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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