&

2009


I ................................................................................................................... 2
II .................................................................................................................. 3
........................................................... 5
DNA:
DNA .............................. 6
............................................. 14


....................................................... 20
:
VIRTUAL CAT ................................................................................................................ 37
........................................................ 59
:
.......................................................................... 60

............................................................................................ 66
.............................................................. 78
:
............. 84

 I

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,
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.
, 1987,



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,

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,
.


. /
1998-1999
.

.
,
,

. .

.
, PhD

1.

()
.

2.

3.

4.

5.

6.

.


. ,

.

7.

1o

DNA: DNA

: .
: ,

DNA
. DNA,
4 * (), (),
(C), (G)+ ( 1),
,
.
DNA ( )
, . ,
,
( cell cycle), 4 (G1, S, G2 ) (
2). DNA
2 .
.

 1. DNA ().
DNA D (1; intercalation), (2; minor groove binding)
(3; covalent binding).

2. (+) (-)
.

 ( / / ),
(checkpoints)
G1, G0
(cell differentiation). ,
(restriction point),
S ( DNA),

( apoptosis) ( 3).

3. .

, 4,
DNA,
(DNA break), (intercalation)
. ,


, DNA (DNA
ladder) ( 5).

4.

(PALA: N---L- , TMP: ).

DNA , in vitro
.
(agarose gel)
DNA *MEL () 562 ()+.

 5.
(agarose gel) DNA
,
DNA ladder (
).
(: DNA
C: DNA
1,5,10: DNA
).

MEL (
control)
(1x10-9 M 1x10-3 M) 37oC 48 .
, DNA
.

: MEL-745PC-4A

: DMEM (Dulbeccos Modified Eagle Medium)

10% v/v 100g/ml (
)

: ()
(incubators)

: 1x105 /ml

: (1x10-9 M
1x10-3 ).

, DNA
, :

1) (~ 1x107 )
.
(0oC).
2) 5 1200 /.
. ( pellet)
10ml (PBS).
3) 5 1200 /.
.
4) 0,5ml (lysis
buffer: 100mM NaCl, 10mM Tris-HCl pH 8, 25mM EDTA pH 8, 0,5% w/v SDS,
0,2mg/ml proteinase-K) eppendorf 1,5ml.
50oC 12-18 .
5)

//

0,5ml

25/24/1).

(
,
).
6) 10 4000 /.
() eppendorf, 0,5ml
// .
7)
eppendorf. 0,1 ( ) 3
CH3COONa 2,5 100% v/v EtOH.
.
8) 5 4000 /
DNA. 0,5ml 70% v/v EtOH
.
9) 5 4000 / EtOH.
5-10 .
10) 0,5-1ml (10mM Tris-HCl pH 8, 1mM
EDTA pH 8) 2. DNA,
4oC 12-24 .
11) DNA
(OD) 260nm, OD260nm /

OD280nm ( 1,8-2
). , 5l DNA 995l
2
260nm 280nm, UV-Vis.
DNA ,
DNA OD260nm = 1 DNA = 50g/ml:

OD260nm x 1ml x 50g/ml = g DNA / l

5l
12) 1% w/v (0,4g 40ml ).
500W 2
2,5l 10mg/ml EtBr.
13)
(wells)
DNA, (gel). ~30-45
( 6).
14) DNA :
10g DNA
2 20l. , 2l
(dye/loading buffer).
15) ,
(DNA markers)
DNA
( 6).
16) 100V ~45 .
17) DNA (UV light)
.
18) .
, 5.

 6. DNA.

1.
.
2.
;
3.
,
() .

2o

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()
,
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.
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()
() .
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, ().
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), ( ),
,
() (),
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(Ach),
.
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N, -

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,
.
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(, ),
, ( 1).

,
,
( - ),
, ,

()

1: ,

o o (..: ,
, , ).
(, )
. (: Smith
and Rennard, Text Book of Pharmacology, W.B.Saunders, 1992, p. 76).

 ()
. ,
,
() ,
( ,
2 ).
Ach,
() ,
,
. , ,
,
.
,

, - Tyrode (0,8% NaCI,
0,02% KCI, 0,2% CaCI2, 0,1% NaHC03, 0,01% MgCI2, 0,005% NaH2P04 0,1%
), (37C), p 7,0-7,2
. Tyrode
,
.

