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세포의 열에 대한 반응시 생기는 생화학적 변화와 apoptosis에 관한 연구

Title
세포의 열에 대한 반응시 생기는 생화학적 변화와 apoptosis에 관한 연구
Other Titles
Studies on the biochemical changes and apoptosis induced by cellular heat shock responses
Authors
朴全義
Issue Date
1996
Department/Major
대학원 약학과
Keywords
세포apoptosis생화학반응변화
Publisher
이화여자대학교 대학원
Degree
Master
Abstract
세포가 mild heat에 노출되었다가 적당한 시간 회복되면 이들은 thermotolerance해지고 heat, oxidative stress, heavy metal, amino acid analogue와 같은 두번째 stress에도 저항성을 가지게 된다. 이러한 thermotolerance는 세포의 죽음을 이해하는데 아주 좋은 system이며 여기에는 heat shock protein이라 부르는 일련의 protein들이 관여한다고 생각되고 있다. 또한 heat에 의해서는 apoptosis라 불리는 programmed cell death가 일어나는데, 이는 necrosis와 구별되는 세포의 죽음으로 세포의 분화, 발생, 항상성 유지에 중요한 역할을 하며, 이것에 대한 연구는 암세포의 사멸, autoimmune disease, neurodegenerative disease, AIDS와 같은 질병에 대한 이해와 치료에 중요한 역할을 하리라 생각된다. 본 논문에서는 세포의 이러한 열에 대한 반응에서 생기는 생화학적인 변화를 살펴보았고, apoptosis를 일으키는 여러 stress에 대해 thermotolerant cell이 apoptosis를 억제하는지를 살펴보았다. Mouse fibroblast cell인 NIH-3T3 cell을 45℃에서 20분 heating 후 37℃에서 24시간 회복시켜 thermotolerant cell을 만들었으며, 이 세포는 heat, diamide와 같은 oxidative stress, methotrexate (MTX)와 같은 anticancer drug에 저항성을 가짐을 cell survival로 확인하였다. 세포에 heat을 주면 단백질 합성이 처음에는 억제되었다가 hsp의 합성이 먼저 시작되면서 다른 단백질들의 합성이 일어나는 것을 [^(35)S]-methionine을 사용하여 합성되는 단백질을 label하여 확인하였으며, hsp70 antibody로 Western analysis 해본 결과 heat과 diamide에 의해서 hsp70이 induction됨을 확인하였다. Heat, diamide, MTX에 의해 NIH-3T3 cell이 apoptosis를 일으킴을 DNA agarose gel, 형광 현미경으로 확인하였으며, 이 현상이 thermotolerant cell에서 억제됨을 DNA agarose gel과 thymidine incorporation으로 확인하였다. 또한 세포 밖에서 넣어 주었던 thymidine이 세포 내로 들어갔다가 heat에 의해 세포 밖으로 excretion 되는 것을 관찰하였으며, 이것은 thermotolerant cell을 다시 heating 했을 때 더 심화됨을 관찰하였다. Thermotolerant cell을 좀 더 characterize 하기 위해 thermotolerant cell에 두 번째 heat을 주고 회복 시간에 따른 단백질 합성 pattern을 [^(35)S]-methionine으로 pulse label하여 control cell과 비교해 보았으며, 이 때 heat shock protein의 양도 hsp70 antibody를 사용하여 Western analysis로 비교해 본 결과, thermotolerant cell은 stress가 와도 이것을 mild하게 인지하므로서 저항성을 가지는 것으로 생각되었다. 이러한 반응 외에 암전이의 억제에 관여한다고 알려진 ㎚23 H1, H2의 mRNA level이 heat, starvation과 같은 stress에 의해 변화함을 Northern analysis로 관찰하였으며, ㎚23 H1 mRNA level은 stress에 의해 양이 감소하였다가 다시 증가했으나, ㎚23 H2 mRNA level은 ㎚23 H1 보다 변화가 적은 것으로 나타났다.;When cells are exposed to mild heat and recovered at 37℃ for various times, they are thermotolerant and resistant to second stresses (heat, oxidative stress, heavy metal, amino acid analogues). Thermotolerance (TT) is a important system for the understanding of the cell death mechanism and the function of heat shock proteins. Apoptosis is a programmed cell death distinguished from necrosis, an important process in cell differentiation, development and homeostasis and also induced by various external stresses including cytokines, viral infection, anticancer dugs, heat and etc. The understanding of the apoptosis gives the clues for therapy of cancer, autoimmune disease, neurodegenerative disease and AIDS. In this study, thermotolerance system was adopted for investigating the cell death by heat, oxidative stress by diamide and anticancer drug MTX treatment. The obtained results were that the thermotolerance induced by mild heat prevented the cell death which was confirmed by DNA fragmentation and chromatin condensation. To explain the mechanism of the resistance of apoptosis in thermotolerant cells, the biochemical changes of thermotolerant cells were examined. Thermotolerant cells were prepared with mild heating at 45℃ for 20 min and recover at 37℃ for 24 hr, and were resistant to heat, diamide and MTX in terms of cell survival. When cells were exposed to heat, the protein synthesis was stopped immediately and slowly recovered : hsp's were appeared at first and the normal protein synthesis patterns were returned afterward. These phenomena were investigated with [^(35)S]-methionine pulse labeling experiment and Western analysis using monoclonal antibody against hsp70. In thermotolerant cells, accumulation of hsp's is an apparent phenomena, however, the function of hsp's in thermotolerant cells should be further studied. Cellular membrane transport changes in thermotolerant cells were investigated with [^(3)H]-thymidine incorporation study. Pumping out of [^(3)H]-thymione by heat in thermotolerant cells was 4 fold greater than that in control cells. This can be a clue of membrane changes in thermotolerant cells, however, the more examination should be peformed. To examine the mechanisms of thermotolerance in cells, the protein synthesis pattern and hsp70 level of control and thermotolerant cells were compared after treatment with the same amount of stress. It turned out that thermotolerant cells had less sensitive to stresses than control cells. This molecular mechanism of resistance to apoptosis in thermotolerant cells should be further investigated. We measure mRNA levels of nm23 H1 and H2 which is known to be associated with cancer metastasis after heating and starvation, the results show nm23 H1 level was changed after stress and recovered, but nm23 H2 level wasn't changed prominently.
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