The Influence of Brain Derived Protein Complex
on the Proliferative
Activity of Hippocampal Progenitor Cells
Giorgi Mosidze, M.S.
Department of Biology,
Faculty of Exact and Natural Sciences,
Iv. Javakhishvili Tbilisi State University,
Tbilisi, Georgia
2012
NEUROGENESIS ?!
Rat’s Hippocampus
1-CA1; 2-CA2; 3CA3 fields;
4-suprapiramidal and
5-infrapiramidal
blades of dentate
gyrus.
6-polymorphic cell
layer(hilus)
(7X3).
Progenitor cells of rats’ dental gyrus
CA1
CA3
DG
Sub granular Layer
Regulation of Neurogenesis
IGF I
ETPC- Endogenous Thermostable Protein Complex
(Investigation was started by Prof. Tumanishvili)
• Ethanol extraction according to the Bullough
method (Bullough et al 1964)
Note: Bullough studied endogenous growth factors and extracts of vertebrate epidermis contain an antimitotic
chemical messenger, called the epidermal chalone, which appears to be of central importance in the control of
epidermal mitosis.
• The termostable complex was received by boiling
at 100 C ;
• Different sources of extraction:
Phylogeneticaly diverse species (Bacteria,
......human);
Different organs of one species (in our case
rat)
Chemical Characterization
Native PAGE
Bovine albumin 66.00 kDa
Fraction I – at
molecular weight
ranged between
45-66 kDa
Egg albumin 45.00kd
Fraction II – at
molecular weight
ranged between
12-17 kDa
Horse myoglobin 17.00kd
Cytochrome C 12.00kd
Brain Heart
Chemical Characterization
hydrophobic-interaction HPLC
The same picture as
at electrophoresis two fractions in the
extracts from
different organs
Fraction I –
corresponds to the
second peak
(hydrophobic)
Fraction II –
corresponds to the
first peak at the
diagram
(hydrophilic);
Influence on Transcriptional
Activity
Intensity of RNA synthesis %
in vitro test system of isolated nuclei
120
100
80
60
40
20
0
1
1. Intact nuclei
2. Nuclei + ETPC
2
Inhibition of Mitotic Index
120
Mitotic Index ‰
100
80
60
40
20
0
1
Note: experiment means the effect of ETPC after 3 hours of injection
2
1.
2.
Control
Experiment
Due to inhibition of transcriptional activity, ETPC
decreases the mitotic index .
Intensity of RNA synthesis %
Specificity
120
80
60
40
20
0
Control
Heart
Liver
Brain
The tissue and species specificity
were studied both at the level of
transcriptional and mitotic activity.
The ETPC is tissue-specific, but it is
not shown to be species - specific.
120
100
80
60
40
20
0
Control Pig Mouce water
cavy
Intensity of RNA synthesis %
100
However, the tissue-specificity reveals only in case of
terminally differentiated cells.
GOAL !
To study the influence of endogenous protein
complex on the proliferative activity of newborn
rats’ brain progenitor cells.
Materials and Methods
 Materials:
• Brain tissue of newborn (5-6 days) rats
• Adult Rats’ brain for the ETPC extraction
 Methods:
• Extraction of protein complex according to the
Bullough method (Bullough et al 1964).
• Histochemical staining (hematoxilin/eosin)
• Imunohistochemical staining ( against Ki 67)
Mitosis
G2 phase
Changing of mitotic activity of dental gyrus cells, in 3 hours after
ETPC injection.
5
4.5
Mitotic index ‰
4
3.5
P<0.002
3
2.5
2
1.5
1
0.5
0
Control
Experimental
Mitotic figures of dentate gyrus of
hippocampus
B
A
7X90
Changing of mitotic activity in CA1 and CA3 fields of hippocampus in 3
hours of ETPC injection.
CA3
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
P>0.05
Control
Experimental
Mitotic index ‰
Mitotic index ‰
CA 1
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
P>0.2
Control
Experimental
Protein complex derived from adult rats,
decreases the mitotic activity of dentate gyrus of
newborn rats’ hippocampus.
Changing of Ki 67 positive cells’ quantity in dentate gyrus, in three
hours after protein complex injection
50
Ki 67 positive cells ‰
45
40
35
P<0.001
30
25
20
15
10
5
0
Control
Experimental
Ki 67 positive cells of dentate gyrus of newborn rats’
hippocampus.
7X40
7X90
Influence of ETPC on the quantity of Ki-67
positive cell
G2
M
S
G1
G0
The increasing of Ki-67 positive
cell quantity possibly defined
by
retention of cells in G2 phase
and by the entering of new
pool of cell into the cell cycle
Is this process reversible or
not?
Mitotic index ‰
The reversed effect of ETPC
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
Equal mitotic index in control and
experiment at 5 hours after injection
Indicates on the reversed effect of ETPC
control
experiment
3
5
7
Hour
What might be the prospective mechanism
of restoration of mitotic index?
Conclusion
 Protein complex derived from adult rats brain decreases the mitotic
activity in homologies tissue in newborn rats.
 This inhibitory effect prolongs only three hours and is reversible
process.
 The cells, which were impeded in G2 phase, maintain ability to
enter into mitosis.
Acknowledgement
G. Tumanishvili †
D. Dzidziguri
N. Giorgobiani
I. Modebadze
E. Bakuradze
L. Rusishvili
M. Rukhadze
T. AslamaziShvili and all members of working group.
Thanks for your attention
Ki -67 positive cells ‰
Influence of ETPC on the quantity of Ki-67
positive cell
60
55
50
45
40
35
30
25
20
15
10
5
0
The quantity of Ki67 positive cells is
increased by the
effect of ETPC
Control
experiment
EPTC in Kidney Tumor
Normal tissue
Transformed tissue
EPTC was extracted
from transformed
cells. The ETPC in
tumor cells was not
identified
The influence of ETPC on the different type of
transformed cells
120
100
80
60
40
20
0
120
Jurkat Cell
MDCK
100
80
60
40
20
120
1
2
0
Erlich ascitic
carcinoma
100
1
80
60
40
20
0
1
2
Note: Transcriptional activity
1. control; 2. experiment ( effect of EPTC on Isolated nuclei)
2
ETCP-Possible using in Cancer Therapy
Two key points, absence of ETPC in transformed cells and
inhibition effect on transcriptional and correspondently
mitotic levels, give us possibility to think of this protein
complex as a potential component of cancer therapy.
The future study
The future study strategy should be focused
on more deep investigation of protein
features, mechanism of acting in cancer
cells.
It will be interesting for our group to check
if this protein complex, works according the
schema advised by our group above.
Ki 67 positive cells ‰
Asymmetric distribution of Ki 67 positive cells in
various fields of hippocampus (intact rats)
60
55
50
45
40
35
30
25
20
15
10
5
0
DG
CA1
CA3
Changing of Ki67 positive cells' quantity in CA1 and CA3 fields in three
hours after protein complex injection
CA3
60
55
50
45
40
35
30
25
20
15
10
5
0
Ki 67 positive cells ‰
Ki 67 positive cells ‰
CA 1
Control
Experimental
60
55
50
45
40
35
30
25
20
15
10
5
0
Control
Experimental