| Mitochondrial Dysfunction and Apoptosis Related Gene Expression in A beta(25-35)-Treated Human Neuroblastoma Cell Line, SK-N-SH |
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Young Sook Choi, Sang Ho Kim |
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Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul, Korea. complt@catholic.ac.kr |
| 사람 신경모세포종에서 아밀로이드베타(Aß25-35) 투여 후 사립체기능 장애와 세포자멸사 관련 유전자들의 발현 |
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| Abstract |
BACKGROUND
Mitochondrial dysfunction plays an important role in Abeta-induced neuronal toxicity in Alzheimer's disease (AD). We measured the membrane potentials of mitochondria (delta psim) and assessed the genetic expressions of A beta(25-35)-induced neurotoxicity in the human neuroblastoma cell line, SK-N-SH cell.
METHODS
SK-N-SH cells were incubated with a single dose of 25 micrometer A beta(25-35) for 0-24 hours, and kinetic study was done. delta psim was measured by flow cytometry. Messenger RNA expressions of cytochrome c oxidase (COX), cytochrome c, succinate dehydrogenase (SDH), amyloid-beta alcohol dehydrogenase (ABAD), caspase 9, and Bcl-2 were measured by quantitative real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR). Cell death rate was measured by MTT reduction assay.
RESULTS
delta psim was reduced at 24 hours. mRNA expression for COX gradually decreased by about 29% (p<0.05) while-expressions for cytochrome c, SDH, ABAD, and caspase 9 increased (p<0.05) progressively during the 24-hour time period. Bcl-2 expression decreased (p<0.05) gradually; and apoptotic cell death rate was about 24% (p<0.01) by 24 hours.
CONCLUSION
Extracellular administration of A beta(25-35) contributes directly to mitochondrial dysfunction in SK-N-SH cells with the enzymatic impairment of the tricarboxylic acid cycle and electron transport chain, and eventually leading to apoptotic cell death. |
| Key Words:
Amyloid beta-protein, Apoptosis, Caspase 9, Cytochrome c, Electron transport complex IV, Membrane potentials, Mitochondria |
| 초 록 |
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연구배경: 사립체 기능부전은 알츠하이머병에서 Aβ에 의하여 유도되는 신경세포독성에 중요한 역할을 담당한다. 본 연구에서는 사람 신경모세포종 세포주인 SK-H-SH에서 Aβ 투여에 의한 사립체 기능과 관련 유전자 및 세포자멸사 유전자들의 발현 정도를 측정하였다. 방법: SK-N-SH에 25 μM의 Aβ25-35를 투여한 후 24시간째에 사립체의 막전위(△Ψm)변동을 flow cytometry로 측정하였고, COX, cytochrome c, SDH, ABAD, caspase 9 및 Bcl- 2 유전자의 발현 정도를 실시간 역전사 중합효소반응으로 0-24시간까지 kinetic study로 정량하였다. MTT reduction assay로 세포생존율을 측정하였다. 결과: △Ψm은 24시간째에 현저히 감소하였다. COX mRNA는 24시간까지 지속적으로 감소하였으며(p<0.05), cytochrome c, SDH, ABAD 및 caspase 9 mRNA는 24시간까지 지속적으로 그 발현이 각각 증가(p<0.05)하였다. Bcl-2 의 발현은 24시간까지 서서히 감소(p<0.05)하였다. 세포사망률은 최대 24%이었다(p<0.01). 결론: 세포 밖 Aβ25-35 투여는 SK-N-SH 세포 내 사립체 기능을 직접 방해하여 세포자멸사를 유발함을 확인할 수있었다. |
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