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세포노화환경에서 단백질 분해 과정 조절에 관한 연구

Title
세포노화환경에서 단백질 분해 과정 조절에 관한 연구
Other Titles
The regulation of protein degradation in cellular senescence condition
Author
장지원
Alternative Author(s)
Jang, Ji Won
Advisor(s)
서혜명
Issue Date
2016-08
Publisher
한양대학교
Degree
Master
Abstract
세포노화는 노화과정에서 여러 기관에서 생물학적인 기능장애를 설명할 수 있는 것으로 알려져 있다. 이전 연구에 따르면 세포노화가 단백질 분해 능력에 중요한 역할 중 하나로 여겨지고 있다. 본 연구에서는 여러 세포노화환경에서 in vitro 노화 연구의 실험적인 환경을 찾고, 그 환경에서의 단백질 분해과정 조절을 연구하고자 하였다. 먼저 Neuro 2a (N2a) 세포에서 세포노화환경으로 저산소 조건, 장기계대배양, cobalt chloride 처리와 hydrogen peroxide 처리를 각각 시행하였다. 여러 세포노화환경에서 세포노화환경의 표지로서 세포생존율과 노화 인자로 알려진 저산소인자-1 알파 (Hypoxia inducible factor-1 alpha, HIF-1α)의 mRNA가 유의성있게 감소하는 것을 확인하였다. 저산소 조건과 장기계대배양에서 단백질분해효소복합체의 키모트립신 유사 활성도가 유의성 있게 감소되었으며, 유비퀴틴이 결합된 녹색형광단백질 기반 분석법을 통하여 유비퀴틴 관련 단백질분해효소복합체인 PA700의 활성도가 증가됨을 확인하였다. 네 가지 세포노화환경에서 자가포식의 표지로서 자가포식포를 형성하는 microtubule-associated protein 1 light chain 3 (LC3)의 단백질 변화를 확인하였다. 또한, 저산소 조건에서 콜린성 뉴런의 세포 크기가 유의성 있게 감소하는 것을 관찰할 수 있었다. 세포노화환경에서 선택한 저산소 조건에서 단백질 분해과정을 조절하여 콜린성 신경세포가 보호됨을 관찰하기 위하여, E3 유비퀴틴 연결효소의 구성요소인 parkin을 N2a 세포에서 과발현시켰다. 저산소 환경에서 Parkin을 과 발현 시켰을 때 세포생존율을 향상시키지 않았다. 그러나 저산소 환경에서 Parkin 과발현이 HIF-1α mRNA의 양을 증가시켰다. 저산소 조건에서 Parkin 과발현에 의하여 키모트립신 유사 활성도와 PA700의 단백질분해효소복합체 활성도를 유의성있게 증가시켰다. 또한 저산소 조건에서 Parkin 과발현이 세포 크기와 신경돌기의 수, 길이를 유의성있게 증가시켰다. 본 연구를 통하여 여러 세포노화환경이 콜린성 뉴런에서 단백질분해 기능을 변화시켰으며 Parkin에 의하여 단백질분해효소복합체 시스템을 변화시켰을 때 저산소 조건에서 콜린성 뉴런이 보호될 수 있음을 확인하였다. | Cellular senescence explains biological malfunction of an organism under aging process. Previous studies have suggested that protein degradation is one of the critical processes of cellular senescence. In this study, we aimed to determine the experimental conditions of in vitro senescence research and to find the regulation of protein degradation in designated senescence condition. First, we examined four different conditions of senescence for neuro-2a (N2a) cells: hypoxia, increase of cell culture passage number, cobalt chloride, or hydrogen peroxide. Under these senescence conditions, we observed the significant decrease of cell viabilities and mRNA expression of hypoxia-inducible factor-1 alpha (HIF-1α) as a marker of cellular senescence. We found significant decrease of chymotrypsin-like proteasome activity in the conditions of hypoxia and increasing passage number and significant increase of ubiquitin-dependent PA700 proteasome activities as shown the degradation of GFP conjugated protein substrate. We found the significant change of microtubule‐associated protein 1 light chain 3 (LC3) protein levels as an autophagy marker in senescence conditions. In addition, we found the significant decrease of cell body areas of cholinergic neurons under hypoxia conditions. To determine the protective effects of the regulation of protein degradation, we overexpressed E3 ubiquitin ligase, parkin in cholinergic neurons. We found that parkin overexpression did not improve cell viability under hypoxia condition. However, parkin overexpression increased the level of HIF-1α mRNA in hypoxia condition. Parkin significantly increased chymotrypsin-like proteasome activities and ubiquitin dependent PA700 proteasome activities in hypoxia condition. In addition, we found significant increase of cell body area, neurite length and neurite number of cholinergic neurons after parkin overexpression in hypoxia condition. Taken together, these data suggest that cellular senescence alters protein degradation function in cholinergic neurons and the modification of proteasome system by parkin can protect cholinergic neurons in hypoxia condition.; Cellular senescence explains biological malfunction of an organism under aging process. Previous studies have suggested that protein degradation is one of the critical processes of cellular senescence. In this study, we aimed to determine the experimental conditions of in vitro senescence research and to find the regulation of protein degradation in designated senescence condition. First, we examined four different conditions of senescence for neuro-2a (N2a) cells: hypoxia, increase of cell culture passage number, cobalt chloride, or hydrogen peroxide. Under these senescence conditions, we observed the significant decrease of cell viabilities and mRNA expression of hypoxia-inducible factor-1 alpha (HIF-1α) as a marker of cellular senescence. We found significant decrease of chymotrypsin-like proteasome activity in the conditions of hypoxia and increasing passage number and significant increase of ubiquitin-dependent PA700 proteasome activities as shown the degradation of GFP conjugated protein substrate. We found the significant change of microtubule‐associated protein 1 light chain 3 (LC3) protein levels as an autophagy marker in senescence conditions. In addition, we found the significant decrease of cell body areas of cholinergic neurons under hypoxia conditions. To determine the protective effects of the regulation of protein degradation, we overexpressed E3 ubiquitin ligase, parkin in cholinergic neurons. We found that parkin overexpression did not improve cell viability under hypoxia condition. However, parkin overexpression increased the level of HIF-1α mRNA in hypoxia condition. Parkin significantly increased chymotrypsin-like proteasome activities and ubiquitin dependent PA700 proteasome activities in hypoxia condition. In addition, we found significant increase of cell body area, neurite length and neurite number of cholinergic neurons after parkin overexpression in hypoxia condition. Taken together, these data suggest that cellular senescence alters protein degradation function in cholinergic neurons and the modification of proteasome system by parkin can protect cholinergic neurons in hypoxia condition.
URI
https://repository.hanyang.ac.kr/handle/20.500.11754/125910http://hanyang.dcollection.net/common/orgView/200000486865
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GRADUATE SCHOOL[S](대학원) > MOLECULAR & LIFE SCIENCE(분자생명과학과) > Theses (Master)
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