| 其他摘要 | 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. |
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