Seminario molecular[1] listo

447 views
381 views

Published on

Published in: Education, Technology
0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total views
447
On SlideShare
0
From Embeds
0
Number of Embeds
1
Actions
Shares
0
Downloads
3
Comments
0
Likes
0
Embeds 0
No embeds

No notes for slide

Seminario molecular[1] listo

  1. 1. THE MOLECULAR MECHANISM OF LEPTIN SECRETION AND EXPRESSION INDUCES BY ARISTOLOCHIC ACID IN KIDNEY FIBROBLASTChung-shi yang; et al.<br />BY: ALEJANDRA ALVAREZ<br />SILVANA ZAPATA <br />Estudiantes medicina UPB<br />III sermestre<br />
  2. 2. OBJETIVE<br />The aim of this study is to explore the effect of AA on leptin production and to dissect the AA-induced downstream signaling in vitro.<br />
  3. 3. INTRODUCTION<br />Leptin is a 16 kDa peptide hormone of cytokine family mainly secreted by adipocyte into blood stream and functions as a central mediator that negatively regulates satiety in hypothalamus1.<br />1: Zhang Y.; et al. 1994.<br />
  4. 4. INTRODUCTION<br />Aristolochic acid (AA) is a famous botanical toxin which has been characterized to associate with the development of aristolochic acid nephropathy (AAN)2<br />2: Vanherweghem JL, et al. (1993) <br />
  5. 5. INTRODUCTION<br />A fibroblast is a type of cell that synthesizes the extracellular matrix and collagen,the structural framework (stroma) for animal tissues, and plays a critical role in wound healing. Fibroblasts are the most common cells of connective tissue in animals3, 4.<br />3: Lan HY.;(2003) .<br />4: Roberts IS.; (1997).<br />
  6. 6. INTRODUCTION<br />Leptin has been considered to play an important role in progressive renal fibrosis. Renal fibroblast, in particular under the progression of fibrosis induced by AA.<br />Findings imply the fibroblast-produced leptin maybe one of the factor which promotes the progression of renal fibrosis induced by AA5.<br />5: Merabet E, et al. (1997)<br />
  7. 7. MATERIALES Y METODOS <br />Cultivo celular : <br />Se obtuvieron fibroblastos NRK-49f a través de una colección de cultivo tipo americano .<br />
  8. 8. 2. La viabilidad celular : <br />Fue evaluada mediante la reducción dependiente de mitocondrias a purpura formazan y la incorporación de BrdU respectivamente .<br />
  9. 9. 3. Medición de la secreción de Leptina:<br />Fue determinada mediante el método de ELISA .<br />
  10. 10. 4. Western blot:<br />Fue utilizado para medir la expresión de la leptina inducida por AA.<br />
  11. 11. 5. RT-PCR y PCR en tiempo real:<br /> La RT-PCR de RNAm a cDNA <br />PCR en tiempo real cuantificar concentración de RNAm.<br />
  12. 12. 6. Transfección: <br />Introducción de siRNA para bloquear la expresión del gen de la leptina.<br />
  13. 13. 7. Inmuno precipitación:<br /><ul><li>Anticuerpo anti-Akt</li></li></ul><li>RESULTADO 2.B<br />(B) Leptinexpression was examined by Western Blot. NRK-49f kidney fibroblasts were treated without or with 12 mM AA for 24 and 48 h.<br />
  14. 14. RESULTADO 2C<br />(C) Expression of leptinmRNA. Fibroblasts were treated with 0, 6 and 12 mM AA for 12 and 24 h, the level of leptin mRNA was determined by RT-PCR<br />
  15. 15. RESULTADO 3A<br />(A) Enhancement of C/EBP a DNA binding activity by AA. Fibroblasts were treated with 0, 6 and 12 mM AA for 3 h, nuclear extracts were isolated and C/EBP a DNA binding activity was analyzed by DAPA.<br />
  16. 16. RESULTADO 3B<br />(B) Knockdown of C/EBP a expression by siRNA. NRK-49f cells were transfected with 0, 90 and 180 nM of C/EBP a siRNA or control scramble siRNA for 8 h, and then cells were treated with 12 mM AA for another 48 h, the expression level of C/EBP a was examined by immunoblotting (upper panel) and RTPCR (lowerpanel).<br />
  17. 17. RESULTADO 4A<br />(A) Examination of Akt activation. NRK-49f cells were incubated with 0 and 12 mM AA for 1, 3 and 6 h. The phosphorylated activation form of Akt was detected by immunoblotting<br />
  18. 18. RESULTADO 4B<br />(B) The levels of phosphorylated PDK1 and PI3K-p85 were evaluated by Western Blot at indicated periods of AA treatment.<br />
  19. 19. RESULTADO 4C <br />(C) The interaction of phosph-PDK1 with Akt. After 1 h of 0 and 12 mM AA treatments, cells were immunoprecipitated with anti-Akt antibody. The immunoprecipitated pellets were further immnoblottedwith anti-phosphoPDK1 or anti-Akt antibody. The input and mouse IgG were served as positive and negative control, respectively.<br />
  20. 20. RESULTADO 5A<br />(A) Knockdown of Akt by siRNA. Cells were transfected with 0, 60 and 120 nM of AktsiRNA or control scramble siRNA for 8 h, and then cells were treated with AA for another 48 h, the expression level of Akt was examined by immunoblotting (upper panel) and RT-PCR (lower panel).<br />
  21. 21. RESULTADO 5C<br />(C) Analysisof Aktactivation. Cells were pretreated with 10 mM LY294002 (PI3K inhibitor) or 50 nMrapamycin (mTOR inhibitor) 1 h prior 12 mM AA addition. After 3 h incubation, level of Akt phosphorylation was determined by immunoblotting.<br />
  22. 22. RESULTADO 5E<br />(E) immunoblotting, respectively.<br />
  23. 23. RESULTADO 5F<br />(F) Expression of leptin mRNA. After the 1 h pretreatment of indicated inhibitors, cells were then treated with AA for another 24 h. RNA was isolated, and the level of leptin mRNA was assessed by RT-PCR.<br />
  24. 24. RESULTADO 6A<br />(A) DAPA<br />
  25. 25. RESULTADO 6B<br />(B) ChIP.<br />
  26. 26. DISCUSION<br />
  27. 27. Mapa silvana zapata <br />
  28. 28. Mapa Alejandra Alvarez<br />
  29. 29. CONCLUSION<br />Leptin is an important hormone in obesity, kidney disease and has implications in different organs of the body.<br />The Molecular Biology is very important in the investigation and diagnosis of different diseases.<br />Leptin is part of the regulation of AA and this is involved in renal fibrosis.<br />Genetic research to deliver various changes to find the solution to many diseases.<br />

×