Hoofdstuk 1 Prepareren en observeren van cellen en weefsels ...............................................................6
Celcultuur, historisch .......................................................................................................................6
Stadia van celcultivering ..............................................................................................................6
Typische celculturen ....................................................................................................................6
Cultuurcondities .........................................................................................................................6
Cultuurrecipiënten ......................................................................................................................6
Oorsprong v cellen, cellijnen ........................................................................................................7
Groeisubstraten en -procedures .......................................................................................................7
Doel............................................................................................................................................7
Celdensiteits-afhankelijke inhibitie ...............................................................................................7
Tumorcellen ................................................................................................................................7
Procedure v celtransfer ................................................................................................................7
Stappen bij celadhesie en -spreiding welke plaatsvinden na losmaken en uitplaten vd cellen ...............8
Apparatuur vo celcultuur ..............................................................................................................8
Vb van celcultuur-exp ..................................................................................................................8
Differentiatie in vitro ....................................................................................................................8
Stamcellen ......................................................................................................................................8
Wat is een stamcel? .....................................................................................................................8
Totipotent....................................................................................................................................8
Pluripotent ..................................................................................................................................8
Multipotent .................................................................................................................................8
Aanmaak muis embyronale stamcellen ........................................................................................8
Aanmaak humane embryonale stamcellen ...................................................................................9
Kwaliteitscontrole........................................................................................................................9
Zuiverheid, Homogeniteit en stabiliteit van celcultures ..................................................................9
Hoofdstuk 1 Prepareren en observeren van cellen en weefsels ............................................................. 10
a) Gebruik van secties nodig om subcellulaire details waar te nemen ........................................... 10
Fixeren ...................................................................................................................................... 10
Inbedden .................................................................................................................................. 10
Snijden ..................................................................................................................................... 10
Ontwikkeling van LM razend snel ................................................................................................ 11
Lichtmicroscopie (LM) ................................................................................................................... 11
Klassieke licht- of klaarveldmicroscoop: ..................................................................................... 11
Speciale lichtmicroscopen voor levende cellen ........................................................................... 12
Principes lichtmicroscopietechnieken ........................................................................................ 12
Kleuren, routine, hematoxyline en eosine (H/E) ................................................................................ 12
1
, Specifieke kleurtechnieken ........................................................................................................ 12
Fluorescentiemicroscoop .......................................................................................................... 13
Elektronenmicroscopie (EM)........................................................................................................... 14
Types van EM ............................................................................................................................. 14
TEM .......................................................................................................................................... 14
SEM .......................................................................................................................................... 15
Speciale EM-technieken ............................................................................................................ 15
Hoofdstuk 2: Bloed ............................................................................................................................ 15
Bloed, plasma, serum .................................................................................................................... 15
Cellen in het bloed ......................................................................................................................... 16
Rode bloedlichaampjes – erythrocyten ....................................................................................... 16
Leukocyten – witte bloedcellen................................................................................................... 18
Thrombocyten (bloedplaatjes) in het bloed ................................................................................. 20
Bloedvorming = hematopoiesis ....................................................................................................... 20
Hoofdstuk 24, MCB: Immuunsysteem ................................................................................................. 20
24.1 Overzicht van afweersystemen in gastheer ............................................................................... 20
3 lagen v immuunafweer v gewervelde dieren ........................................................................ 21
