Vai ai contenuti. | Spostati sulla navigazione | Spostati sulla ricerca | Vai al menu | Contatti | Accessibilità

| Crea un account

Grisafi, Davide (2008) Possibile impiego delle cellule staminali del fluido amniotico per la riparazione del danno polmonare in un modello animale per broncodiplasia: valutazione in vitro e in vivo. [Tesi di dottorato]

Full text disponibile come:

[img]
Anteprima
Documento PDF
7Mb

Abstract (inglese)

Introduction.
Since our knowledge on the replication and differentiating capability of the respiratory tract is still limited, the therapeutic potential of this system is quite unexplored. The respiratory disease in pediatric age concerns several pathologies among which broncopulmonary dysplasia (BPD) in neonate and premature newborns, asthma and cistyc fibrosys are the most studied because of their diffusion among children and their infaust prognosis.. In the last few years some studies have shown the possibility of deriving progenitors with various potential from the amniotic fluid. Amniocentesis is a widely accepted method for prenatal diagnosis; it is associated with low risk both for the mother and the fetus and overcomes the ethical problems commonly associated to other sources. Recently, it has been described that amniotic fluid stem (hAFS) cells, for their ability to differentiate to various lineages, could represent a good candidate for therapeutic applications. The recent characterization of hAFS and the consolidation of the techniques for intratracheal transplantation have shown new perspectives for gene and cell therapy applications. In particular, for these purposes hAFS cells should be genetically modified with a therapeutic gene and delivered systematically or injected directly into the tissue of interest.

Materials and methods
The in vitro phase has evaluated for the first time the possibility to infect hAFS with first generation E1-deleted adenoviral vectors, and the mantainence of the stemness and differentiating capability even after transduction with foreign gene sequences.
In the in vivo phase of the project we verified the pulmonary homing and the eventual engraftment of hAFS cells, after intratracheal administration, in a 60% O2 rat model presenting a respiratory disease similar to the one observed in human patients affected by broncopulmonary dysplasia (BPD) and cystic fibrosis. The symptoms were reproduced by using OXYCYCLER, with two pressurized rooms in which animals are exposed at controlled percentages of O2 and CO2. In this trial, hAFS cells have been infected AdHCMVsp1LacZ, a first generation E1 deleted viral vector transducing LacZ, the β-gal specific gene used as marker.

Results
At first, we investigated the feasibility of transducing hAFS cells with adenoviral vectors and to determine whether transduced stem cells retain the ability to differentiate into different lineages. Herein, we showed that hAFS cells could be efficiently infected by first generation adenovirus vectors. In addition, we demonstrated that infection and expression of two different marker genes, LacZ and EGFP, have no effect on cells phenotype and differentiation potential. In particular, on undifferentiated status, hAFS cells continued to express both the transgenes and stemness cell markers OCT4 and SSEA4 (stage-specific embryonic antigen 4). When cultured under mesenchymal conditions, infected cells could still differentiate into osteocytes and adipocytes expressing lineage specific genes.
Differently to what observed in embryonic stem cells, the amniotic fluid stem cells easily infect very efficiently. This could represent an excellent starting point for gene therapy studies in which a transient expression would be a necessary condition to the therapeutic approach.
In the in vivo phase we transplanted hAFS cells with an intratracheal administration in a rat model generated exposing newborns at 60% O2 for two weeks, reproducing in this way the chronic damage that can be seen in human patients affected by BPD. The results show that the model for chronic lung damage has been properly implemented; specific staining for lacZ performed three weeks post-transplant confirmed for hAFS cells a bronchiolar homing. After four weeks transplantation LacZ positive cells have been detected inside alveolis. Finally, an important phenomenon of damage repair was observed in the treated animals as compared to untreated controls.


