Giorgi Mosidze, M.S.
The Influence of Brain Derived Protein Complex on the Proliferative
Activity of Hippocampal Progenitor Cells
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; 3-CA3 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
Egg albumin 45.00kd
Horse myoglobin 17.00kd
Cytochrome C 12.00kd
Brain Heart
Fraction I – at molecular weight ranged between 45-66 kDa
Fraction II – at molecular weight ranged between 12-17 kDa
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
in vitro test system of isolated nuclei
1 20
20
40
60
80
100
120
Inte
nsity
of R
NA
synt
hesi
s %
1. Intact nuclei2. Nuclei + ETPC
Inhibition of Mitotic Index
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Mito
tic I
ndex
‰
1. Control 2. ExperimentNote: experiment means the effect of ETPC after 3 hours of injection
Due to inhibition of transcriptional activity, ETPC decreases the mitotic index .
Specificity
0
20
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60
80
100
120
Inte
nsity
of R
NA
synt
hesi
s %
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.
Con-trol
Pig Mouce water cavy
020406080
100120
Inte
nsity
of R
NA
synt
hesi
s %
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)
G2 phase
Mit
osis
Changing of mitotic activity of dental gyrus cells, in 3 hours after ETPC injection.
Control Experimental0
0.51
1.52
2.53
3.54
4.55
Mito
tic in
dex
‰
P<0.002
Mitotic figures of dentate gyrus of hippocampus
7X90
A B
Changing of mitotic activity in CA1 and CA3 fields of hippocampus in 3 hours of ETPC injection.
Cont
rol
Expe
rimen
tal012345
CA 1
Mito
tic
inde
x ‰
Control Experimental0
0.51
1.52
2.53
3.54
4.55
CA3
Mito
tic in
dex
‰P>0.05 P>0.2
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
Control Experimental0
5
10
15
20
25
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35
40
45
Ki 6
7 p
ositi
ve c
ells
‰
P<0.001
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
G1
S
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?
The reversed effect of ETPC
3 5 70
0.51
1.52
2.53
3.54
4.5
controlexperiment
Hour
Mito
tic in
dex
‰
Equal mitotic index in control and experiment at 5 hours after injection Indicates on the reversed effect of ETPC
What might be the prospective mechanism of restoration of mitotic index?
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.
Conclusion
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
Influence of ETPC on the quantity of Ki-67 positive cell
Control experiment05
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Ki -
67 p
ositi
ve c
ells
‰
The quantity of Ki-67 positive cells is increased by the effect of ETPC
EPTC in Kidney Tumor
EPTC was extracted from transformed cells. The ETPC in tumor cells was not identified
Normal tissue
Transformed tissue
The influence of ETPC on the different type of transformed cells
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1 2
MD
CK
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1 2
Erl
ich
asci
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carc
inom
a
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1 2
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Note: Transcriptional activity 1. control; 2. experiment ( effect of EPTC on Isolated nuclei)
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 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.
The future study
Asymmetric distribution of Ki 67 positive cells in various fields of hippocampus (intact rats)
DG CA1 CA305
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Ki 6
7 po
sitiv
e ce
lls ‰
Changing of Ki67 positive cells' quantity in CA1 and CA3 fields in three hours after protein complex injection
Contro
l
Expe
rimen
tal0
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CA 1
Ki 6
7 po
sitiv
e ce
lls ‰
Contro
l
Expe
rimen
tal0
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CA3
Ki 6
7 po
sitiv
e ce
lls ‰
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