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Effect and mechanism of reduced glutathione treatment on lipopolysaccharide-induced cellular injury : 내독소유발 세포손상에 대한 환원형 글루타치온 투여의 효과 및 작용 기전

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dc.contributor.advisor권운용-
dc.contributor.author김병현-
dc.date.accessioned2017-07-19T10:38:19Z-
dc.date.available2017-07-19T10:38:19Z-
dc.date.issued2017-02-
dc.identifier.other000000142627-
dc.identifier.urihttps://hdl.handle.net/10371/132958-
dc.description학위논문 (석사)-- 서울대학교 대학원 : 의학과, 2017. 2. 권운용.-
dc.description.abstract활성산소물질(reactive oxygen species)의 과량 발생에 의한 산화스트레는 패혈증에서 세포의 손상 및 사멸을 일으키고 결과적으로 다발성 장기손상을 초래하게 되어 패혈증 환자의 사망률을 높이는 원인 중 하나이다. 환원형 글루타치온 (reduced glutathione, GSH)은 자연적으로 존재하는 트리 펩타이드(glutamyl–cysteinyl -glycine)로 체내에서 글루타치온 산화 환원 싸이클(glutathione redox cycle)을 통하여 과산화수소를 제거하는 대표적인 항산화물질이다. 본 연구는 사람 폐미세혈관 내피세포에 내독소(lipopolysaccharide)를 처리하여 인위적으로 세포에 산화스트레스를 만든 후 글루타치온의 항산화효과로 인한 세포 보호효과를 측정할 목적으로 시행되었다. 10 μg/mL의 내독소를 처리한 세포에 각각 0, 0.4, 0.8mM 의 글루타치온을 처리 후 12시간 뒤 환원형 글루타치온(GSH), 산화형 글루타치온(GSSG), 환원형/산화형 글루타치온 비, 과산화수소농도 및 nuclear factor (NF)-κB p65 DNA -binding activity 측정하였다. 또한 세포 내 NF-κB 분자작용신호에 관련된 세포질 내 phosphorylated inhibitor κB-α (p-IκB-α), phosphorylated Akt (p-Akt), 및 핵내 nuclear factor erythroid 2-related factor 2 (Nrf2) 발현 정도를 측정하였다. 처리한 글루타치온의 농도가 높아짐에 따라 배양된 세포의 글루타치온 농도 및 환원형/산화형 글루타치온 비가 상승하였고, 세포 생존률은 상승하였다. 이와 더불어 Akt 생존분자작용신호에 관계된 세포질 내 p-Akt 및 핵 내 Nrf2 발현이 촉진되며 염증관련 사이토카인이 줄어드는 것을 관찰 할 수 있었다. 결론적으로 환원형 글루타치온은 내독소 투여로 비롯된 세포의 산화손상을 지연시키며 이러한 항산화효과의 분자수준 작용기전은 Akt-Nrf2 경로의 활성화와 관련되어 있었다.-
dc.description.abstractReactive oxygen species (ROS) are chemically reactive materials including radical and non-radical oxygen species formed by the partial reduction of oxygen. Oxidative stress caused by excess ROS is one of many mechanisms by which multi-organ failure occurs in sepsis. Reduced glutathione (GSH) is a tripeptide formed by glutamic acid, cysteine, and glycine, which acts as an antioxidant via the glutathione redox cycle in eliminating hydrogen peroxide (H2O2). This study aimed to prove that GSH has protective effects against oxidative stress in the lipopolysaccharide (LPS)-exposed cell. We induced oxidative stress in human lung microvascular endothelial cells (HMVEC-L) by incubating them with various concentrations of LPS. In 10 μg/mL LPS-exposed cells, cell viability and the dose related effects of GSH were assessed 24 hours after treatment with 0, 0.4, and 0.8 mM of GSH, respectively. After a twelve hour incubation period, GSH level, glutathione disulfide (GSSG) level, the GSH/GSSG ratio, and H2O2 level were measured. For measuring the effects of GSH on intracellular protective mechanisms against oxidative stress, we measured expression levels of phosphorylated inhibitor κB-α (p-IκB-α), cytoplasmic phosphorylated Akt (p-Akt), and nuclear factor erythroid 2–related factor 2 (Nrf2) using western blots. As the concentration of GSH increased, the intra cellular GSH level and the GSH/GSSG ratio increased along with a decreasing level of H2O2 and proinflammatory cytokines. Cell viability increased as GSH concentration increased and this result was associated with an increase of cytoplasmic p-Akt expression and Nrf2 expression. In conclusion, GSH treatment attenuated LPS-induced cell injury by up-regulating Nrf2 signaling in the Akt survival pathway.-
dc.description.tableofcontentsI. Introduction 1
II. Materials and Methods 3
2.1 Materials 3
2.2 Clinically Relevant Doses of reduced Glutathione 3
2.3 Cell Culture and Drug Treatment 3
2.4 Antioxidant and Oxidative Stress Measurements 4
2.5 Nuclear and Cytoplasmic Extracts 4
2.6 Real-Time Polymerase Chain Reaction 5
2.7 NF-κB p65 DNA–Binding Activity Measurement 5
2.8 Western Blotting 6
2.9 Statistical Analysis 6
III. Results 7
3.1 Toxicity of LPS on HMVEC-L 7
3.2 Dose Related Effect of GSH on LPS-Exposed Cells 9
3.3 Antioxidant and Oxidative Stress in LPS-Exposed Cells 11
3.4 Intracellular Proinflammatory Cytokine Levels 13
3.5 Intracellular Signaling in LPS-Exposed Cells 15
IV. Discussion 18
References 21
요약(국문초록) 26
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dc.formatapplication/pdf-
dc.format.extent674982 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subject글루타치온-
dc.subject글루타치온 산화환원 싸이클-
dc.subject활성산소물질-
dc.subject산화스트레스-
dc.subjectAkt survival pathway-
dc.subjectNrf2-
dc.subject.ddc610-
dc.titleEffect and mechanism of reduced glutathione treatment on lipopolysaccharide-induced cellular injury-
dc.title.alternative내독소유발 세포손상에 대한 환원형 글루타치온 투여의 효과 및 작용 기전-
dc.typeThesis-
dc.contributor.AlternativeAuthorByunghyun Kim-
dc.description.degreeMaster-
dc.citation.pages27-
dc.contributor.affiliation의과대학 의학과-
dc.date.awarded2017-02-
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