24.2 Immunoglobulines: structuur en functie .................................................................................. 21
De bloedsomloop en lymfatische systemen ................................................................................ 21
24.3 Verkrijgen van antilichaam diversiteit en B-cel ontwikkeling ...................................................... 22
Initiatie vd adaptieve immuunrespons in lymfeklieren .................................................................. 22
24.4 De MHC en antigen presentatie .............................................................................................. 22
24.5 T cellen, T-cel receptoren, en T-cel ontwikkeling ...................................................................... 22
Drie routes van complementactivering ........................................................................................ 22
Cellulaire afweer ........................................................................................................................... 24
Humorale afweer ........................................................................................................................... 24
Hoofdstuk 3: Organellen ..................................................................................................................... 26
a) Biomembranen ..................................................................................................................... 26
Vries-breek en Vries-ets technieken ............................................................................................ 27
Myelinehuls............................................................................................................................... 27
Biomembranen: verschillen van vorm ......................................................................................... 27
b) Mitochondriën....................................................................................................................... 27
Structuur .................................................................................................................................. 27
Functies .................................................................................................................................... 28
Evolutie vanuit bacteriën ............................................................................................................ 28
Fusie en fission.......................................................................................................................... 29
2
, Reacties.................................................................................................................................... 29
c) Endoplasmatisch reticulum ................................................................................................... 29
Eiwitsynthese ............................................................................................................................ 30
Topologie vd secretieweg ........................................................................................................... 30
Belangrijkste gebeurtenissen bij cotranslationeel import ............................................................. 30
Afsplitsing v signaalpeptide in RER door signaalpeptidase ............................................................ 31
Stappen in cotranslationeel import in RER................................................................................... 31
smooth endoplasmatisch reticulum (SER) .................................................................................. 33
d) Golgi complex/ apparaat ........................................................................................................ 34
Structuur en fct ......................................................................................................................... 34
Vesikulair transport van ER nr het Golgi-apparaat ........................................................................ 34
Regio’s in golgi apparaat ............................................................................................................. 34
Eiwitglycosilatie......................................................................................................................... 34
Functionele verschillen tussen de Golgi-cisternen ....................................................................... 35
2 modellen voor het intra-Golgi transport .................................................................................... 35
sortering en export vanuit Golgi .................................................................................................. 35
overzicht secretie en endocytose (secretory en endocytic pathway).............................................. 35
Mechanismen in transporten ...................................................................................................... 36
Anterograad/ retrograad transport: recuperatie v lipiden .............................................................. 36
e) Cytosomen: Lysosomen ........................................................................................................ 36
De wegen naar het lysosoom ...................................................................................................... 36
Autofagocytose ......................................................................................................................... 36
Transport v golgi nr lysosoom ..................................................................................................... 36
Transport vh Golgi en plasmamembraan nr lysosoom .................................................................. 37
Lysosomale opslagziekten ......................................................................................................... 37
f) Cytosomen: Peroxisomen ...................................................................................................... 37
g) Extracellulaire vesikels (EVs) .................................................................................................. 38
Hoofdstuk 5: cytoskelet ...................................................................................................................... 38
a) Actine- of microfilamenten .................................................................................................... 38
In een oogopslag: ...................................................................................................................... 38
Typische fcts van actine-filamenten: ........................................................................................... 39
Actine-polymerisatie en soorten v actine .................................................................................... 39
Microfilament-bundeling in verschillende variaties ...................................................................... 40
Microvilli op basis v stabiele microfilamenten ............................................................................. 41
Het belang vh cytoskelet in celadhesie ....................................................................................... 41
Stappen in mobiliteit, lamellipodium .......................................................................................... 42