, .
:

. (
,
).
.
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: Tyrode (5-6 ), - HCI (1 mg/ml), (1mg/ml),

l (0,2 mg/ml)

.
,
,
( , )
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 ,
.
.
.
, ,
, .
:
(37C) -
( ),
, (isotonic myograph
detecting head)
(isotonic myograph transducer),
(physiograph MK-III-S Narco).
,
,
,
.
,
.
,
.
(transducer),
,
,
,
, .

1) 1-2 min.
2) .
, ,
1-1,5 min,
,
.

, ,
.

3) ,
,
.
,
.
4) , .

II
.. , , 2001, /

3o

: Ph.D.,
: . , /

- SDS-PAGE
1.

. ,
, , . ,
pH, (-) (+).
, ,
,
. ,

.

,
. ,
:

, pH

.
(DNA, RNA),
.


, SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel
electrophoresis) Laemmli (1).

.
/ :
(CH2=CHCONH2) ,
(N,N-methylene bisacrylamide, CH2=CHCONHCH2NHCOCH=CH2).

1.
,
.

H .
TEMED (,,,, tetramethylethylenediamine, (CH3)2NCH2CH2N(CH3)2), (APS,
ammonium persulfate, O3SOO SO3). APS
SO4, TEMED.
.
SDS-PAGE :
1. (stacking gel, 2)
: ,
Tris-HCl pH 6.8.
,
, .
(wells,
). ,
( 2 3).
2. (separating gel, 2F)
Tris-HCl pH 8.8
, .
(%) ,
.

(kDa)

50500

10

20300

12

10-200

15

3-100

( ),
(running buffer, pH 8.3)

 / (sample buffer, pH 6.8), SDSPAGE SDS.

( SDS ), 1.
( ) 2.

.

.
(
pH 6.8
Cl-
). pH 8.8

Cl- ,
. 2
SDS-PAGE.
(sample loading buffer)
1. Tris-HCl pH 6.8 (pH
) 2.
(front) ( bromophenol blue, pyronin Y) 3. ,

4. (DTT, dithiothreitol) -,
(
)
SDS .
,
Coomassie Brilliant Blue ( >100ng)
(10 ).

, . .

.

FF

2. (slab gel apparatus). , D:

, : ,
C: .
(stacking gel, E)
(separating resolving gel, F)
.

2.
.
mini protean III, BIORAD,
/ , ,

3. -
(running buffer): 25mM Tris, 250mM , 0,1%
SDS.
5x (sample buffer): 250 mMTris/HCI (pH 6.8), 10% (w/v) SDS, 50%
(v/v) 15% (v/v) 2-.
/ : 0,25% Coomassie Brilliant Blue R250 45% ,
10% dH2O.
: 45% , 10% dH2O.
1,5 M Tris pH 8.8 dH2O, 1,0 M Tris pH 6.8
dH2O, 10% w/v SDS dH2O, 100% , 10% w/v
dH2O,

6
3

2
5

4
1

3. E mini protean III BIORAD.

1:
. (spacers)
2:
, 3: 2
.
4:
, 5:

, 6: .

.
, 7:
(
).

29:1 w/v : dH2O.

:
.
.
(), .

4.
1. .
,
(spacers) ( 1.0-1.5 mm).
.
2. 10 ml 12% :

3,3 ml dH2O

4,0 ml 29:1 w/v : dH2O

2,5 ml Tris-HCl 1,5M pH 8.8

100 l SDS 10% w/v dH2O

100 l APS 10% w/v dH2O

4 l TEMED

,
. APS TEMED
, .
3. ,
.
4. - - .

,
. 60 min. H

.
5.
dH2O dH2O.
6. 4 ml , :

2,7 ml dH2O

0,67 ml 30% w/v (dH2O)

0,5 ml Tris-HCl 0,625 M pH 6.8

40 l SDS 10% w/v dH2O

40 l APS 30% w/v dH2O

4 l TEMED

 ,
.
APS TEMED ,
.
7. dH2O

.

,
(wells) .
8. ,
. (15-45 min).
9. 3 .
10.
.
11. .
12. (~60 min, 160V).
13.
Coomassie Brilliant Blue
Western.
5.

