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Short report of visiting
research course
By: Kambiz Hassanzadeh
Supervisor: Professor Sandra Ceccatelli
Karolinska Institute, Stockholm 2009
I was involved in two projects
1)Evaluation the effect of perflurooctanesulfonic
acide (PFOS) on proliferation and differentiation
of cortical neural stem cells isolated from
embryo of mice.
2) Effect of gender and age difference on the
reactivity of human neurospheres to different
toxicant stimulus.
Techniques
In order to be involved in cell culture
projects, I learned the different techniques
which are needed for working with cells
such as (seeding, splitting, harvesting,
freezing,…..).
Also I learned the test of viability of cells
(Trypan blue), immunocytochemistry and
histochemical methods to investigate the
proliferation and differentiation of cortical
neural stem cell pathways.
Additionally I learned gene expression assay
& analysis using Real-Time PCR method.
Project 1Methods:
1) Preparation of cortical neural stem cells from
embryo of mice
2) Exposing the cells to different doses of PFOS
(12.5, 25, 50 100 nM)
3) Harvesting the cells and Trypan blue test
4) Evaluation of different factors which are
involved in proliferation and differentiation
using immunocytochemistry ( Nestin, SOX2,
Tuj1, Ki 67, BDNF, GFAP, TrkB, ….).
Project 2 method:
Cell culture: Cryopreserved human neural spheres
were cultured at 37°C and 5% CO2 as a single cell
suspension culture in proliferation medium consisting
of Dulbecco’s modified Eagle medium (DMEM) and
Hams F12 (3:1) supplemented with B27, 20 ng/mL
epidermal growth factor, and 20 ng/mL recombinant
human fibroblast growth factor. Single cell suspension
was prepared for each experiment. Differentiation was
initiated by growth factor withdrawal in differentiation
medium [DMEM and Hams F12 (3:1) supplemented
with N2 supplement and plated onto poly-dlysine/laminin–coated cover glasses.
Chemical exposure:
The cells were exposed to:
1) MeHg (2.5, 10, 25, 100 nM)
2) H2O2 (5, 25, 50, 100 μM)
3) Staurosporin (5, 10, 15 nM)
in proliferation and differentiation medium.
Immunohistochemistry:
Proliferating or differentiating single cell
suspension was fixed in 4% paraformaldehyde
for 30 min. After washing the cells in
phosphate-buffered saline (PBS), they were
incubated with primary antibody for evaluation
of differentiation overnight at 4°C. Afterward,
cell suspension was incubated with secondary
antibody for 1 hour and then nuclei were
counterstained with Hoechst. Antibodies for
staining was β(III)tubulin (1:200; Sigma
Aldrich) which is a marker for neurons
Individuals
Individuals
Identification
Number
503
585:2
Gender
Developmental
Quality
stage
(Gestational Week)
♂
16
Very good in proliferation
♀
8.5
Very good in proliferation
557:4
♂
6
Very week in proliferation
9 1/2
♀
9.5
Good in proliferation
603:3
♀
8
Week in proliferation
Study Design for Immunocytochemistry
1. H2O2 ± GF
5, 25, 50, 100µM
Apoptosis: 24hrs in culture
Staining
Differentiation: Exposure
Fixing and Staining
2. MeHg ± GF
Exposure
24hrs
Fixing and
4 days in culture
2.5, 10, 25, 100nM
Apoptosis & Differentiation:
Exposure
4 Days in culture
Fixing and Staining
3. Staurosporine ± GF 5, 10, 25nM
Apoptosis
24hrs in culture
Exposure
24hrs
Fixing and Staining
Gene expression analysis
In order to find the possible mechanisms we
examined the bellow genes involving in
differentiating pathways.
NOS3, VEGF, ENO2, Hes family genes, Jag1,
Notch1, Mash1, DKK1, SOX2, PAX6,…..
Method:
Cells in culture 24 hrs Exposure to H2O2 or
MeHg for 4, 24, 48 hrs
Harvesting
Conclusion
Our results showed that there are differences in
susceptibility of different individuals to toxicant
stimulus based on their sex and age. This study
is continuing now and for confirming, some
other individuals are being studied by our
colleagues in lab.
Meetings & Seminars
Also I have attended in group meetings and
journal clubs as well as seminars organised by
Karolinska Institute.