3
, Basisanatomie vd dwarsgestreepte skeletspier ........................................................................... 43
b) Intermediaire filamenten ....................................................................................................... 44
In een oogopslag: ...................................................................................................................... 44
fct en samenstelling .................................................................................................................. 44
Basis: ....................................................................................................................................... 44
Polymerisatie van monomeer tot filament ................................................................................... 44
IF-eiwitfamilies .......................................................................................................................... 44
Keratine expressie in epithelia .................................................................................................... 45
Weefselspecificiteit v keratine expressie ..................................................................................... 45
Rol van intermediaire-filamenten in cel-celadhesie ..................................................................... 45
c) Microtubuli ........................................................................................................................... 45
In een oogopslag: ...................................................................................................................... 45
Functies .................................................................................................................................... 46
Samenstelling ........................................................................................................................... 46
Verschijningsvormen ................................................................................................................. 46
Microtubulair voorkomen en polariteit in diverse celtypes/celstadia/celorganellen ........................ 46
Morfologie vh centrosoom: primaire MTOC in dierlijke cellen ........................................................ 47
Groei v microtubuli .................................................................................................................... 47
Dynamiek v microtubuli ............................................................................................................. 48
Structuur en dynamiek v microtubuli .......................................................................................... 48
Microtubulaire inhibitoren .......................................................................................................... 48
Transport in een axon: in vivo experiment .................................................................................... 48
Overzicht vd 2 types van microtubulaire motor-eiwitten ............................................................... 48
Intracellulair transport van organellen: associatie en dissociatie .................................................. 50
Coöperatieve rol v myosines en kinesines aan de celcortex .......................................................... 50
Contrasterende/coöperatieve rol v MT en MF geassocieerde motoreiwitten bij organeltransport ..... 50
Varianten van microtubulaire monomeren en superorganisaties ................................................... 50
Cilia en Flagellen ....................................................................................................................... 51
Centrosoom-duplicatie tijdens mitose ........................................................................................ 52
Stadia mitose: kritische rol vd spoelfiguur en centrosomen, beide MT ........................................... 52
Soorten cytoskeletten: opbouw en typisch IF-beeld ......................................................................... 53
Hoofdstuk 6: Celadhesie .................................................................................................................... 54
De belangrijkste juncties ................................................................................................................ 54
De verschillende intercellulaire juncties .......................................................................................... 54
Rol bij uitsortering van verschillende celtypes .................................................................................. 54
1. Intercell juncties vd gepolariseerde epitheelcel als representatieve organisatie ........................ 55
4
, 1.1. Tight junction / zonula occludens (ZO) ............................................................................ 55
1.2. Adherens junction (AJ) / zonula adherens (ZA) / belt desmosome ..................................... 55
Belang v cel-cel adhesie tijdens de embryonale ontwikkeling ....................................................... 55
E-cadherine .............................................................................................................................. 56
Model vo ontstaan v cel-cel adhesies.......................................................................................... 56
E-cadherine = invasie-supressor molecule .................................................................................. 56
1.3. Desmosomen of spot desmosomen of macula adherens ................................................ 57
1.4. Gap juncties / GJs ......................................................................................................... 58
Structuur v Gap junctie .............................................................................................................. 58
1.5. Nanobuisjes, cel nr cel communicatie ............................................................................ 58
2. Families van cel-cel adhesiemoleculen .................................................................................. 58
Cel-adhesie moleculen .............................................................................................................. 58
3. Inflammatie als illustratie vd belangrijke rol v intercellulaire adhesieprocessen ......................... 59
Rol selectines en integrines bij extravasatie v leukocyten ............................................................. 59
Rol diverse adhesiemoleculen bij stapsgewijze extravasatie van leukocyten .................................. 60
Hoofdstuk 7: extracellulaire matrix ...................................................................................................... 60
Chapter 20 in Lodish: cellen integreren in weefsels .............................................................................. 60
1. Cel-cel en cel-extracell matrix adhesie: overzicht ................................................................... 60
2. Cel-cel- en cel-extracell-matrixverbindingen en hun adhesiemoleculen................................... 61
Eiwitten vd extracellulaire matrix ................................................................................................ 61
