Σημειώσεις Εργαστηρίου Φαρμακολογίας Ι Και ΙΙ
description
Transcript of Σημειώσεις Εργαστηρίου Φαρμακολογίας Ι Και ΙΙ
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&
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:
2009
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I ................................................................................................................... 2
II .................................................................................................................. 3
........................................................... 5
DNA:
DNA .............................. 6
............................................. 14
....................................................... 20
:
VIRTUAL CAT ................................................................................................................ 37
........................................................ 59
:
.......................................................................... 60
............................................................................................ 66
.............................................................. 78
:
............. 84
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DNA
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(C), (G)+ ( 1),
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DNA ( )
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2). DNA
2 .
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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. .
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DNA,
(DNA break), (intercalation)
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-
, 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.
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electrophoresis) Laemmli (1).
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/ :
(CH2=CHCONH2) ,
(N,N-methylene bisacrylamide, CH2=CHCONHCH2NHCOCH=CH2).
1.
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TEMED (,,,, tetrame-
thylethylenediamine, (CH3)2NCH2CH2N(CH3)2), (APS,
ammonium persulfate, O3SOO SO3). APS
SO4, TEMED.
.
SDS-PAGE :
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: ,
Tris-HCl pH 6.8.
,
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(wells,
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2. (separating gel, 2F)
Tris-HCl pH 8.8
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10 20300
12 10-200
15 3-100
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PAGE SDS.
( SDS ), 1.
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.
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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
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2
4 1
7
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6
5
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4.
1. .
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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
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3. ,
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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
-
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APS TEMED ,
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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.
, .
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Rf (retention factor), :
(cm)
(cm)
Rf
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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)
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(.. PBS TBS pH 7.4 0,1 % Tween 20)
,
(.. rabbit anti-mouse
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conjugate .
, ( ).
.
(alkaline
phosphatase, AP) horseradish peroxidase, HRP.
: NBT (nitro-blue tetrazolium chloride) BCIP 5-bromo-4-
chloro-3-indolylphosphate p-toluidine salt) (DAB).
(
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6. E mini trans-blot cell, BIORAD.
1:
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9. -
(transfer buffer)
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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% -
.
.
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( ) HRP-
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10.
1.
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2. PVDF 1min 100%
.
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3. ,
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5. sandwich ,
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.
6.
.
7. 100V 2h,
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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
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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).
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, (1ml
/ ).
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1x10-9 1x10-3 .
,
(control).
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(Luvox, Fevarin, Movox), (Paxil, Seroxat, Aropax, Rexetin,
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Venlafaxine
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17 (.. ),
Paul Ehlrich
. ,
1935
Domagk. , Fleming (1929)
Chain Florey (1940)
.
, (.. -,
..).
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(, ..)
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( ,
, . , 2001)
.
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,
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:
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1).
() (, , ..)
()
() (, , Pneumonocystis carinii)
1.
(.. ) (.. )
.
. A580: 580nm.
(
) ,
.
1.
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,
.
Gram(+) Gram(-)
.
( 2):
()
(, , , ,
)
()
(, , , , )
() (,
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() (,
, 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),
() , () ,
, ,
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. :
() :
.
-
.
(tolerance)
,
.
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()
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(transposable elements),
.
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..
2.
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,
..
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:
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.
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(
-
). (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)
- + ( )
++ (