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I. 서론 13
II. 연구배경 16
1. 알츠하이머 16
2. 알츠하이머 발병 원인 16
가. Amyloid 전구 단백질(Amyloid precusor protein; APP) 17
나. Presenilin(PS) 17
다. Apolipoprotein E4 (ApoE4) 18
3. 알츠하이머와 혈관성 질환 18
III. 연구 재료 및 방법 20
1. PC12 cell 20
가. PC12 cell의 배양 20
나. 세포 생존율 20
다. qRT-PCR 21
2. 실험동물의 사육 24
가. 실험의 적용 24
나. 실험동물의 관리 24
다. 실험동물의 알츠하이머 치매 유도 25
3. 실험동물의 식이 25
가. 인도 유향 (Boswellia serrata) 26
나. 결명자 (Cassia obtusifolia L.) 26
다. 번행초 (Tetragonia tetragonoides) 26
라. 황금 (Scutellaria baicalensis Georgi) 27
마. 연교 (Forsythiasuspensa (Thunb.) Vahl) 28
4. 식이섭취량 28
5. 식이효율 (Food efficiency ratio; FER) 28
6. Oral glucose tolerance test (OGTT) 29
7. 인슐린 저항성 검사(ITT ; Insulin tolerance test) 29
8. Dual energy X-ray absorptiometry (DEXA) 30
9. 인지력 검사 30
가. 수동 회피 반응 실험(Passive avoidance test) 30
나. Y자 미로 실험 (Y-maze test) 31
다. 수중 미로 실험 (Water maze test) 31
10. 조직 채취 32
11. 생화학 검사 32
가. 혈청 32
나. 조직 32
다. Western blot 34
라. 뇌 beta-amyloid 축적 35
12. 통계처리 35
IV. 실험 결과 41
1. PC12 cell 41
가. PC12 cell MTT assay 41
나. PC12 Cell Rt-PCR 50
2. 식이 섭취량 및 체중 증가 55
가. 식이 섭취량 55
나. 체중 증가 55
다. 활동량 56
3. 혈당 63
가. 주차 별 혈당의 변화 63
나. Oral glucose tolerant test (OGTT) 63
다. 혈청 내 인슐린 64
라. Insulin tolerant test (ITT) 65
4. 체구성 성분의 변화 71
가. 지방 무게 71
나. Dual energy X-ray absorptiometry ; DEXA 72
5. 인지 능력 83
가. 수동 회피 실험 (Passive avoidance test) 83
나. Y자 미로 실험 (Y-maze) 83
다. 수중 미로 실험 (Water-maze) 84
6. 생화학 검사 90
가. 혈청 생화학 검사 90
나. 조직 생화학 검사 98
다. 뇌 형광염색 105
라. Western blot 107
V. 결론 및 고찰 113
VI. 참고문헌 117
ABSTRACT 122
Table 01. 중앙 치매센터, 치매 유병 현황 (2017) 15
Table 02. Primer square used for RT-PCR 36
Table 03. The composition of experimental diet 40
Figure 01. Experimental design at PC12 cell 37
Figure 02. Experimental grouping 38
Figure 03. Experimental design 39
Figure 04. Change of cell viability treat amyloid-β... 43
Figure 05. Improvement of PC12 cell viability with... 44
Figure 06. Improvement of PC12 cell viability with... 44
Figure 07. Improvement of PC12 cell viability with... 45
Figure 08. Improvement of PC12 cell viability with... 45
Figure 09. Improvement of PC12 cell viability with... 46
Figure 10. Improvement of PC12 cell viability with... 47
Figure 11. Improvement of PC12 cell viability with... 47
Figure 12. Improvement of PC12 cell viability with... 48
Figure 13. Improvement of PC12 cell viability with... 48
Figure 14. Improvement of PC12 cell viability with... 49
Figure 15. Result of BDNF sequence level used qRT-PCR... 52
Figure 16. Result of CNTF sequence level used qRT-PCR... 53
Figure 17. Result of Tau sequence level used qRT-PCR... 54
Figure 18. Change of food intake during experimental... 57
Figure 19. Average diet intake during experimental period 58
Figure 20. Change of weight during experimental period 59
Figure 21. Weight gain during experimental period 60
Figure 22. Feeding efficiency ratio during experimental... 61
Figure 23. Result of mobile activity time ratio total... 62
Figure 24. Change of blood glucose during experimental... 66
Figure 25. Change of serum glucose levels at oral... 67
Figure 26. Area under the curve of serum glucose levels... 68
Figure 27. Change of serum insulin levels oral glucose... 69
Figure 28. Change of serum glucose level insulin... 70
Figure 29. Fat weight at the end of experimental 74
Figure 30. Result of bone mass density of pelvis during... 75
Figure 31. Change of lean mass at pelvis during... 76
Figure 32. Change of fat mass at pelvis during... 77
Figure 33. Body composition component increase ratio of... 78
Figure 34. Change of Bone mass density at left femur... 79
Figure 35. Change of Lean mass at left femur during... 80
Figure 36. Change of fat mass at left femur during... 81
Figure 37. Body composition component increase ratio of... 82
Figure 38. Result of short-term memory measured by... 85
Figure 39. Result of short-term spatial memory measured... 86
Figure 40. Result of latancy to first into the zone 3... 87
Figure 41. Result of frequency entering into the zone 3... 88
Figure 42. Result of duration stay into the zone 3 during... 89
Figure 43. Result of serum triglyceride levels 92
Figure 44. Result of serum total cholesterol levels 93
Figure 45. Result of serum HDL-Cholesterol levels 94
Figure 46. Result of serum ratio HDL-cholesterol levels... 95