Statistiche Download - Aggiungi a RefWorks
Tipo di EPrint:Tesi di dottorato
Relatore:Maurizio, Scarpa
Correlatore:Tomanin, Rosella - Zaramella, Patrizia
Dottorato (corsi e scuole):Ciclo 20 > Scuole per il 20simo ciclo > MEDICINA DELLO SVILUPPO E SCIENZE DELLA PROGRAMMAZIONE > GENETICA BIOCHIMICA E MOLECOLARE
Data di deposito della tesi:24 Gennaio 2008
Anno di Pubblicazione:24 Gennaio 2008
Informazioni aggiuntive:tesi con argomento terapia genica-terapia cellulare
Parole chiave (italiano / inglese):terapia genica, cellule staminali, adenovirus, gene therapy, stem cells, cellule staminali del fluido amniotico, broncodisplasia
Settori scientifico-disciplinari MIUR:Area 06 - Scienze mediche > MED/10 Malattie dell'apparato respiratorio
Struttura di riferimento:Dipartimenti > Dipartimento di Pediatria
Codice ID:324
Depositato il:24 Ott 2008
Simple Metadata
Full Metadata
EndNote Format

Bibliografia

I riferimenti della bibliografia possono essere cercati con Cerca la citazione di AIRE, copiando il titolo dell'articolo (o del libro) e la rivista (se presente) nei campi appositi di "Cerca la Citazione di AIRE".
Le url contenute in alcuni riferimenti sono raggiungibili cliccando sul link alla fine della citazione (Vai!) e tramite Google (Ricerca con Google). Il risultato dipende dalla formattazione della citazione.

1. Abman SH. Recent advances in the pathogenesis and treatment of persistent pulmonary Cerca con Google

2. hypertension of the newborn. Neonatology. 2007;91(4):283-90. Epub 2007 Jun 7. Cerca con Google

3. Albertine KH, Wang ZM, Michael JR. Expression of endothelial nitric oxide synthase, inducible nitric oxide synthase,and endothelin-1 in lungs of subjects who died with ARDS. Chest. 1999 Jul;116(1 Suppl):101S-102S. Cerca con Google

4. Albertine KH, Jones GP, Starcher BC, Bohnsack JF, et al. Chronic lung injury in preterm lambs. Disordered respiratory tract development. Am J Respir Crit Care Med. 1999 Mar;159(3):945-58. Cerca con Google

5. Allen J, Zwerdling R, Ehrenkranz R, Gaultier C, et al. Statement on the care of the child with chronic lung disease of infancy and childhood. Am J Respir Crit Care Med. 2003 Aug 1;168(3):356-96. Cerca con Google

6. Atala A. Tissue engineering and regenerative medicine: concepts for clinical application. Cerca con Google

7. Rejuvenation Res. 2004 Spring;7(1):15-31. Cerca con Google

8. Bancalari E, Claure N, Sosenko IR. Bronchopulmonary dysplasia: changes in pathogenesis, epidemiology and definition. Semin Neonatol. 2003 Feb;8(1):63-71. Review. Cerca con Google

9. Belik J, Pan J, Jankov RP, Tanswell AK. Bronchial epithelium-associated pulmonary arterial muscle relaxation in the rat is absent in the fetus and suppressed by postnatal hypoxia. Am J Physiol Lung Cell Mol Physiol. 2005 Feb;288(2):L384-9. Cerca con Google

10. Bland RD, Albertine KH, Carlton DP, Kullama L, et al. Chronic lung injury in preterm lambs: abnormalities of the pulmonary circulation and lung fluid balance. Pediatr Res. 2000 Jul;48(1):64-74. Cerca con Google

11. Blanco LN, Frank L. The formation of alveoli in rat lung during the third and fourth postnatal weeks:effect of hyperoxia, dexamethasone, and deferoxamine. Pediatr Res. 1993 Sep;34(3):334-40. Cerca con Google

12. Bokodi G, Treszl A, Kovács L, Tulassay T, et al. Dysplasia: a review. Pediatr Pulmonol. 2007 Oct;42(10):952-61. Cerca con Google

13. Boström H, Willetts K, Pekny M, Levéen P, et al. PDGF-A signaling is a critical event in lung alveolar myofibroblast development and alveogenesis. Cell. 1996 Jun 14;85(6):863-73. Cerca con Google

