| Epidermal stem cells are a kind of tissue specificity stem cells which are located in basal layer of epidermis. They are important to epidermal renewal, and they are also the main source for generation, reparation and rebuilding of skin and its appendant organ. The differentiation and proliferation of epidermal stem cells is determined not only by the genomes inside themselves, but also by the environment around the stem cells, this environment is also called"Niche". Wnt family is one of the most important parts of"Niche". They take part in the process of cell differentiation, polarity, migration and proliferation. Wnt-1 displays a very important role in development and differentiation of epithelium tissue, such as neuroepithelial development and breast cancer generation. Wnt-1 expression concords to development of skin and its appendant organ in phase: Wnt-1 expressed actively in embryo period, but rested in adult; human epidermal stem cells assembled in primary epidermal ridges in 16th week, and formed matured glandular organs in 24th week, but, no any glandular organ reproductions were observed in adult. These demonstrated that, development of skin and its appendant organ relates to Wnt-1. So, we here studied the influence of Wnt-1 to the differentiation and proliferation of epidermal stem cells.In this topic, based on the thought of"function is determined by structure", we first analyzed the amino acids sequence, secondary structure and physico-chemical property of Wnt-1 to get an allround recognize of this protein. And then, using recombinant adenovirus gene introduction technology, we expressed Wnt-1 in epidermal stem cells. This is because Wnt-1 is a kind of unstable protein and is very easy to be degradated. When Wnt-1 were added in the culture medium of epidermal stem cells, it is difficult to maintain the concentrationof Wnt-1, and to result in causing inter class and intra class errors. At the last, the proliferation, marker molecular, and matrix metalloproteinase-2 and 7, [Ca2+]i were detected to analysis the influence of Wnt-1 to differentiation and proliferation of epidermal stem cells. First Part, Bioinformatics Analysis for Wnt-1 Protein1. Objective: To get an allround recognize with bioinformatics characters of Wnt-1 protein.2. Methods: Phyletic evolution scheme of Wnt family was deawed by Phylip and Treeview softwares. Amino acid composition, isoelectric point, molecular weight, signal peptide, cleavage site and titration curve were analysis by related modules from ExPASy and Anthroprot software; Secondary structure of Wnt-1 were predicted by GOR4, HNN and SOPMA. Hydrophilicity, polarity and refractivity were analysized by related modules from ExPASy.3. Results: Wnt-16, Wnt-4 and Wnt-11 exist solitarily in their family. Wnt-1 conjugates with Wnt-6 for it has similar amino acids sequences to Wnt-6; Wnt-1 is classed to be a kind of positive charged protein and a kind of unstable protein; its theoretical isoelectric point (pI) is about 9; the signal peptide of Wnt-1is a short peptide including 27 amino acids. Regions 50-60, 112-172, 222-238, 272-360 were recognized as random coil and (or) extended structure region of Wnt-1 by GOR4, HNN and SOPMA. Regions 50-60, 112-172, 222-238, 272-360 have the values higher than threshold in hydrophilicity, polarity and refractivity analysis.4. Conclusion: Wnt-1 conjugate with Wnt-6, it is unstable in physiological condition; regions 150-160 and 335-342 accord to both spatial structure element and physico-chemical property element, have the most possibility to be the functional domain of Wnt-1 protein.Second Part, Construction of Wnt-1 Recombinant Adenovirus1. Objective: To construct Wnt-1 recombinant adenovirus particles.2. Methods: The recombinant shuttle plasmids which include Wnt-1 gene named pAdTrack-CMV/Wnt-1 were got from another researcher. These plasmids were linearized and cotransduced into Ecoli. BJ5183 with adenovirus frame plasmids to construct recombinant adenovirus plasmids pAdTrack-CMV/Wnt-1. Then, pAdTrack-CMV/Wnt-1 were linearized by PacI and transferred into QBI-293A cells to form recombinant adenovirus particles. Finally, those adenovirus particles were amplificated to a great quantity for use later.3. Results: Recombinant adenovirus plasmids pAdEasy-1/Wnt-1 could be digested by restriction enzyme PacI to two specificity straps, the posterior is far great than 15Kb and the anterior is about 4.5Kb, in coincidence with theory. 36 hours after QBI-293A cells were transduced with recombinant adenovirus plasmids pAdEasy-1/Wnt-1, green fluorescent could be observed. It means that recombinant adenovirus particles were generated correctly. Finally, we got enough recombinant adenovirus particles amplification to be used for next experiment.4. Conclusion: Wnt-1 recombinant adenovirus particles were constructed correctly.Third Part, Study on Influence of Wnt-1 to Differentiation and proliferation of Epidermal Stem Cells1. Objective: To investigate the influence of Wnt-1 to differentiation and proliferation of epidermal stem cells.2. Methods: The epidermal stem cells were got from foreskin of health child, and were infected by recombinant adenovirus particles which take along with Wnt-1 gene. Then, the expression of green fluorescent was observed; the existing of Wnt-1 mRNA were detected by RT-PCR. Finally, the quantity and diameter of those cells were measured by CASYexcell system; the expression of K5,K6,K7,K8,K10,K18,K19,CD44,CEA,ER and PR were detected by immunohistochemistry; the concentration of matrix metalloproteinase-2 and 7 in cell culture medium were measured by ELISA; [Ca2+]i was dected by confocal laser scanning microscope.3. Results: Epidermal stem cells began to proliferate quickly after cultivated for about 4 days, and reach the platform for about 9 days. After epidermal stem cells were infected by recombinant adenovirus particles who take along with Wnt-1 gene, green fluorescent could be observed. Existing of Wnt-1 mRNA could be detected by RT-PCR. CASYexcell system showed that, those infected cells proliferated more quickly, but their diameter had not changed. Keratin 10 expressing became positive, keratin 18 expression enhanced significantly, keratin 19 expression attenuated significantly. ELISA showed that MMP-2,MMP-7 expression in cell culture medium became positive. Confocal laser scanning microscope showed that [Ca2+]i raised enormously.4. Conclusion: Wnt-1 has the ability to induce human epidermal stem cells different to glandular epithelium-like cells.
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