Coomassie Brilliant Blue

1. (fixing)
~15 min.
2. (staining)
2 , .
3.
(destain).
.

.
,
(15-20 min)

 .
1g.
6.

, .
.
Rf (retention factor), :
(cm)

Rf =
(cm)
Rf
. .
Rf
.

4. 12%
() E. coli ( 1). -
Coomassie Brilliant Blue R250. Rf
1

( cm).

7. Western - Western blotting

Western (2) (Western blot, immunoblot).
( )

.
( SDS-PAGE) ( 5)

.
(PVDF, polyvinylidene fluoride).

Whatman

Whatman

5:

.
sandwich , ( ):
, 3 Whatman, , , 3 Whatman
( ).

 ,
:

(blocking) (BSA, bovine

serum albumin)

( )

(.. PBS TBS pH 7.4 0,1 % Tween 20)

,
(.. rabbit anti-mouse
).
conjugate .
, ( ).

(alkaline
phosphatase, AP) horseradish peroxidase, HRP.
: NBT (nitro-blue tetrazolium chloride) BCIP 5-bromo-4chloro-3-indolylphosphate p-toluidine salt) (DAB).

(
).
.

. ,

.

8.
:

mini

trans-blot

cell,

BIORAD,

/ , , -shaker,
, Whatman 3 ,
PVDF , ,
sandwich , .

3
4

6. E mini trans-blot cell, BIORAD.

1:
, 2:,
sandwich
3: 4:
, (1)
( ).

9. -
(transfer buffer)
: 25 mM Tris base, 192 mM , 0,037% w/v SDS
20% v/v . SDS
.


.
PBS (phosphate buffered saline) pH 7.4, 0,1% Tween 20.
PBS : 137 mM NaCl, 10 mM Na2PO4, 2,7
mM KCl, 1,8 mM KH2PO4.
blocking: 3% (BSA) 5% PBST (PBS 0,1%
Tween 20).
: 100 mM Tris-Cl/100 mM NaCl/5 mM MgCl2 pH 9,5.
NBT ( -): 75mg/ml 70% -
30% dH2O.
BCIP (5--4--3-- -): 50mg/ml 100% .
.
.
( ) HRP-
( ) .

10.

1.
. ,
sandwich , PVDF
Whatman 3M .
2. PVDF 1min 100%
.
.
3. ,
5min.
4. sandwich : 1 , 3 Whatman
3M, , PVDF, 3 Whatman 3M 1

. sandwich
. sandwich
.
5. sandwich ,
.
.
.
6.
.
7. 100V 2h,
.
8. PBST.
Coomassie Brilliant Blue .

PBST 4C .
Western .
1. blocking 1h .
2. blocking 1h
.
.
3. 4x PBST 10min .
4. blocking
1h .
..
.
5. 3.
6. 3x 5min .

,
. NBT-BCIP.

15ml AP pH 9,5 66l NBT 50l

BCIP ( ).

 .
5-30min.
d2
.

11.

Rf.
kDa : 1:175, 2:83, 3:62, 4: 47,5, 5: 32,5, 6:25.

!!! !!!
.

1
2
3

2
1

1
1
2
3

12.
1. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature. 227, 680-685
2. Towbin, H., et al. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to
nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76,
4350-4354.
3. Berg, J. M., J. L. Tymoczko, et al. (2002). Biochemistry. New York, W. H. Freeman and Co.
4. Cooper, G., Ed. (2000). The Cell - A molecular approach. Sunderland, Sinauer Associates.
5. Janeway, C. A., P. Travers, et al., Eds. (2001). Immunobiology: The Immune System in
Health & Disease. New York, Garland Science Publishing.
6. Lodish, H., A. Berk, et al. (2000). Molecular Cell Biology. New York, W. H. Freeman & Co.
7. Wilson, K. and J. Walker, Eds. (2000). Principles and Techniques of Practical Biochemistry.
Cambridge, Cambridge University Press.

4o
:
VIRTUAL CAT

: Ph.D.,
: . , /

1.

. ,
,
. -
.

,

, ,
.
,
, .

in vitro , -
,
,
, ,
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in vivo ,
,
.