Figure 47. Result of serum GOT levels 96
Figure 48. Result of serum GPT levels 97
Figure 49. Result of liver triglyceride levels 100
Figure 50. Result of liver glycogen levels 101
Figure 51. Result of brain triglyceride levels 102
Figure 52. Result of brain total cholesterol levels 103
Figure 53. Result of brain glycogen levels 104
Figure 54. Fluorescence staining of β-amyloid plaques in... 106
Figure 55. Result of western blot at hippocampus 109
Figure 56. Result of AKT and phosphorylation AKT... 110
Figure 57. Result of GSK-3-β and phosphorylation... 111
Figure 58. Result of Tau and phosphorylation Tau level... 112
초록보기 더보기
The number of elderly people is growing worldwide, as is the rate of dementia. Likewise, Korea is now becoming an aged society, with 10% of the elderly being diagnosed with dementia. Dementia is classified as Alzheimer's, vascular dementia, and other dementias. Among them, Alzheimer's disease is most prevalent, accounting for more 60% of all dementia patients. Also, most people who develop premature dementia, before the age of 65, are known to have Alzheimer's disease. Alzheimer's disease is characterized by memory loss and decreased cognitive ability due to the accumulation of β-amyloid plaques in the brain and neurofibrillary tangles (NFT) in the cell due to the phosphorylation Tau protein. Alzheimer's disease has been widely studied, but to this day it is still unclear what causes it and it treatment is still largely ineffective. Therefore, we explored the etiology and potential interventions for Alzheimer's disease by inducing apoptosis using β-amyloid using an in vitro experimental model in PC12-cells. The efficacy of natural substances that significantly increase cell survival were explored. We then evaluated the substances that showed promise in the in vitro study in and animal model of Alzheimer's disease.
When inducing β-amyloid apoptosis in PC12 cell, Boswellia serrata, Cassia obtusifolia L., Tetragonia tetragonoides, Scutellaria baicalensis Georgi, Forsythiasuspensa (Thunb.) Vahl extracts were selected as natural products with significantly increased cell survival. Using pRT-PCR in PC12 cells, we found that the five extracts significantly increased the activity of the BDNF and CNTF, and decreased the activity of Tau. The extract of each natural product was administered at a dosage of 0.5g/kgB·W to rats fed a high-fat diet and with Alzheimer's disease induced by injecting β-amyloid into rat's brain chamber through ICV.
Groups that ate the Boswellia serrata extracts and Cassia obtusifolia L. extracts showed increased insulin resistance in blood glucose metabolism and they did not affect the improvement of Alzheimer's pathology. However, for groups that received Boswellia serrata, there was a significantly an improvement in weight loss and metabolism in body fat.
Groups that ate the Tetragoniatetragonoidesextractsexperienced significant improvements in their cognitive ability and less phosphorylation of the tau proteins. Although Alzheimer's pathology was improved, fat metabolism and blood glucose metabolism were not improved, making it difficult for Tetragonia tetragonoides extracts to be applied to the prevention and treatment Alzheimer's disease.
Groups that ate the Scutellaria baicalensis Georgi extracts and Forsythiasuspensa (Thunb.) Vahl extracts showed significantly lower blood glucose in OGTT. and the insulin in the blood was not increased, so it was judged to have decreased insulin resistance. Cognition, as evaluated using the passive avoidance test, Y-maze and water-maze experiments, showed significant improvements in cognitive ability. Also, the rats exhibited a low level of β-amyloid accumulation in the hippocampus, and the Western blot shows that AKT phosphorylation activity significantly less phosphorylation of the Tau protein by activating the phosphorylation GSK-3-β in the brain. Therefore, Scutellaria baicalensis Georgi extracts and Forsythiasuspensa (Thunb.) Vahl extracts do not increase insulin resistance, but improve congnitive ability, and reduce the accumulation of β-amyloid and phosphorylation of tau in the hippocampus. By improving the pathology of Alzheimer's they are considered effective for preventing Alzheimer's disease in rats.
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