Variatie inde relatieve dichtheid v cellen en ECM ......................................................................... 61
Functies v ECM .......................................................................................................................... 61
3. Extracellulaire matrix I: Basale lamina .................................................................................... 62
Belangrijke eiwitcomponenten vd basale lamina ......................................................................... 62
4. Extracellulaire matrix II: bindweefsel....................................................................................... 63
Biosynthese v fibrillaire collagenen door fibroblasten .................................................................. 63
Interacties v fibrillaire collagenen met fibrilgeassocieerde collagenen .......................................... 64
Glycosaminoglycanen (GAG’s) ................................................................................................... 64
Structuur v fibronectine (FN) ...................................................................................................... 64
Elastische en collageenvezels in bindweefsels ............................................................................ 65
5. Adhesieve interacties in beweeglijke en nt-beweeglijke cellen .................................................. 65
Integrines vormen vers complexen .............................................................................................. 65
Conformaties v integrines .......................................................................................................... 65
Dystrofine glycoproteïne complex ............................................................................................... 66
5
, Hoofdstuk 1 Prepareren en observeren van cellen en weefsels
Doel: cellen/weefsels observeerb maken vo LM of EM
- Probleem: weinig licht/e- doorlatend, weinig contrast in biol materiaal
- Methodes: fixeren, inbedden en snijden, kleuren of contrasteren
- Eerste cellen die groeien
Celcultuur, historisch
- Tissue culture: zenuw kikkerembryo in lymfe op glasplaatje met depressie 3D
- 2D monolaagcultures (Earle): simuleren 3D organen/weefsels, oorspronkelijk bestaan uit
orgaanexplanten in EMEM (Earle’s minimum essential medium)
- Later: stabiele cellijnen: onbeperkte levensduur, door tumorale oorsprong, door ‘crisis’ (selectie)
v cultures, die na verouderen sterven (senescense)
o Niet echt stabiel, na aantal keer splitsen veranderen ze toch
Stadia van celcultivering
Stukje orgaan/tumor: cellijn maken (onderzoek eenvoudiger maken zo)
Stappen:
- Weefsel enzymatich behandelen
- Cellen zakken of gecentrifugeerd nr bodem
- Uitgeplaat in een groeirecipient (bv petridish)
- Hopelijk overleven + uitgroeien, indien confluente laag v cellen, losmaken en
splitsen
Typische celculturen
Links: cellen sterk vergroot (witte lijn = celrand)
Rechts: lage vergroting, kolonie ° uit 1 cel, bestaat uit meerdere
cellen
Cultuurcondities
Cultuurcondities moeten juist zijn want cellen die groeien in vitro
moeten vglbaar zijn met die in vivo
MEM = minimum essential medium
Bestaat uit:
- Isotonische zouten
- Energiebron (glucose)
- Essentiële (nt zelf maakbaar) aminozuren + vitamines
- Serum (bijna essentieel, duur natuurproduct) (paar of kalfsserum)
Cultuurrecipiënten
- Gesloten flessen (falcons): CO2 toevoegen en dicht draaien (voor warme kamers)
- Open platen (voor in CO2 ovens)
- Rollerflessen in warme kamer voor groot opp met cellen maar weinig medium
- CO2 oven waar cultuurplaten in gaan (goede vochtigheid: incubator)
6
Celcultuur, historisch .......................................................................................................................6
Stadia van celcultivering ..............................................................................................................6
Typische celculturen ....................................................................................................................6
Cultuurcondities .........................................................................................................................6
Cultuurrecipiënten ......................................................................................................................6
Oorsprong v cellen, cellijnen ........................................................................................................7
Groeisubstraten en -procedures .......................................................................................................7
Doel............................................................................................................................................7
Celdensiteits-afhankelijke inhibitie ...............................................................................................7
Tumorcellen ................................................................................................................................7
Procedure v celtransfer ................................................................................................................7
Stappen bij celadhesie en -spreiding welke plaatsvinden na losmaken en uitplaten vd cellen ...............8
Apparatuur vo celcultuur ..............................................................................................................8
Vb van celcultuur-exp ..................................................................................................................8
Differentiatie in vitro ....................................................................................................................8
Stamcellen ......................................................................................................................................8
Wat is een stamcel? .....................................................................................................................8
Totipotent....................................................................................................................................8
Pluripotent ..................................................................................................................................8
Multipotent .................................................................................................................................8
Aanmaak muis embyronale stamcellen ........................................................................................8
Aanmaak humane embryonale stamcellen ...................................................................................9
Kwaliteitscontrole........................................................................................................................9
Zuiverheid, Homogeniteit en stabiliteit van celcultures ..................................................................9
Hoofdstuk 1 Prepareren en observeren van cellen en weefsels ............................................................. 10
a) Gebruik van secties nodig om subcellulaire details waar te nemen ........................................... 10
