Σημειώσεις Εργαστηρίου Φαρμακολογίας Ι Και ΙΙ

95
Α Ρ Ι Σ Ο Σ Ε Λ Ε Ι Ο Π Α Ν Ε Π Ι Σ Θ Μ Ι Ο Θ Ε Α Λ Ο Ν Ι Κ Θ Σ Μ Θ Μ Α Φ Α Ρ Μ Α Κ Ε Τ Σ Ι Κ Θ Ε Ρ Γ Α Σ Θ Ρ Ι Ο Φ Α Ρ Μ Α Κ Ο Λ Ο Γ Ι Α ΕΡΓΑΣΘΡΙΑΚΕ ΑΚΘΕΙ ΦΑΡΜΑΚΟΛΟΓΙΑ Ι & ΦΑΡΜΑΚΟΛΟΓΙΑ ΙΙ ΑΣΕΡΙΟ . ΣΙΦΣΟΓΛΟΤ ΘΕΟΔΩΡΟ ΚΛΑΒΙΑΔΘ ΙΩΑΝΝΘ ΒΙΗΙΡΙΑΝΑΚΘ ΛΕΤΚΟΘΕΑ ΠΑΠΑΔΟΠΟΤΛΟΤ Τπεφκυνοσ Εργαςτθρίων: Ιωάννθσ Παςπαλτςισ ΘΕΑΛΟΝΙΚΘ 2009

description

gg

Transcript of Σημειώσεις Εργαστηρίου Φαρμακολογίας Ι Και ΙΙ

  • &

    .

    :

    2009

  • I ................................................................................................................... 2

    II .................................................................................................................. 3

    ........................................................... 5

    DNA:

    DNA .............................. 6

    ............................................. 14

    ....................................................... 20

    :

    VIRTUAL CAT ................................................................................................................ 37

    ........................................................ 59

    :

    .......................................................................... 60

    ............................................................................................ 66

    .............................................................. 78

    :

    ............. 84

  • I

    II

    , .

    .

    ,

    , , ,

    ( ), (, ..)

    ,

    .

    , 1987,

    .

    .

    , ,

    .

    ,

    .

    , ,

    ,

    .

    . , PhD

  • II

    ,

    .

    ,

    .

    ,

    .

    . /

    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

    :

    : , /

    ()

    ,

    , ,

    , ,

    , , ()

    .

    , .

    ()

    () .

    ,

    , .

    , ().

    (

    ), ( ),

    ,

    () (),

    .

    , .

    (Ach),

    .

    , Ach.

    N, -

  • , oo ,

    ,

    .

    , Ach, ,

    (, ),

    , ( 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

    ,

    .

    , .

    : . (

    ,

    ).

    .

    . , , ,

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

    l (0,2 mg/ml)

    .

    ,

    ,

    ( , )

    Tyrode (37C), ,

  • ,

    .

    .

    .

    , ,

    , .

    :

    (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 (,,,, tetrame-

    thylethylenediamine, (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)

    7 50500

    10 20300

    12 10-200

    15 3-100

    ( ),

    (running buffer, pH 8.3)

  • / (sample buffer, pH 6.8), SDS-

    PAGE 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 ).

    , . .

    .

  • 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,

    F

    F

    A

  • 3. E mini protean III BIORAD.

    1:

    . (spacers)

    2:

    , 3: 2

    .

    4:

    , 5:

    , 6: .

    .

    , 7:

    (

    ).

    29:1 w/v : dH2O.

    :

    .

    .

    (), .

    2

    4 1

    7

    3

    6

    5

  • 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)

    (cm)

    Rf

    . .

    Rf

    .

    4. 12%

    () E. coli ( 1). -

    Coomassie Brilliant Blue R250. Rf

    1

    ( cm).

    1

    Rf =

  • 7. Western - Western blotting

    Western (2) (Western blot, immunoblot).

    ( )

    .

    ( SDS-PAGE) ( 5)

    .

    (PVDF, polyvinylidene fluoride).

    5:

    .

    sandwich , ( ):

    , 3 Whatman, , , 3 Whatman

    ( ).

    Whatman

    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-4-

    chloro-3-indolylphosphate p-toluidine salt) (DAB).

    (

    ).

    .

    . ,

    .

  • 8.

    :

    mini trans-blot cell, BIORAD,

    / , , -shaker,

    , Whatman 3 ,

    PVDF , ,

    sandwich , .

    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

    .

    2

    1

    3

    4

  • .

    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

    1 2 3 4 5 6 1 2 3 4 5

    1

    2

    3

    4

    5

    6

    1 2

    3

    4

    5

    6

  • 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 , -

    ,

    ,

    , ,

    . ,

    in vivo ,

    ,

    .

  • Strathclyde .

    Virtual Cat ( 2.5.6).

    .

    2.

    ,

    , ,

    .

  • .

    , .

    .

    ( ),

    . ,

    () .

    .

    ( )

    .

    , .

    .

    .

    ( ), ,

    .

