Abstract
Diabetes mellitus is the most common disease of the metabolism in humans. Despite the insulin treatment, diabetic patients have to struggle with a lot of complications. Beta cell transplantation can restore the functional beta cell mass in diabetic patients but it is seriously hampered by donor shortage. This problem could be solved by finding ways to generate more beta cells from the available tissue. Our research group has recently demonstrated that primary acinar cells of the exocrine rat pancreas can transdifferentiate in culture into endocrine beta cells. During this process acinar cells loose their phenotypical characteristics before the endocrine markers appear. Therefore it is very hard to demonstrate transitional cells expressing phenotypic markers of the original acinar cells and of the final beta cells. In this study we aim to develop a method for genetic tracing by which we can unambiguously demonstrate the exocrine-endocrine transdifferentiation. Hereby we make use of two different tracing models.In the first tracing model we use lenti-Pelastase-EGFP. PCR-analysis showed that the elastase-promoter remains active during a long time in the transdifferentiating acinar cells. From this we hypothesize that lenti-Pelastase-EGFP is a useful tracing vector. Immediately after isolation we transduce the rat exocrine fraction with the vector whereby only the transduced, elastase-expressing cells will be EGFP positive. The cells are brought under conditions that induce the exocrine-endocrine transdifferentiation. The presence of EOFP+, insulin+ cells on day 8 would prove that the acinar cells (EGFP+) obtain a beta cell phenotype (insulin+). Before performing this transduction, we optimized first the multiplicity of infection (MOI), this is the ratio of the amount of virus to the amount of cells. We concluded that we could successfully transduce acinar cells with lentiviral vectors. We obtained a high transduction efficiency (87,9 :t 2,4%) while using a high MOI. Upon transduction of rat exocrine fraction with lenti-Pelastase-EGFP we see that EGFP
remains present till day 8 in culture. But after fixation and immunocytochemical staining for insulin, we could not detect the EGFP anymore. During visualization of EGFP with a polyclonal anti-GFP, we could see an aspecific binding of the antibody. Until now we were not able to visualize EGFP together with insulin. In the near future we will test other antibodies to GFP.
In case the previous model doesn't work, we will use a second tracing model, namely a Cre/Lox system. Hereby the rat exocrine fraction will be transduced by two constructs, Pamy-Cre-Ires-ECFP and R26-Lox-Red-Lox-Ires-Nuc-EGFP. These constructs are first cloned and then they are inserted in lenti- or adenoviral vectors. Immediately after isolation, rat exocrine fraction will be transduced by both vectors. The amylase promoter will be producing Cre and ECFP only in acinar cells, while all other transduced cells will be positive for Red and EGFP. In acinar cells Cre will remove the sequence between the Lox-sites (Red). Because of this, the acinar cells will be ECFP+, EGFP+ and Red-. Non-acinar cells (amylase-negative) won't produce Cre and ECFP. Because of this, these cells will be ECFP-, EOFP+ and Red+. During tracing the presence of EGFP+, Red-, insulin+ cells would indicate the exocrine-endocrine transdifferentiation. During cloning of the two constructs we experienced a lot of problems. We succeeded in cloning the plasmid pBS6-R26-Lox-Red2-Lox-Ires2-Nuc-EGFP-intronbetaglobin-polyAbetaglobin whose functionality has been demonstrated. Until now we did not succeed in the cloning of the plasmid Pamy-Cre-Ires-ECFP. In the near future we will finish the cloning of the constructs and insert them in lenti- and/or adenoviral vectors.
| Date of Award | 28 Jun 2005 |
|---|---|
| Original language | Dutch |
| Supervisor | Luc Bouwens (Promotor) & Harry Heimberg (Co-promotor) |
Keywords
- transdifferentiation