命名为κ-SLPTX-Ssm1a。利用Edman降解法测定κ-SLPTX-Ssm1a的成熟肽序列。它由51个氨基酸残基组成，MALDI-TOF鉴定其分子量为6050.2 Da。并解析了其二硫键配对模式（1-5、2-4、3-6）和液相三维结构。κ-SLPTX-Ssm1a的二硫键配对模式与已知的ICK (1-4、2-5、3-6)，DDH（1-3、2-5、4-6）和Kunitz家族（1-6、2-4、3-5）不同。 κ-SLPTX-Ssm1a抑制Kv1.3通道电流IC50为61.5±8.3 nM。κ-SLPTX-Ssm1a抑制Kv4.1通道电流IC50为3.16±0.5 μM，其IC50是Kv1.3的60倍。κ-SLPTX-Ssm1a可抑制人T细胞Kv1.3 电流，IC50为39.2 ± 14.1 nM。κ-SLPTX-Ssm1a通过抑制T细胞上的Kv1.3，从而抑制T细胞的增殖和相关细胞因子，如TNF-α, IL-8, IL-2, IL-17, IFN-γ和 IL-22的分泌水平，但不影响T细胞上Kv1.3的表达量。 κ-SLPTX-Ssm1a对K14-VEGF转基因小鼠的治疗作用结果表明κ-SLPTX-Ssm1a对K14-VEGF小鼠的耳朵变红、棘层加厚、角化过度和伴角化不全等银屑病症状均有改善, 且炎性细胞的浸润和血管增殖现象减轻。经κ-SLPTX-Ssm1a处理后的K14-VEGF小鼠血液中TNF-α, IFN-γ, IL-2, IL-12, IL-22和IL-23的水平也逐渐降至正常水平。 综上所述我们从少棘蜈蚣中分离出一个 Kv1.3离子通道的多肽抑制剂κ-SLPTX-Ssm1a选择性抑制Kv1.3通道，并可以。它对银屑病动物模型K14-VEGF转基因小鼠有明显的治疗作用。这些工作为离子通道和神经生物学研究提供了新的工具分子，也为银屑病的治疗提供了新的理论依据。 |
| 英文摘要 | Psoriasis is a common inflammatory autoimmune condition in which environment factors and genetic predisposition contribute to the development of disease. Over-expression or over-activation of Kv1.3 channels have been proved to be involved in the mechanism of psoriasis and specifically inhibitors of Kv1.3 may be new approaches for the treatment of psoriasis. A novel neurotoxin-like peptide inhibitor of Kv1.3, κ-SLPTX-Ssm1a, was purified from Scolopendra subspinipes mutilans venoms by a two-step purification protocol including one step of gel filtration and one step of RP-HPLC. We studied the structure-function of κ-SLPTX-Ssm1a and the therapeutical effect of it on psoriasis in K14-VEGF mice was tested as well. Edman degradation method was used for the determination of its amino acid sequence. κ-SLPTX-Ssm1a was composed of 51 amino acids with a molecular weight of 6050.2 Da. Furthermore, we analyzed its NMR structure and disulfide bond model (1-5, 2-4, 3-6) which was different from the known ICK (1-4, 2-5, 3-6), DDH (1-3, 2-5, 4-6) and Kunitz family (1-6, 2-4, 3-5). κ-SLPTX-Ssm1a was a inhibitor of Kv1.3 and Kv4.1 and the IC50 values were 61.5±8.3 nM and 3.16±0.5 μM（n=5）respectively. κ-SLPTX-Ssm1a inhibited Kv1.3 on T cells with a IC50 value of 39.2±14.1 nM（n=5）. As a blockade of Kv1.3, κ-SLPTX-Ssm1a prevented the over activation/proliferation and cytokine secretion of T cells, such as NF-α, IL-8, IL-2, IL-17, IFN-γ and IL-22, but not the express of Kv1.3 itself. Therapeutic effects of κ-SLPTX-Ssm1a in K14-VEGF transgenic mice showed that psoriatic symptoms of K14-VEGF mice, as red ears, spinout layer thickening, hyperkeratosis and diversification were improved. At the same time, the infiltration of inflammatory cells and vascular proliferation were reduced. The levels of TNF-α, IFN-γ, IL-2, IL-12, IL-22 and IL-23 in K14-VEGF mice blood after treatment with κ-SLPTX-Ssm1a were gradually decreased to normal. In summary, κ-SLPTX-Ssm1a, a novel inhibitor of Kv channel purified from Scolopendra subspinipes mutilans venoms, had an obvious therapeutic benefit on the K14-VEGF mice. This work provided not only new tools for the studies of ion-channels and neurobiology but also new theoretical basis for the treatment of psoriasis. |
| 语种 | 中文 |
| 公开日期 | 2014-06-18 |
| 源URL | [http://159.226.149.42:8088/handle/152453/7903]  |
| 专题 | 昆明动物研究所_动物毒素室 |
| 推荐引用方式 GB/T 7714 | 康迪. 少棘蜈蚣Kv1.3离子通道抑制剂的结构与功能及其对转基因小鼠银屑病模型的作用[D]. 北京. 中国科学院研究生院. 2014. |
入库方式： OAI收割
来源：昆明动物研究所
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