    .

    .

    .

    .

    ,

    .

  • , -

    ,

    .

    .

    (1) .

    (1).

    ,

    (mAchR) ,

    .

    :

    ,

    ,

    .

    -

    (Adr) ,

    .

  • ,

    .

    (nAchR).

    - (1)

    ,

    .

    (1),

    , .

    . .

    .

    - ,

    .

    , ,

    - (1) ,

    .

    .

  • ,

    , .

    ,

    (AchR) ,

    .

    ,

    (

    ).

    3.

    - .

    , stress, .

    stress,

    . ,

    ,

    ,

    .

    ,

    , . ,

    ,

    . ,

    ,

    , , .

  • . , ,

    , .

    ,

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

    G ,

    . G

    ,

    .

    ,

    ,

    . , 1

    , ,

    Na+ . 2

    .

    1

    , .

    2 (.. ,

    ),

    , ,

    , . 3

    .

    2

    1,

    .

    ,

    ,

    .

    .

  • .

    .

    . ,

    .

    ,

    (..).

    , .

    .

    , , , ,

    .

    , ,

    .

    , ,

    :

    -

    ( ).

    ... ,

    .

    ... ,

    , .

    :

  • ,

    ,

    ,

    ( )

    C, :

    ,

    ()

    .

    ,

    ,

    .

  • .

    .

    , ,

    ,

    .

    ,

    .

    , , ,

    .

    .

    ,

    (ATP ADP )-, ,

    (cAMP).

    ,

    . ,

    .

    . (A1, A2A, A2B

    A3). , G.

    .

    . 1

    , ,

    cAMP , + .

    .

    .

    .

  • ,

    . ,

    , .

    , V.

    . ,

    . ,

    .

    Atropa belladonna

    .

    , .

    ( ).

    ( , ),

    .

    . ,

    ,

    .

    .

    .

    , ,

    .

    , , ,

    .

  • .

    .

    ,

    .

    ,

    .

    . ,

    . .

    , .

    .

    ,

    .

    .

    , .

    ,

    . , ,

    .

    .

    ,

    .

    .

    ,

    ,

    .

    ,

    .

  • Parkinson, , , ,

    , ,

    .

    .

    .

    .

    ,

    ( 8 4

    ).

    , .

    ,

    .

    .

    , .

    .

    .

    .

    ,

    .

    .

    .

    ,

    , .

  • . ,

    . ,

    .

    ,

    ,

    . ,

    .

    .

    (1, 2, 3, 4).

    :

    1 , ,

    , ,

    ,

    (

    )

    2

    3

    ()

    ,

    , ,

    4 , ,

    ,

  • ,

    .

    .

    .

    , .

    ,

    .

    ,

    .

    , ,

    , ,

    .

    ,

    .

    ,

    .

    (

    ).

    , ,

    .

    .

    ,

    .

    ,

    .

    .

    1 .

  • , ,

    .

    .

    1 ,

    .

    .

    , .

    ,

    ,

    (, -, ).

    .

    ,

    .

    , , .

    G

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

    .

    ,

    , , , ,

    .

    .

    . ,

    +. Ca+2

    ,

    .

    ,

    .

    .

    . ,

    , , .

  • , ,

    , ,

    .

    , .

    ( ), (

    ).

    ,

    ,

    .

    .

    , .

    , ,

    , .

    ,

    , ().

    , .

    ,

    .

    . ,

    . , ,

    .

    -

    , - .

    ,

    .

    ,

    ,

    .

    ,

    per os , .

  • ,

    .

    ,

    .

    , .

    (AchE),

    . AchE

    .

    ,

    AchE

    , AchE

    .

    ,

    . , AchE

    ,

    ,

    .

    , AchE.

    AchE,

    - ,

    (t1/2 ~60 min, t1/2 - ~1 min).

    , AchE

    .

    , .

    AchE ,

    .

    .

    .

  • ,

    , .

    .

    .

    , , , .

    .

    . ,

    (, , .)

    (, ).

    .

    .

    .

    ,

    .

    .

    ,

    (

    , , ),

    .

    .

    L.

    .

    .

    ,

    .

  • , ,

    .

    ,

    , , , .

    . ,

    , ,

    .

    .

    ,

    .

    ,

    .

    ,

    .

    ,

    .

    .

    ,

    .

    , ,

    .

    ,

    .

  • ,

    , .

    .

    8-SPT (8-parasulphophenyltheophylline)

    8-SPT .

  • /

  • 1

    :

    : Ph.D., .

    : , /

    , ,

    . ,

    , , ,

    .

    ,

    ,

    .

    : )

    , )

    )

    ( )

    .

    , in vivo

    , (tissue factor),

    .

    (Hageman factor)

    .

    ,

  • (clot).

    ,

    .

    , , ,

    .

    :

    . ( )

    - - II, VII,

    IX X. . : , LMWH:

    , PL:

    ( : Rang and Dales,

    Pharmacology 6th

    edition, 2007).

