大蹼铃蟾（Bombina maxima）皮肤分泌物非晶状体-晶状体蛋白与三叶因子复合物细胞生物学活性和分子作用机制
文献类型：学位论文
| 作者 | 何英英 |
| 学位类别 | 博士 |
| 答辩日期 | 2007-07 |
| 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 | 非晶状体betagamma-晶状体蛋白 三叶因子蛋白 核定位 细胞凋亡 伤口修复 转录调节 |
| 其他题名 | Cellular Functions and MolecularMechanisms of Non-lens -crystallin and Trefoil Factor Complex from Bombina maxima skin secretion |
| 学位专业 | 动物学 |
| 中文摘要 | 非晶状体betagamma-晶状体蛋白质(non-lens betagamma-crystallins)在脊椎动物中以一簇的基因家族的形式存在，在各种上皮细胞中广泛表达，但对其在体内承担的功能，人们几乎一无所知。三叶因子蛋白(trefoil factors, TFFs)主要分布在胃肠道上皮和两栖动物皮肤表面，许多研究表明该家族的蛋白质，在粘膜保护，损伤修复和肿瘤抑制中具有重要的作用；但是自发现以来，TFFs一直作为孤儿配基存在，对于其作用的机制了解得很少。人们从来没有把两个家族的蛋白质联系在一起来考虑过。 本实验室从中国特有的两栖动物大蹼铃蟾(Bombina maxima)皮肤分泌物中，分离到这两个家族的蛋白质的天然结合在一起的复合物，并命名为：非晶状体betagamma-晶状体蛋白和三叶因子蛋白复合物（non-lens betagamma-crystallin and treifol factor complex,betagamma-CAT）。尽管在低剂量下，betagamma-CAT已对小鼠，大鼠和兔有致死活性，但其结构与人源的non-lens betagamma-crystallins和TFFs的同源性，提示了它可能在正常的生理功能中起到重要作用，也为揭示两类重要的蛋白质的功能和作用机制提供了可能性。本研究工作，在多种细胞株中，对betagamma-CAT的生物学活性作了详细的研究，并对其作用机制进行了进一步深入的探讨。 我们原代培养了人脐静脉血管内皮细胞(HUVEC)，兔主动脉内皮细胞(RAEC)，兔心内皮细胞(REEC)；培养了多种肿瘤细胞株。betagamma-CAT能够引起多种贴壁细胞的脱落。betagamma-CAT在高剂量下能够引起这些细胞的凋亡；但是在不同的细胞株中，发生凋亡的通路可能是不同的。在低剂量下，betagamma-CAT能够促进细胞的迁移，对HUVEC具有诱导伤口修复的活性。 以HUVEC细胞为模型，我们探讨了betagamma-CAT的作用机制。在激光共聚焦显微镜下，观察到betagamma-CAT诱导HUVEC发生囊泡化，这个效应是剂量依赖的；囊泡化的发生不依赖于NH4Cl的存在，但是NH4Cl能够增大囊泡化的效应。在荧光染料Cy3直接标记betagamma-CAT时，观察到betagamma-CAT被定向运输到细胞核上。荧光染料FITC分别标记betagamma-CAT的轻链和重链的多克隆抗体，免疫荧光染色发现，在较短的时间(5 min)内, betagamma-CAT已经进入细胞，并有部分分子被运输到细胞核上。随着时间增加到30 min，轻链和重链在核上的比例也渐增加；到2小时，bg-CAT的重链全部集中在细胞核上，而betagamma-CAT的轻链则退出核外，主要聚集在细胞核周围。核定位显示betagamma-CAT分子进入HUVEC细胞核，可能在基因的转录调节中发挥作用。我们运用基因芯片检测了加药处理前后细胞蛋白质表达水平的变化。在四组重复实验中均显示，加入betagamma-CAT处理后，有121个基因发生上调，这些基因在调节细胞的生存和死亡中具有复杂的功能。其中核受体蛋白质家族（NR4A1等）的变化最为明显。而其他的基因包括调节细胞早期生长，凋亡，炎症反应的相关基因和金属蛋白酶。同时，加入betagamma-CAT处理后，仅有2个基因发生下调，包括胶原I，这为解释细胞发生脱落和调亡提供了分子基础。 本研究工作揭示了非晶状体betagamma-晶状体蛋白和三叶因子蛋白的相互作用，共同定位到细胞核上，并调节基因的转录，首次提示非晶状体betagamma-晶状体蛋白可能参与一条全新的细胞信号调节途径，在组织平衡，肿瘤发生和胚胎发育中起到重要的作用；同时也为三叶因子蛋白的分子作用机制的解析提供了一种新的可能性。 |
| 英文摘要 | Non-lens betagamma-crystallin was found in the vertebrates as a gene family. It was expressed in the epidermis and distributed in other tissues, such as heart, lung, stomach and so on. But people nearly know nothing about its functions. Treifol factor family protein was mainly distributed in the epithelial cells of intestine and gastric tract in mammals and it also identified from the skin of the amphibians. Many research works on the trefoil factor protein illustrated its importance in mucusal protection, wound healing and tumorgenesis. As it was found in 1982, trefoil factors had been an orphan ligand and many people devoted themselves to finding its receptors. Until now we knew little about its function mechanisms according to play its important roles. In the past twenty years, nobody had related the proteins belonging to the two different families together. The Chinese red belly frog (Bombina maxima) is an endemic amphibian in the mountainous regions of southwestern China. A protein composed of two subunits, which is responsible for its lethal toxicity on mice, rat and rabbit, was identified