Fixeren ...................................................................................................................................... 10
Inbedden .................................................................................................................................. 10
Snijden ..................................................................................................................................... 10
Ontwikkeling van LM razend snel ................................................................................................ 11
Lichtmicroscopie (LM) ................................................................................................................... 11
Klassieke licht- of klaarveldmicroscoop: ..................................................................................... 11
Speciale lichtmicroscopen voor levende cellen ........................................................................... 12
Principes lichtmicroscopietechnieken ........................................................................................ 12
Kleuren, routine, hematoxyline en eosine (H/E) ................................................................................ 12
1
, Specifieke kleurtechnieken ........................................................................................................ 12
Fluorescentiemicroscoop .......................................................................................................... 13
Elektronenmicroscopie (EM)........................................................................................................... 14
Types van EM ............................................................................................................................. 14
TEM .......................................................................................................................................... 14
SEM .......................................................................................................................................... 15
Speciale EM-technieken ............................................................................................................ 15
Hoofdstuk 2: Bloed ............................................................................................................................ 15
Bloed, plasma, serum .................................................................................................................... 15
Cellen in het bloed ......................................................................................................................... 16
Rode bloedlichaampjes – erythrocyten ....................................................................................... 16
Leukocyten – witte bloedcellen................................................................................................... 18
Thrombocyten (bloedplaatjes) in het bloed ................................................................................. 20
Bloedvorming = hematopoiesis ....................................................................................................... 20
Hoofdstuk 24, MCB: Immuunsysteem ................................................................................................. 20
24.1 Overzicht van afweersystemen in gastheer ............................................................................... 20
3 lagen v immuunafweer v gewervelde dieren ........................................................................ 21
24.2 Immunoglobulines: structuur en functie .................................................................................. 21
De bloedsomloop en lymfatische systemen ................................................................................ 21
24.3 Verkrijgen van antilichaam diversiteit en B-cel ontwikkeling ...................................................... 22
Initiatie vd adaptieve immuunrespons in lymfeklieren .................................................................. 22
24.4 De MHC en antigen presentatie .............................................................................................. 22
24.5 T cellen, T-cel receptoren, en T-cel ontwikkeling ...................................................................... 22
Drie routes van complementactivering ........................................................................................ 22
Cellulaire afweer ........................................................................................................................... 24
Humorale afweer ........................................................................................................................... 24
Hoofdstuk 3: Organellen ..................................................................................................................... 26
a) Biomembranen ..................................................................................................................... 26
Vries-breek en Vries-ets technieken ............................................................................................ 27
Myelinehuls............................................................................................................................... 27
Biomembranen: verschillen van vorm ......................................................................................... 27
b) Mitochondriën....................................................................................................................... 27
Structuur .................................................................................................................................. 27
Functies .................................................................................................................................... 28
Evolutie vanuit bacteriën ............................................................................................................ 28
Fusie en fission.......................................................................................................................... 29
2
, Reacties.................................................................................................................................... 29
c) Endoplasmatisch reticulum ................................................................................................... 29
Eiwitsynthese ............................................................................................................................ 30
Topologie vd secretieweg ........................................................................................................... 30
Belangrijkste gebeurtenissen bij cotranslationeel import ............................................................. 30
Afsplitsing v signaalpeptide in RER door signaalpeptidase ............................................................ 31
Stappen in cotranslationeel import in RER................................................................................... 31
smooth endoplasmatisch reticulum (SER) .................................................................................. 33
d) Golgi complex/ apparaat ........................................................................................................ 34
