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학제간연구 과학기술학 뇌인지과학 기술정책 기타 복합학

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논문 기본 정보

자료유형
학위논문
저자정보

김지항 (강원대학교, 강원대학교 대학원)

발행연도
2016
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강원대학교 논문은 저작권에 의해 보호받습니다.

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Current diabetic therapies are based on many medications, such as synthetic drugs and hypoglycemic agents. However, it has frequently side effects and high cost. For this reason, there is a continuous need to develop new and better pharmaceuticals as alternatives for the management and treatment of the disease. Therefore, it is necessary to investigate natural antidiabetic material without side effects. Mushroom has been used extensively as a folk remedy in the world. Among them, it is known that the mushroom Inonotus obliquus has antioxidant pigments. It will protect beta-cells, because of reduced reactive oxygen species (ROS). The purpose of this study was to elucidate the cytoprotective effects of a polysaccharide isolated from Inonotus obliquus, which was extracted from the fruiting body of I. obliquus (PFIO) and liquid culture broth of I. obliquus (PLIO). The cytoprotective effects of PFIO and PLIO on H2O2-induced apoptosis of RINm5F pancreatic β-cells were comparatively investigated using an MTT assay, immunofluorescent staining, flow cytometry, and western blot analyses in vitro. The results showed that PFIO and PLIO treatment decreased DNA fragmentation and the rate of apoptosis. In addition, pretreatment of cells with PFIO and PLIO before H2O2 exposure resulted in increased insulin secretion and scavenging activity for intracellular ROS compared with treatment with H2O2 alone. Thus, the results of this study suggest that PFIO and PLIO may have protective effects against H2O2-induced oxidative stress via regulation of MAPKs, NF-κB, and apoptotic protein, and have potential merit as a medicinal food for the prevention of diabetes.
#초경량 무인비행장치 #무인비행기 #드론 #공간정보 #정사영상 #3D 모델

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CONTENTS
Abstract ----------------------------------------------------------------- ⅰ
Contents ----------------------------------------------------------------- ⅲ
List of Figures ---------------------------------------------------------- vi
Abbreviations ----------------------------------------------------------- ix
Ⅰ. Introduction ------------------------------------------------------- 1
1. 1. Introduction to the reactive oxygen species (ROS) --- 1
1. 2. Introduction to the Inonotus obliquus ------------------- 3
1. 3. Introduction to the diabetes mellitus ------------------- 4
1. 4. The objectives in this study ----------------------------- 6
Ⅱ. Materials and Methods ------------------------------------------- 10
2. 1. Materials ---------------------------------------------------- 10
2. 2. Sample preparation ----------------------------------------- 11
2. 2. 1. Extraction procedure of polysaccharide from fruiting body of I. obliquus ------------------------- 11
2. 2. 2. Extraction procedure of polysaccharide from liquid culture broth of I. obliquus ------------------
11
2. 3. Cell culture and treatments -------------------------------- 14
2. 3. 1. Culture condition ------------------------------------- 14
2. 3. 2. Hydrogen peroxide treatment ---------------------- 14
2. 4. Cell viability assay------------------------------------------ 14
2. 4. 1. Cytotoxicity of PFIO and PLIO --------------------- 15
2. 4. 2. Cytotoxicity of hydrogen peroxide ---------------- 15
2. 4. 3. Protective effect analysis --------------------------- 15
2. 5. Inhibitory effect of oxidative stress induced apoptosis- 16
2. 5. 1. Intracellular ROS scavenging activity ------------- 16
2. 5. 2. Anti-apoptotic efficacy ----------------------------- 16
2. 5. 2. 1. Annexin V/propidium iodide staining -------- 16
2. 5. 2. 2. Hoechst 33342 staining ---------------------- 17
2. 5. 2. 3. Caspase-3 activity ---------------------------- 18
2. 6. Western blot analysis -------------------------------------- 18
2. 6. 1. Western blot ----------------------------------------- 18
2. 6. 2. Nuclear protein extraction -------------------------- 19
2. 6. 3. Preparation of fractional proteins ------------------ 19
2. 7. Insulin secretion analysis ---------------------------------- 19
2. 8. Statistical analysis ------------------------------------------ 20
Ⅲ. Results and Discussion ------------------------------------------ 21
3. 1. Optimal concentration of PFIO, PLIO, and H2O2 in RIN m5F β-cells and protective effects on cell viability---
21
3. 1. 1. Cytotoxicity of PFIO and PLIO --------------------- 21
3. 1. 2. Cytotoxicity of hydrogen peroxide ---------------- 21
3. 1. 3. Cyto-protective effect of PFIO and PLIO -------- 27
3. 2. Anti-oxidative effects of PFIO and PLIO --------------- 27
3. 3. Anti-apoptosis effects of PFIO and PLIO --------------- 33
3. 3. 1. Hoechst 33342 staining ----------------------------- 33
3. 3. 2. Annexin V/PI staining ------------------------------- 33
3. 4. Western blot analysis ------------------------------------ 36
3. 4. 1. Expression of MAPKs and apoptotic-associated protein by PFIO and PLIO -------------------------
36
3. 4. 2. Activation of caspase-3 by PFIO and PLIO ------ 41
3. 4. 3. Translocation of NF-κB by PFIO and PLIO ----- 41
3. 5. Insulin secretion -------------------------------------------- 45
3. 5. 1. Effect of PFIO and PLIO on H2O2-induced insulin secretion ---------------------------------------------- 45
Ⅳ. Conclusion -------------------------------------------------------- 47
References -------------------------------------------------------------- 49
Abstract in Korean ----------------------------------------------------- 59

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