14. Coalson JJ, Kuehl TJ, Prihoda TJ, deLemos RA. Diffuse alveolar damage in the evolution of bronchopulmonary dysplasia in the baboon. Pediatr Res. 1988 Sep;24(3):357-66. Cerca con Google

15. Coalson JJ, Winter V, Yang F. Site specificity of surfactant protein expression in airways of baboons during gestation. Anat Rec. 1998 Mar;250(3):300-15. Cerca con Google

16. Chen Z, Chintagari NR, Guo Y, Bhaskaran M, et al. Gene expression of rat alveolar type II cells during hyperoxia exposure and early recovery. Free Radic Biol Med. 2007 Aug 15;43(4):628-42. Cerca con Google

17. De Coppi P, Bartsch G Jr, Siddiqui MM, Xu T, et al. Isolation of amniotic stem cell lines with potential for therapy. Nat Biotechnol. 2007 Jan;25(1):100-6. Epub 2007 Jan 7. Cerca con Google

18. Fairweather DV, Lucas M. Four years' experience of antenatal chromosome diagnosis. Eur J Obstet Gynecol Reprod Biol. 1978 Aug;8(4):205-9. Cerca con Google

19. Frank L. Oxygen toxicity in neonatal rats: the effect of endotoxin treatment on survival during and post-O2 exposure. Pediatr Res. 1987 Feb;21(2):109-15. Cerca con Google

20. Goto H, Senba T, Sato M, Minami T. Intratracheal administration of bleomycin via a catheter in unanesthetized rats. Exp Anim. 2004 Apr;53(2):113-9. Cerca con Google

21. Grover TR, Zenge JP, Parker TA, Abman SH. Vascular endothelial growth factor causes pulmonary vasodilation through activation of the phosphatidylinositol-3-kinase-nitric oxide pathway in the late-gestation ovine fetus. Pediatr Res. 2002 Dec;52(6):907-12. Cerca con Google

22. Han RN, Han VK, Buch S, Freeman BA, Post M, Tanswell AK. Insulin-like growth factor-I and type I insulin-like growth factor receptor in 85% O2-exposed rat lung. Am J Physiol. 1996 Jul;271(1 Pt 1):L139-49. Cerca con Google

23. Karjalainen MJ, Neuvonen PJ, Backman JT. Tolfenamic acid is a potent CYP1A2 inhibitor in vitro but does not interact in vivo: correction for protein binding is needed for data interpretation. Eur J Clin Pharmacol. 2007 Sep;63(9):829-36. Epub 2007 Jul 6. Cerca con Google

24. Kawabata K, Sakurai F, Koizumi N, Hayakawa T, Mizuguchi H. Adenovirus vector-mediated gene transfer into stem cells. Mol Pharm. 2006 Mar-Apr;3(2):95-103. Cerca con Google

25. Kazzi SN, Kim UO, Quasney MW, Buhimschi I. Polymorphism of tumor necrosis factor-alpha and risk and severity of bronchopulmonary dysplasia among very low birth weight infants. Pediatrics. 2004 Aug;114(2):e243-8. Cerca con Google

26. Kinsella JP, Greenough A, Abman SH. Bronchopulmonary dysplasia. Lancet. 2006 Apr 29;367(9520):1421-31. Cerca con Google

27. Jankov RP, Johnstone L, Luo X, Robinson BH, et al. Macrophages as a major source of oxygen radicals in the hyperoxic newborn rat lung. Free Radic Biol Med. 2003 Jul 15;35(2):200-9. Cerca con Google

28. Jiang Y, Vaessen B, Lenvik T, Blackstad M, et al. Multipotent progenitor cells can be isolated from postnatal murine bone marrow, muscle, and brain. Exp Hematol. 2002 Aug;30(8):896-904. Cerca con Google

29. Jobe AH. Glucocorticoids, inflammation and the perinatal lung. Semin Neonatol. 2001 Aug;6(4):331-42. Review. Cerca con Google

30. Lemons JA, Bauer CR, Oh W, Korones SB, et al. Very low birth weight outcomes of the National Institute of Child health and human development neonatal research network, January 1995 through December 1996. Pediatrics. 2001 Jan;107(1):E1. Cerca con Google

