» arhiv
Možnosti:
[Prikaži v obliki za tiskanje]
[Naloga še ni vpisana v zbirko Cobiss]
ID naloge: 163 Letnik: 2003 Predmet: patologija in histologija
POVECANO IZRAŽANJE KASPAZE-9 SPROŽI PROCES APOPTOZE IN VIVO
Avtor: Mirjam Druškovic
Mentor: doc. dr. Irina Milisav
IZHODIŠCE: Apoptoza ali programirana celicna smrt je proces pri katerem celice aktivno podležejo samounicenju. S procesom apoptoze se v organizmu uravnavata število in kakovost celic. Okvare regulacije se odražajo v številnih patoloških stanjih, kot so npr. maligna obolenja in nevrodegenerativne bolezni. Pri proženju in izvedbi procesa imajo kljucno vlogo specificne proteaze-kaspaze, ki se aktivirajo s cepljenjem neaktivne oblike- prokaspaze.
NAMEN: Kaspaza-9 je osrednji prožilec procesa apoptoze, saj lahko npr. po ishemiji tkiva sproži proces apoptoze ali pa ga ojaca, ce se je zacel z aktivacijo drugih kaspaz. Za razliko od ostalih kaspaz lahko povecana koncentracija kaspaze-9 v razmerah in vitro sproži njeno aktivacijo. V tej nalogi smo želeli preveriti ali lahko samo povecanje koncentacije kaspaze-9 sproži proces celicne smrti v živih celicah.
HIPOTEZE: Postavili smo naslednje hipoteze:
1. Povecano izražanje kaspaze-9 sproži celicno smrt.
2. Celice odmrejo v procesu apotoze.
3. Proženje apoptoze s povecanim izražanjem kaspaze-9 poteka pri normalnih in pri maligno transformiranih celicah.
METODE: Pri opazovanju apoptoze v celicnih kulturah smo uporabili celicne kulture podganjih melanotrofov in astrocitov ter humanih celic HeLa. Nato smo kaspazo-9 oznacili s fluorescencno beljakovino EGFP, ki smo jo v celico vnesli kot konstrukt DNA. V celicah se je DNA prepisala v fuzijsko beljakovino, ki je fluorescirala. Fluorescenco smo zaznavali s konfokalnim mikroskopom. Število apoptotskih in nekrotskih celic smo dolocali z barvanjem s fluorescencno oznacenim aneksinom V in z barvilom Sytox Green ter nato izracunali delež apoptotskih celic glede na vse celice na krovniku. V vseh vzorcih smo upoštevali vsaj 5 neodvisnih poskusov (n=5), ki smo jih statisticno obdelali z dvosmernim neparametricnim Studentovim t-testom.
REZULTATI: Po vnosu konstrukov v celice se je zmanjšalo število fluorescencnih celic v primeru vnosa konstrukta kaspaze-9 oznacene z zeleno fluorescencno beljakovino (EGFP) v primerjavi s kontrolno skupino pri vseh celicnih kulturah. Po dolocanju števila apoptotskih in nekrotskih celic, smo ugotovili, da je vecina celic umrla v procesu apoptoze in ne nekroze. Kolicina izražene kaspaze-9 je sorazmerna s številom apoptotskih celic.
ZAKLJUCKI: Potrdili smo vse zastavljene hipoteze.
1. Povecano izražanje kaspaze-9 sproži celicno smrt.
2. Celice odmrejo v procesu apoptoze in ne nekroze po povecanem izražanju kaspaze-9.
3. Proženje apoptoze s povecanim izražanjem kaspaze-9 poteka v primarnih celicnih kulturah normalnih podganjih melanotrofov in astrocitov, ohranjeno pa je tudi pri celicah HeLa, ki so maligno transformirane.
«»
[Abstract / English version]
POVECANO IZRAŽANJE KASPAZE-9 SPROŽI PROCES APOPTOZE IN VIVO
Author: Mirjam Druškovic
Mentor: doc. dr. Irina Milisav
BACKGROUND: Apoptosis or programmed cell death is a process by which the cells are actively destroyed. It controls the number and the quality of the cells. Malfunctions of the apoptosis are manifested in malignancies and in neurodegenerative disorders. Caspases are proteases, which have a central role in triggering and executing apoptosis. They are synthesized as inactive procaspases and are activated through proteolytic processing.
AIM: Caspase-9 is the central activator of apoptosis (e.g. after the tissue ischemia) or it can amplify the apoptosis, when it has been started by other caspases. It was shown that the increased concentration of caspase-9 can induce its activation in vitro. Our goal was to find out whether the increased concentration of caspase-9 triggered the cell death in vivo.
HYPOTHESES: The hypotheses were:
1. Over expression of caspase-9 triggered the cell death.
2. The cells dyed from apoptosis.
3. The apoptosis because of the caspase-9 over expression was triggered in normal and malignantly transformed cells.
METHODS: To observe the process of apoptosis in cultured mammalian cells we used the primary cultures of rat melanotrophs and astrocytes and the human cell line HeLa. The cells were transfected with the DNA encoding the fluorescent fusion protein between the caspase-9 and green fluorescent protein (EGFP). The fluorescent cells were detected by the confocal microscope. We determined the number of apoptotic and necrotic cells for fluorescent and non-fluorescent cells by a fluorescent analogue of annexin V and a dye Sytox Green. Then we calculated the percentage of apoptotic cells in relation to all cells on cover slips. For all samples we performed at least 5 independent experiments (n=5) and subjected the results to the statistical analysis by Student's t-test.
RESULTS: Over expression of caspase-9 reduced the number of cells in comparison to the cells over expressing cytochrome c. Almost all cells dyed of apoptosis. The number of apoptotic cells was proportional to the levels of over expression of caspase-9.
CONCLUSIONS: We confirmed all the hypotheses.
1. Over expression of caspase-9 triggers cell death.
2. Cells die through apoptosis and not necrosis.
3. The apoptosis due to the caspase-9 over expression is triggered in normal and malignantly transformed cells.
|
|
 |
Išči po nalogah
Brskaj po nalogah
|