  • 1.

    :

    ) , ,

    ( ) ,

    - (1:1)

    1000 .

    . ,

    , .

    ) ( ):

    ,

    ( , VII, IX, X C S).

    -

    . -

    1 . ,

    1 2,3- .

    1 () 2,3- 1

    .

    , 1,

    2,3- 1

    1 (), 1

    .

    1 . ,

    2-4

    .

  • .

    .

    2.

    ,

    .

    ,

    .

    . ,

    c-AMP .

    .

    Von-Willebrand .

    3.

    ,

    .

    ,

    . ( )

    , ,

    . tPA( tissue plasminogen activator)

    .

    6

    , .

    .

    ,

    (.. - ).

  • ,

    ,

    (

    Von-Willebrand).

    ,

    .

    ) (PTT)

    ) (aPTT, activated partial thromboplastine time)

    ) ()

    1. ,

    2. 38% w/v

    3. CaCl2 (2,8 g/l)

    4. (thromboplastine - symplastine excel, Biomrieux)

    5. ( - Platelin LS, Biomrieux)

    )

    , V,

    VII, X, .

    : 0,1 ml 0,2 ml .

    37 C

    ,

    . ~13 sec, .

    (.. ).

  • ) (aPTT)

    , VII

    XIII.

    : 0,1 ml 0,1 ml (..

    ) 37 C 1-3 min. 0,1

    ml CaCl2

    .

    ( ).

    36-38 sec.

    .. .

    .

    PT aPTT

    : ) , ) , ) , )

    1,

    .

    ) ()

    ,

    . .

    .

    .

  • 2o

    : .

    : ,

    ,

    , ,

    . ,

    .

    ( , , , , , ,

    , , , ..)

    (neoplastic cell

    transformation), (neoplasms)

    (cell proliferation),

    (cell survival), (cell differentiation)

    (programmed cell death apoptosis). ,

    ,

    (metastasis),

    .

    .

    . ,

  • .

    : 1) , 2) , 3)

    , 4) , 5) , 6) , 7)

    ( ), 8) , 9)

    , 10) 11)

    .

    (cell proliferation cycle)

    ( 1). , ,

    2

    . , ,

    DNA, .

    4 : G1, G2, S ( 1).

    G0 ( ). G1/G0

    /

    ,

    ,

    .

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

  • . , G1

    DNA, S,

    G2

    . ,

    . ,

    G1/G0,

    .

    , ,

    , , Vinca .

    ,

    , cis-platin, .

    :

    1) : DNA 7

    2 2 DNA,

    (cross-linking) DNA,

    . , ,

    , .

    2) :

    ()

    DNA. ,

    * (), 5- (5-FU), (Ara-C)].

    3) :

    DNA, (intercalation)

    2 ( D,

    , , ).

    .

    4) : ,

    .

  • 5) :

    (, ).

    6) :

    (chemical inducers).

    ,

    .

    7) :

    .

    8) (tyrosine kinase inhibitors):

    (, ).

    9) (inducers of apoptosis):

    .

    10) (monoclonal antibodies - mAbs):

    .

    ,

    2 ,

    : ) ,

    . ,

    )

    .

    ,

    (..

    , , ..),

    , .

  • (cell tissue culture) in vitro

    ,

    (primary tissue) (primary cell line)

    (clone) ( ) ,

    . (3D)

    in vivo.

    , ,

    (monolayer cell culture) (suspension cell culture).

    (culture medium) , , , ,

    (growth factors)

    (.. ). , (..

    ),

    . 2 ,

    (monolayer) () (suspension) ().

    8. () ().

    in vitro ()

    , : 1)

    , 2)

    ,

    , 3)

  • , 4) ,

    5)

    . ,

    : 1)

    , , 2)

    ( 10

    ), 3)

    ,

    .

    , in vitro

    ,

    ,

    .

    ,

    ( )

    (cell kinetics curve)

    (foci), (

    plating efficiency).

    , (doubling time),

    (lag period), (log

    period),

    (plateau period),

    .

    3.

    , ,

    ,

    ()

  • (hemocytometer)

    , .

    (%), (

    ) 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 ,

    9 1mm ( 5).

    () 0,1mm. ,

    : 1mm x 1mm x 0,1mm = 0,1mm3 = 10-4cm3. 1cm3

    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.

    .

    ;

  • 3

    : 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 .

    GRAM

    . : : -, () B, C, G

    - , viridans, , D ,

    (), : ( ),

    aureus ( )

    . : , ( )

    GRAM

    . :

    . : Veillonella

    GRAM .

  • 1. : , subtilis

    2. : , ,

    (diphtheroids),

    .

    1.: ,

    (botulinum), (Welchii), difficile

    2. :

    GRAM

    .

    , , Bordetella

    .

    Fusobacterium, (Bacteroides-fragilis, melaninogenicum, )

    .

    (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/)

    . coli

    30

    30

    10

    10

    5

    ++++ ( >15mm)

    - + ( )

    ++ (