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| Author(s): |
Ilana Gabanyi
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ) |
| Defense date: | 2010-11-12 |
| Examining board members: |
Mari Cleide Sogayar;
Maria Rita dos Santos e Passos Bueno;
Deborah Schechtman
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| Advisor: | Mari Cleide Sogayar |
| Abstract | |
Diabetes Mellitus type 1 (DM1) is caused by an auto-imunne destruction of the pancreatic β cells, found in the endocrine portion of the pancreas, known as pancreatic islets. These β cells are responsible production and release of insulin, a hormone which promotes glucose internalization by cells. Along with other hormones, insulin is a major regulator of blood glucose levels (glycemia). One of the therapeutical strategies used to treat DM1 is pancreatic islet transplantation. One of the major problem related to this therapy is the lack of adequate cell mass to be infused into the pacients. An attempt to solve this problem is the development of an alternative source of insulin-producing cells by differentiation of stem cells, which display this differentiating potential. Pax4 is one of the transcription factors responsibles for β cell differentiation, being essential for its proper development and maturation, therefore being a good candidate to induce stem cell differentiation into insulin producing cells in vitro. A promising alternative to avoid the alterations of the differentiated cells genome due to its undesirable effects of integrating vectors, but yet allowing the Pax4 to act in diferentiation within the cells are the proteins with a transduction domain (PTDs), which would have the ability to lead the Pax4 protein directly into the cells. The Pax4 could thus act in the nucleus and generate specific transcriptional responses. The PTDs are small peptide sequences which allow translocation of proteins across cell membranes and their internalization into target cells. One of the most commonly studied PTDs is the VP22, a product of the UL49 gene from Herpes Simplex vírus type I. Therefore, the VP22.Pax4 fusion protein would transduce Pax4 into the stem cells, thus allowing the transcription activation of certain genes by Pax4, leading to improvement in the process of stem cells differentiation into insulin-producing cells. To this end, we cloned the Pax4 cDNA from RINm5f murine insulinoma cells, constructed the pVP22.Pax4 vector and transfected this construct into CHO cells, which then produced the VP22.Pax4 fusion protein. Upon verifying that VP22 was also able to transduce proteins into stem cells, by confocal microscopy analysis, after the treatment of these cells with the fusion protein VP22.eGFP, we incorporated the fusion protein VP22.Pax4 to one of the steps of the protocol used for stem cells diferentiation into insulin producing cells in our lab, by co-culturing with CHO cells producing VP22.Pax4. We observed that the addition of Pax4 led to the formation of a higher number of insulin producing cell clusters, therefore we conclude that VP22 may be used as a tool to internalize proteins into stem cells, and that the addition of Pax4 may improves protocols seeking the production of insulin-producing cell (AU) | |