Successfully reported this slideshow.
Regulation of neurogenesis and gliogenesis of retinoic acid-induced P19 embryonal
carcinoma cells by P2X2 and P2X7 recepto...
Prochain SlideShare
Chargement dans…5
×

Poster final-yuhasi et al

72 vues

Publié le

.

Publié dans : Sciences
  • Soyez le premier à commenter

  • Soyez le premier à aimer ceci

Poster final-yuhasi et al

  1. 1. Regulation of neurogenesis and gliogenesis of retinoic acid-induced P19 embryonal carcinoma cells by P2X2 and P2X7 receptors studied by RNA interference Analyzed by : Ait Belcaid H , Boudjafad Z, Freyssin A, Lguensat A, Omari Z, Rachid M The development of the nervous system requires complex series of cellular programming and intercellular communication events that lead from the early neural induction to the formation of a highly structured central and peripheral nervous system. The switch from neurogenesis to gliogenesis is the result of changes in stem cell properties that are controlled by both extrinsic and intrinsic factors that promote or inhibit neurogenesis and gliogenesis. This study investigated how down-regulation of P2X2 and P2X7 receptor expression by RNA interference (RNAi) affects neural differentiation and phenotype specification of P19 embryonal carcinoma cells. Introduction Experimental procedure 1. Culture of P19 Cells 2. Construction of lentiviral vector expressing sh RNA P19 cells Sh RNA P2X2 Sh RNA P2X7 3. RTPCR  shRNA were evaluated for their efficiency in silencing mRNA expression of P2X2 and P2X7 receptor 4. Determination of cell proliferation by flow cytometry 5. Western-blot 6. Flow cytometry quantification of neural and glial markers expression P2X2 P2X7 Β actin antiβ tubuline anti GFAP 2nd Antibody Flow cytometry quantification Brdu P19 cells Ab anti Brdu 2nd Ab (Alexa) Flow cytometry analysis P19 cells Cell Lysis 2nd Anibodies P2X2 R P2X7 R β-actin Ab anti Brdu 2nd Ab (Alexa) Protein P2X2 P2X7 Flow cytometry analysis of β-3 tubulin expression Flow cytometry analysis of GFAP expression Flow cytometry analysis of β-3 tubulin expression Flow cytometry analysis of GFAP expression Results I- Effects of silencing P2X2 receptor expression on gliogenesis and neurogenesis RT-real time PCR analysis of β-3 tubulin gene expression RT-real time PCR analysis of GFAP gene expression Silencing P2X2 induces the reduction of neurogenesis through the decrease of neural marker beta-3 tubulin and an increase of gliogenesis through the increase of the glial marker GFAP. II- Effects of silencing P2X7 receptor expression on gliogenesis and neurogenesis RT-real time PCR analysis of β-3 tubulin gene expression RT-real time PCR analysis of GFAP gene expression Silencing P2X7 induces no significant effect on neurogenesis, because of the non significant increase of beta-3 tubulin, and a decrease of gliogenesis due to a marked decrease of GFAP. The results confirm the relevance of purinergic signaling in determining the phenotypic fate of neural differentiation, in which P2X2 and P2X7 receptors promote neurogenesis and gliogenesis, respectively. Conclusion To improve the performance of this study, we propose to use other technics like: • Using of : a bacterial plasmid, all receptors except P2X1, other stem cells, fluorescence microscopy (or confocal), immunohistochemistry techniques, another marker HNK-1. • Consider coexistence between different subtypes of P2X receptors. • Limitations related to the markers used : • GFAP-positive cells may be still immature and capable to neuronal differentiation => non specific marking. • III beta-tubulin : immunodetected in tumours of non-neuronal origin such as squamous cell carcinoma => The use of embryonal carcinoma cells may generate non specific marking. • RNAi technique may not work in later stages of mammalian development as well as that technique may activate the interferon response • No experiment had been realised to determine the exact mechanism by which each receptor promots or inhibits neuro/gliogenesis limitations Futur work

×