Strathclyde .
Virtual Cat ( 2.5.6).
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2.

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(1) .

(1).

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Ph.D.,

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:
. ( )
- - II, VII,
IX X. . : , LMWH:
, PL:
( : Rang and Dales,
th
Pharmacology 6 edition, 2007).

1.

:
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1000 .
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) ( ):
,
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1.

2.

38% w/v

3.

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4.

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5.

( - Platelin LS, Biomrieux)

)
, V,
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37 C
,
. ~13 sec, .


(.. ).

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XIII.
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) 37 C 1-3 min. 0,1
ml CaCl2
.
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2o

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( , , , , , ,
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transformation), (neoplasms)
(cell proliferation),
(cell survival), (cell differentiation)
(programmed cell death apoptosis). ,
,
(metastasis),
.

.

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.


: 1) , 2) , 3)
, 4) , 5) , 6) , 7)
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(cell proliferation cycle)
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DNA, .
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/
,
,
.

7. (G1, S, G2, M),

.


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3) :
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.
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.

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(, ).
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(chemical inducers).

.
7) :

.
8) (tyrosine kinase inhibitors):

(, ).
9) (inducers of apoptosis):

.
10) (monoclonal antibodies - mAbs):
.

,
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(cell tissue culture) in vitro
,
(primary tissue) (primary cell line)
(clone) ( ) ,
. (3D)
in vivo.
, ,
(monolayer cell culture) (suspension cell culture).
(culture medium) , , , ,
(growth factors)
(.. ). , (..
),
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8. () ().

in vitro ()
, : 1)
, 2)
,
, 3)

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5)
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( 10
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,
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,
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(cell kinetics curve)
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plating efficiency).
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period),
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.
3.
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,
()

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, .

(%), (
) 100%.

9. (),
.

MEL-745PC-4A (Murine ErythroLeukemia cells)
.
(suspension culture) ,
(incubators) (37oC)
5% v/v CO2, ( 4).

MEL

, 48
(1x10-9 1x10-3 ) .

 50%
(IC50).

: MEL-745PC-4A

: DMEM (Dulbeccos Modified Eagle Medium)

10% v/v 100g/ml (
)

: 24 (24-well plates)
(incubators) ( 4)

: 1x105 /ml

: (1x10-9
1x10-3 ).

10. () (laminar flow hood).

(B) (incubator)
. () 24 (24-well plate).


1. MEL 1x105 /ml,
, (1ml
/ ).
2.

1x10-9 1x10-3 .
,

(control).
3. 48 (incubators).
4. (
Neubauer) .
5.

.
6. ( IC50
),
.

( Neubauer)

( Neubauer) ,
(
), .
2 ,
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() 0,1mm. ,
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1ml, :
(/ml) = / x 104

( ).

.
2
,
.

A, B, (
5).

:
(A+B++) x 104 = / ml
4

 11. .
100x. 1mm.


4.
.
5.
; (drug screening assay)
.
6.
50%
(IC50).
;
7.
.
;

: Ph.D.,
: . , /
1.
,

,

,
,
.

.
.
.
,

.
, ,
.

, ,
.
, ,
.


, ,
,
.


,
. ,
, , , ,
,
.
,
40% 30% .
: ,
, , , (
) ...

,
:

/
/



/
, ,

, ,
,
.
,
.
( )
,
, , .
(- )
,
.
,

, , .

,
.
()
, .
, ,
.

2.

(Selective

Serotonin

Reuptake

Inhibitors, SSRIs)

.

,
,
.
,
.

, .
, , .

(Celexa, Cipramil, Dalsan, Emocal, Sepram, Seropram), ,
(Lexapro, Cipralex, Esertia), (Prozac, Fontex, Seromex, Seronil, Depres),
(Luvox, Fevarin, Movox), (Paxil, Seroxat, Aropax, Rexetin,
Xetanor, Paroxat), (Zoloft) (Zelmid, Normud).
Bupropion
, ,
,
, .
,

 .
.
,
.
, .
Venlafaxine

(Serotonin-Norepinephrine
Reuptake Inhibitor, SNRI). , ,
. ,
,
.
,
.
Atomoxetine

,
(Attention-Deficit Hyperactivity Disorder, ADHD).
.