31. Levin DL, Heymann MA, Kitterman JA, Gregory GA, et al.. Persistent pulmonary hypertension of the newborn infant. J Pediatr. 1976 Oct;89(4):626-30. Cerca con Google

32. Liu B, Harvey CS, McGowan SE. Retinoic acid increases elastin in neonatal rat lung fibroblast cultures. Am J Physiol. 1993 Nov;265(5 Pt 1):L430-7. Cerca con Google

33. Northway WH Jr, Rosan RC, Porter DY. Pulmonary disease following respirator therapy of hyaline-membrane disease. Bronchopulmonary dysplasia. N Engl J Med. 1967 Feb 16;276(7):357-68. Cerca con Google

34. O'Brodovich HM, Mellins RB. Bronchopulmonary dysplasia. Unresolved neonatal acute lung injury. Am Rev Respir Dis. 1985 Sep;132(3):694-709. Review. Cerca con Google

35. Ortiz LA, Gambelli F, McBride C, Gaupp D, et al. Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects. Proc Natl Acad Sci U S A. 2003 Jul 8;100(14):8407-11. Epub 2003 Jun 18. Cerca con Google

36. Pantalitschka T, Poets CF Inhaled drugs for the prevention and treatment of bronchopulmonary dysplasia. Pediatr Pulmonol. 2006 Aug;41(8):703-8. Cerca con Google

37. Pelkonen AS, Hakulinen AL, Hallman M, Turpeinen M. Effect of inhaled budesonide therapy on lung function in schoolchildren born preterm. Respir Med. 2001 Jul;95(7):565-70. Cerca con Google

38. Pierce RA, Albertine KH, Starcher BC, Bohnsack JF, et al. Chronic lung injury in preterm lambs: disordered pulmonary elastin deposition. Am J Physiol. 1997 Mar;272(3 Pt 1):L452-60. Cerca con Google

39. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999 Apr 2;284(5411):143-7. Cerca con Google

40. Plötz FB, Slutsky AS, van Vught AJ, Heijnen CJ. Ventilator-induced lung injury and multiple system organ failure: a critical review of facts and hypotheses. Intensive Care Med. 2004 Oct;30(10):1865-72. Cerca con Google

41. Riordan JR, Rommens JM, Kerem B, Alon N, et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science. 1989 Sep 8;245(4922):1066-73. Cerca con Google

42. Rova M, Haataja R, Marttila R, Ollikainen V, et al. Data mining and multiparameter analysis of lung surfactant protein genes in bronchopulmonary dysplasia. Hum Mol Genet. 2004 Jun 1;13(11):1095-104. Epub 2004 Apr 21. Cerca con Google

43. Shaffer SG, O'Neill D, Bradt SK, Thibeault DW. Chronic vascular pulmonary dysplasia associated with neonatal hyperoxia exposure in the rat. Pediatr Res. 1987 Jan;21(1):14-20. Cerca con Google

44. Shulenin S, Nogee LM, Annilo T, Wert SE, Whitsett JA, Dean M. ABCA3 gene mutations in newborns with fatal surfactant deficiency. N Engl J Med. 2004 Mar 25;350(13):1296-303. Cerca con Google

45. Stenmark KR, Balasubramaniam V. Angiogenic therapy for bronchopulmonary dysplasia: rationale and promise. Circulation. 2005 Oct 18;112(16):2383-5. Cerca con Google

46. Speer CP. New insights into the pathogenesis of pulmonary inflammation in preterm infants. Cerca con Google

47. Biol Neonate. 2001;79(3-4):205-9. Cerca con Google

48. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, et al. Embryonic stem cell lines derived from human blastocysts. Science. 1998 Nov 6;282(5391):1145-7. Cerca con Google

49. Warner BB, Stuart LA, Papes RA, Wispé JR. Functional and pathological effects of prolonged hyperoxia in neonatal mice. Am J Physiol. 1998 Jul;275(1 Pt 1):L110-7. Cerca con Google

Download statistics

Solo per lo Staff dell Archivio: Modifica questo record