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Abstract
국문초록
목차
I. 서론 12
II. 실험 재료 및 방법 14
1. 세포와 세포배양 14
2. 배지와 저 산소 환경 14
3. 세포 처치 (treatment) 15
4. 세포 증식 속도 (Cell doubling time) 15
5. 세포 생존 측정 (Clonogenic survival assay) 15
6. 세포 주기 분석 (Flow cytometric analysis) 16
7. 단백질 추출 (Cell lysate preparation) 16
8. 단백질 분석 (Western blot analysis) 17
9. 면역 침강 (Immunoprecipitation ) 18
10. 면역 복합체 효소 분석 (immunocomplex Kinase assay) 19
11. RNA 분리 (RNA isolation) 19
12. cDNA chip microarray 20
III. 결과 21
1. 세포 증식 시간 21
2. 세포 생존율 22
3. 세포 주기 변화 22
4. Cyclin B/cdc2와 Cyclin D/cdk4의 활성 23
5. ATM의 활성과 p53 단백질의 발현 24
6. 새로운 유전자의 발현 25
IV. 고찰 27
V. 참고문헌 49
Table 1. Doubling time of the human colon adenocarcinoma RKO. Cells were treated with normoxia or hypoxia condition containing neutral pH7.5 or acidic pH 6.6 medium. Cells were harvested with trypsin and counted the number with trypan blue stain. 45
Table 2. Number of genes of either 2 increased or 2 decreased in RKO cells. Cells were incubated in normoxia pH 7.5, normoxia pH 6.6, hypoxia pH 7.5 and hypoxia pH 6.6 for 0-72 hours. 46
Table 3. Gene lists of up regulation on RKO cells. Cells were incubated in normoxia pH 7.5, normoxia pH 6.6, hypoxia pH 7.5 and hypoxia pH 6.6 for 0-72 hours. Cells were harvested with trypsin and performed RNA isolation by using 47
Table 4. Gene lists of down regulation on RKO cells. Cells were incubated in normoxia pH 7.5, normoxia pH 6.6, hypoxia pH 7.5 and hypoxia pH 6.6 for 0-72 hours. Cells were harvested with trypsin and performed 48
Figure 1. Schematic diagram of hypoxia development.(15) 32
Figure 2. Clonogenic surviving fraction in RKO cell. Cells were treated in hypoxia pH 7.5, normoxia pH 7.5, hypoxia pH 6.6 and normoxia pH 6.6 for 24, 48, 72hours, and than cultured with pH 7.5 medium in a 5% CO2 incubator for 7-10 days. 33
Figure 3. Changes in percentage of cell cycle distribution in RKO cells. Cells were incubated in normoxia pH 7.5(A), normoxia pH 6.6(B), hypoxia pH 7.5(C) and hypoxia pH 6.6(D) for 0-72 hours. 34
Figure 4. Changes in percentage of apoptosis distribution in RKO cells. Cells were incubated in normoxia pH 7.5(●), normoxia pH 6.6(□), hypoxia pH 7.5(▲) and hypoxia pH 6.6(◇) for 0-72 hours. 36
Figure 5. Changes in percentage of G1 phase distribution in RKO cells. Cells were incubated in normoxia pH 7.5(●), normoxia pH 6.6(□), hypoxia pH 7.5(▲) and hypoxia pH 6.6(◇) for 0-72 hours. 38
Figure 6. DNA histogram of cell cycle in RKO cells. Cells were incubated in normoxia pH 7.5 (A), normoxia pH 6.6 (B), hypoxia pH 7.5 (C) and hypoxia pH 6.6 (D) for 0-72 hours. 40
Figure 7. Activation of Cyclin B in RKO cells. A.Cells were incubated in nornoxia pH 7.5, normoxia pH 6.6, hypoxia pH 7.5 and hypoxia pH 6.6 for 0-72 hours. Cells havested with trypsin and prepared the lysates. 41
Figure 8. Activation of Cyclin D in RKO cells. A. Cells were incubated in normoxia pH 7.5, normoxia pH 6.6, hypoxia pH 7.5 and hypoxia pH 6.6 for 0-72 hours. Cells havested with trypsin and prepared the lysates. Activity of 42
Figure 9. Activation of ATM in RKO cells. A. Cells were incubated in normoxia pH 7.5, normoxia pH 6.6, hypoxia pH 7.5 and hypoxia pH 6.6 for 0-72 hours. Cells havested with trypsin and prepared the lysates. 43
Figure 10. Activation of p53, phosphor-p53 and p21 in RKO cells. Cells were incubated in normoxia pH 7.5, normoxia pH 6.6, hypoxia pH 7.5 and hypoxia pH 6.6 for 0-72 hours. 44
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