,
.
,
. ,
,
, , ,
, ...
. ,
, , , , , ...

Modafinil
,
,
. ,
, .
Divalproex
1:1
,
. ,
-
.

, ,
. ,

,
.

, .
, .
GABAA ,
.

4o

: .
: ,

17 (.. ),
Paul Ehlrich
. ,
1935
Domagk. , Fleming (1929)
Chain Florey (1940)

.
, (.. -,
..).
( )

.

.

(, ..)
, .

 ( ,
, . , 2001)
.
(),
,
.

:
() ( )
( ),
( ) Gram (Gram - Gram + ,
1).
() (, , ..)
()
() (, , Pneumonocystis carinii)

1.

(.. ) (.. )
.
. A580: 580nm.

(
) ,
.
1.

,
.

Gram(+) Gram(-)
.

( 2):
()
(, , , ,
)
()
(, , , , )
()

(,

, , )
() (,
, D, , )
() :
,

..

DNA,

(, , )

2. - .

 ()

,

, .


5-20%.

.


.
.. .
(resistance)
( )
.
(

I.
, , Gram .
. : : -, () B, C, G
- , viridans, , D ,
GRAM
(), : ( ),

aureus ( )
. : , ( )
GRAM . :

. : Veillonella

GRAM

1. : , subtilis
2. : , ,
(diphtheroids),
.
1.: ,
(botulinum), (Welchii), difficile
2. :
.
, , Bordetella
.
Fusobacterium, (Bacteroides-fragilis, melaninogenicum, )
.

GRAM
(ENTEROBACTERIACEAE)

(Escherichia coli), , :
(vulgaris): Retgeri (Providencia rettgeri), Morgani (Morganella morgani)
(mirabilis), (Enterobacter),
(Citrobacter), flexneri, sonnei, boydii,
cholerasuis, enteritidis
aeruginosa, maltophila, Cepacia (Acinetobacter),

() , () ,

, ,

)
. :

() :

.


.
(tolerance)
,

.
() :
()
(, , )
(transposable elements),
.
, :
1. -
..

2.
.. .
,
..
.

..
, .. Gram (-)


.

.
( )
( )
(

). (MIC,
minimal inhibitory concentration) (MBC,
minimal bactericidal concentration) .
,

.

(Disc-Agar Diffusion Method)

,

. ,

( Mller-Hinton agar LB agar).
,
.
,
()
16-18 .
( )
3. (
) (tetr), (pen), (chlor), (pol),
(gent), (nal), (crph),
(strep) (amp)
.

 3.

. (Amp),
(Ceph), (Nal), (Strep) (Gent)
() (Pol), (Chlor),
(Tetr) (Pen).

Mller-Hinton

(Whatman No3,
6-7 mm) .
37C 16-18
,
( )
.
.

. ()
.

-
.

.

1.
Bunsen, 37C,
Mller-Hinton ()*, L-Broth 5ml,
Pasteur, , ,
,
, , , ,
, ,

.

* Mller-Hinton 3g
, 17g , 2
121C 20min.
50C,
25ml .
LB (Luria
Bertani broth). LB 10g
, 5g , 5g NaCl 17g .
.

2.
. coli (Gram -),

.
3.

. coli. -

().


. ,
. (

 )

37C.
16-18 , . ,
,
.
.
(++++)
>15 mm
(-) (+).

1. Jawetz ., Melmick J.L. and Adelbers: Review of Medical Microbiology, p. 117,

1980, Lange Medical Publications, Los Altos Califomia.
2. Krupp .., Chatton M.J. and Trerney .. jr.:
() ', .. ,
1987, . 1381.
3. Disk diffusion method: Acar J.F. (1980): Antibiotics in Laboratory Medicine
(Lorian V., Editor), Williams and Vilkins, Baltimore, pp 24-54.
4.

Disk diffusion method: Barry .., Thornsberry C. (1980): Manual of Clinical

Microbiology (Editors: Lenntte, Balows, Hausler and Traunt) merican Society
for Microbiology, pp 463-474.

5.

, .. , 2001 .

6.

, , 1987 .

(g/)

30

30

10

10

. coli

++++ ( >15mm)
- + ( )
++ ( <15mm)

1.
2.
3.