Structuur en fct ......................................................................................................................... 34
Vesikulair transport van ER nr het Golgi-apparaat ........................................................................ 34
Regio’s in golgi apparaat ............................................................................................................. 34
Eiwitglycosilatie......................................................................................................................... 34
Functionele verschillen tussen de Golgi-cisternen ....................................................................... 35
2 modellen voor het intra-Golgi transport .................................................................................... 35
sortering en export vanuit Golgi .................................................................................................. 35
overzicht secretie en endocytose (secretory en endocytic pathway).............................................. 35
Mechanismen in transporten ...................................................................................................... 36
Anterograad/ retrograad transport: recuperatie v lipiden .............................................................. 36
e) Cytosomen: Lysosomen ........................................................................................................ 36
De wegen naar het lysosoom ...................................................................................................... 36
Autofagocytose ......................................................................................................................... 36
Transport v golgi nr lysosoom ..................................................................................................... 36
Transport vh Golgi en plasmamembraan nr lysosoom .................................................................. 37
Lysosomale opslagziekten ......................................................................................................... 37
f) Cytosomen: Peroxisomen ...................................................................................................... 37
g) Extracellulaire vesikels (EVs) .................................................................................................. 38
Hoofdstuk 5: cytoskelet ...................................................................................................................... 38
a) Actine- of microfilamenten .................................................................................................... 38
In een oogopslag: ...................................................................................................................... 38
Typische fcts van actine-filamenten: ........................................................................................... 39
Actine-polymerisatie en soorten v actine .................................................................................... 39
Microfilament-bundeling in verschillende variaties ...................................................................... 40
Microvilli op basis v stabiele microfilamenten ............................................................................. 41
Het belang vh cytoskelet in celadhesie ....................................................................................... 41
Stappen in mobiliteit, lamellipodium .......................................................................................... 42
3
, Basisanatomie vd dwarsgestreepte skeletspier ........................................................................... 43
b) Intermediaire filamenten ....................................................................................................... 44
In een oogopslag: ...................................................................................................................... 44
fct en samenstelling .................................................................................................................. 44
Basis: ....................................................................................................................................... 44
Polymerisatie van monomeer tot filament ................................................................................... 44
IF-eiwitfamilies .......................................................................................................................... 44
Keratine expressie in epithelia .................................................................................................... 45
Weefselspecificiteit v keratine expressie ..................................................................................... 45
Rol van intermediaire-filamenten in cel-celadhesie ..................................................................... 45
c) Microtubuli ........................................................................................................................... 45
In een oogopslag: ...................................................................................................................... 45
Functies .................................................................................................................................... 46
Samenstelling ........................................................................................................................... 46
Verschijningsvormen ................................................................................................................. 46
Microtubulair voorkomen en polariteit in diverse celtypes/celstadia/celorganellen ........................ 46
Morfologie vh centrosoom: primaire MTOC in dierlijke cellen ........................................................ 47
Groei v microtubuli .................................................................................................................... 47
Dynamiek v microtubuli ............................................................................................................. 48
Structuur en dynamiek v microtubuli .......................................................................................... 48
Microtubulaire inhibitoren .......................................................................................................... 48
Transport in een axon: in vivo experiment .................................................................................... 48
Overzicht vd 2 types van microtubulaire motor-eiwitten ............................................................... 48
Intracellulair transport van organellen: associatie en dissociatie .................................................. 50
Coöperatieve rol v myosines en kinesines aan de celcortex .......................................................... 50
Contrasterende/coöperatieve rol v MT en MF geassocieerde motoreiwitten bij organeltransport ..... 50
Varianten van microtubulaire monomeren en superorganisaties ................................................... 50
Cilia en Flagellen ....................................................................................................................... 51