and purified. Surprisingly, sequence determination established that its alpha-subunit is a non-lens betagamma-crystallin member, while its beta-subunit is a three-domain trefoil factor. The protein was thus named betagamma-crystallin and trefoil factor complex, abbreviated betagamma-CAT. The high homologue in structure suggest that it may play very important role in the normal life of the frog, and it gives us a chance to recover the mechanism of the crystallin and treifol factor. In our research work, we try to find out the cellular functions and molecular mechanisms on various cells, including the primary cultured HUVEC, RAEC, REEC and several tumor cell lines in intestine and stomach and breast cancer. Induction of cell detachment and cell death was observed after treatment with various dosages of betagamma-CAT. And in different cell lines there may be different pathways and machenisms. Under low dosages (pM), betagamma-CAT can stimulation HUVEC to migrate on the collagen-precoated matrix and promote the activity of wound healing. Using the HUVEC as the model, we studied the machenisms of the mutiple functions of betagamma-CAT. We clearly observed betagamma-CAT induced HUVEC vacuolation with dose-depedant manners under the confocal microscopy. The process to vaculate was independent with the absence of NH4Cl, but the presence of NH4Cl will amplify the vacuolation result. Directedly labbed with cy3 fluorescent dye, betagamma-CAT was detected to traffic to cellular nuleus. The alpha-unit and beta-unit were labbed with another fluorescent dye FITC. In 5 minutes, betagamma-CAT was observed to uptake by HUVEC and partial was sent the nucleus in the 30 minutes. The alpha-subunit was concentrated on the nuleus, but the beta-unit equipped the nucleus and mainly distributed around the nucleus at the end of 2 hours. Traffic to nucleus suggests the protein may be a regulator in gene transcription. Microarray assay demonstrated a number of genes, which are consistent with the biological processes involved in tissue repair and tissue homeostasis, were regulated. Among these, 121 gene was upregulated, which encoded transcription factors, inflammation related cytokines, as well as cell growth and apoptosis regulatory proteins and metalloproteinases, among which members of orphan nuclear receptor subfamily 4 were the most prominent. Only two of these are downregulated, including collagen I, which supported for the cell detachment treated with betagamma-CAT. Our findings demonstrate how members of non-lens betagamma-crystallins and trefoil factors are likely to function in the regulation of cell migration, survival and apoptosis, suggesting novel initial regulators and a new pathway involved in tissue repair, tissue homeostasis and in development. |
| 语种 | 中文 |
| 公开日期 | 2010-10-14 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6125]  |
| 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 | 何英英. 大蹼铃蟾（Bombina maxima）皮肤分泌物非晶状体-晶状体蛋白与三叶因子复合物细胞生物学活性和分子作用机制[D]. 北京. 中国科学院研究生院. 2007. |
入库方式： OAI收割
来源：昆明动物研究所
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