Centrosoom-duplicatie tijdens mitose ........................................................................................ 52
Stadia mitose: kritische rol vd spoelfiguur en centrosomen, beide MT ........................................... 52
Soorten cytoskeletten: opbouw en typisch IF-beeld ......................................................................... 53
Hoofdstuk 6: Celadhesie .................................................................................................................... 54
De belangrijkste juncties ................................................................................................................ 54
De verschillende intercellulaire juncties .......................................................................................... 54
Rol bij uitsortering van verschillende celtypes .................................................................................. 54
1. Intercell juncties vd gepolariseerde epitheelcel als representatieve organisatie ........................ 55
4
, 1.1. Tight junction / zonula occludens (ZO) ............................................................................ 55
1.2. Adherens junction (AJ) / zonula adherens (ZA) / belt desmosome ..................................... 55
Belang v cel-cel adhesie tijdens de embryonale ontwikkeling ....................................................... 55
E-cadherine .............................................................................................................................. 56
Model vo ontstaan v cel-cel adhesies.......................................................................................... 56
E-cadherine = invasie-supressor molecule .................................................................................. 56
1.3. Desmosomen of spot desmosomen of macula adherens ................................................ 57
1.4. Gap juncties / GJs ......................................................................................................... 58
Structuur v Gap junctie .............................................................................................................. 58
1.5. Nanobuisjes, cel nr cel communicatie ............................................................................ 58
2. Families van cel-cel adhesiemoleculen .................................................................................. 58
Cel-adhesie moleculen .............................................................................................................. 58
3. Inflammatie als illustratie vd belangrijke rol v intercellulaire adhesieprocessen ......................... 59
Rol selectines en integrines bij extravasatie v leukocyten ............................................................. 59
Rol diverse adhesiemoleculen bij stapsgewijze extravasatie van leukocyten .................................. 60
Hoofdstuk 7: extracellulaire matrix ...................................................................................................... 60
Chapter 20 in Lodish: cellen integreren in weefsels .............................................................................. 60
1. Cel-cel en cel-extracell matrix adhesie: overzicht ................................................................... 60
2. Cel-cel- en cel-extracell-matrixverbindingen en hun adhesiemoleculen................................... 61
Eiwitten vd extracellulaire matrix ................................................................................................ 61
Variatie inde relatieve dichtheid v cellen en ECM ......................................................................... 61
Functies v ECM .......................................................................................................................... 61
3. Extracellulaire matrix I: Basale lamina .................................................................................... 62
Belangrijke eiwitcomponenten vd basale lamina ......................................................................... 62
4. Extracellulaire matrix II: bindweefsel....................................................................................... 63
Biosynthese v fibrillaire collagenen door fibroblasten .................................................................. 63
Interacties v fibrillaire collagenen met fibrilgeassocieerde collagenen .......................................... 64
Glycosaminoglycanen (GAG’s) ................................................................................................... 64
Structuur v fibronectine (FN) ...................................................................................................... 64
Elastische en collageenvezels in bindweefsels ............................................................................ 65
5. Adhesieve interacties in beweeglijke en nt-beweeglijke cellen .................................................. 65
Integrines vormen vers complexen .............................................................................................. 65
Conformaties v integrines .......................................................................................................... 65
Dystrofine glycoproteïne complex ............................................................................................... 66
5
, Hoofdstuk 1 Prepareren en observeren van cellen en weefsels
Doel: cellen/weefsels observeerb maken vo LM of EM
- Probleem: weinig licht/e- doorlatend, weinig contrast in biol materiaal
- Methodes: fixeren, inbedden en snijden, kleuren of contrasteren
- Eerste cellen die groeien
Celcultuur, historisch
- Tissue culture: zenuw kikkerembryo in lymfe op glasplaatje met depressie 3D
- 2D monolaagcultures (Earle): simuleren 3D organen/weefsels, oorspronkelijk bestaan uit
orgaanexplanten in EMEM (Earle’s minimum essential medium)
- Later: stabiele cellijnen: onbeperkte levensduur, door tumorale oorsprong, door ‘crisis’ (selectie)
v cultures, die na verouderen sterven (senescense)
o Niet echt stabiel, na aantal keer splitsen veranderen ze toch
Stadia van celcultivering
Stukje orgaan/tumor: cellijn maken (onderzoek eenvoudiger maken zo)
Stappen:
- Weefsel enzymatich behandelen
- Cellen zakken of gecentrifugeerd nr bodem
- Uitgeplaat in een groeirecipient (bv petridish)
- Hopelijk overleven + uitgroeien, indien confluente laag v cellen, losmaken en
splitsen
Typische celculturen
Links: cellen sterk vergroot (witte lijn = celrand)
Rechts: lage vergroting, kolonie ° uit 1 cel, bestaat uit meerdere
cellen
Cultuurcondities
Cultuurcondities moeten juist zijn want cellen die groeien in vitro
moeten vglbaar zijn met die in vivo
MEM = minimum essential medium
Bestaat uit:
- Isotonische zouten
- Energiebron (glucose)
- Essentiële (nt zelf maakbaar) aminozuren + vitamines
- Serum (bijna essentieel, duur natuurproduct) (paar of kalfsserum)
Cultuurrecipiënten
- Gesloten flessen (falcons): CO2 toevoegen en dicht draaien (voor warme kamers)
- Open platen (voor in CO2 ovens)
- Rollerflessen in warme kamer voor groot opp met cellen maar weinig medium
- CO2 oven waar cultuurplaten in gaan (goede vochtigheid: incubator)
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