표제지
Acknowledgement
Contents
ABBREVIATIONS 12
Abstract 13
Introduction 14
Material and methods 17
Confirmation of Optimized Hypoxic Condition through Nitrogen Gas Injection 17
hESCs Culture and Formation of hEBs 17
Reverse Transcription-Polymerase Chain Reaction (RT-PCR) 18
Real-Time Quantitative RT-PCR (Taqman method) 19
Immunocytochemistry Analysis 19
Results 21
Setup of Optimized Hypoxic Condition using N2 Gas Injection 21
The culture of human embryoid body in normoxic and hypoxic condition 21
The pattern of Oct4 expression in Hypoxic and Normoxic Condition 22
The pattern of MAP2 expression in Hypoxic and Normoxic Condition 23
The pattern of AFP expression in Hypoxic Condition and in Normoxic Condition 24
The pattern of Brachyury and vessel lineage expression in Hypoxic and Normoxic Condition 24
Discussion 27
Reference 30
Table 1. Sequences of RT-PCR Primers. 33
Table 2. Sequences of Real Time PCR Primers and Probes. 34
Figure 1. Scheme of hEBs culture for comparison two kinds of conditions(normoxic and hypoxic conditions) 35
Figure 2. Experimental diagram of two kinds of methods for hypoxic conditiona medium and demand oxygen value changing graph of two kinds of methods. (A) Experimental diagram for media group treated with hypoxia incubator(3% O tension) (B) Experimental diagram for media group treated with N2 gas bubbling... 36
Figure 3. The morphology of hEBs cultured in normoxic or hypoxic condition (A) hEBs cultured for 7 days in normoxic condition (B) hEBs cultured for 14 days in normoxic condition (C) hEBs cultured for 21 days in normoxic condition (D) hEBs cultured for 7 days in hypoxic condition (E) hEBs cultured for 7 days in hypoxic... 37
Figure 4. Characterization of Oct4 expression pattern in human embryoid body cultured in normoxic and hypoxic condition. (A) RT-PCR result. (B) Realtime PCR result 38
Figure 5. Immunofluorescence results of Oct4 expressed in the human embryoid body (A) hEBs cultured for 7 days in normoxic condition (B) hEBs cultured for 14 days in normoxic condition (C) hEBs cultured for 21 days in normoxic condition (D) hEBs cultured for 7 days in hypoxic condition (E) hEBs cultured for 7 days in... 39
Figure 6. Characterization of MAP2 expression pattern in human embryoid body cultured in normoxic and hypoxic condition. (A) RT-PCR result. (B) Realtime PCR result 40
Figure 7. Immunofluorescence results of MAP2 expressed in the human embryoid body (A) hEBs cultured for 7 days in normoxic condition (B) hEBs cultured for 14 days in normoxic condition (C) hEBs cultured for 21 days in normoxic condition (D) hEBs cultured for 7 days in hypoxic condition (E) hEBs cultured for 7 days in... 41
Figure 8. Characterization of AFP expression pattern in human embryoid body cultured in normoxic and hypoxic condition. (A) RT-PCR result. (B) Realtime PCR result 42
Figure 9. Immunofluorescence results of AFP expressed in the human embryoid body (A) hEBs cultured for 7 days in normoxic condition (B) hEBs cultured for 14 days in normoxic condition (C) hEBs cultured for 21 days in normoxic condition (D) hEBs cultured for 7 days in hypoxic condition (E) hEBs cultured for 7 days in... 43
Figure 10. Realtime PCR result of Brachyury expression pattern in human embryoid body cultured in normoxic and hypoxic condition 44
Figure 11. Immunofluorescence results of Brachyury expressed in the human embryoid body (A) hEBs cultured for 7 days in normoxic condition (B) hEBs cultured for 14 days in normoxic condition (C) hEBs cultured for 21 days in normoxic condition (D) hEBs cultured for 7 days in hypoxic condition (E) hEBs... 45
Figure 12. Realtime PCR result of PECAM expression pattern in human embryoid body cultured in normoxic and hypoxic condition 46
Figure 13. Immunofluorescence results of PECAM expressed in the human embryoid body (A) hEBs cultured for 7 days in normoxic condition (B) hEBs cultured for 14 days in normoxic condition (C) hEBs cultured for 21 days in normoxic condition (D) hEBs cultured for 7 days in hypoxic condition (E) hEBs 47
Figure 14. Realtime PCR result of VE-Cad expression pattern in human embryoid body cultured in normoxic and hypoxic condition 48
Figure 15. Immunofluorescence results of VE-Cad expressed in the human embryoid body (A) hEBs cultured for 7 days in normoxic condition (B) hEBs cultured for 14 days in normoxic condition (C) hEBs cultured for 21 days in normoxic condition (D) hEBs cultured for 7 days in hypoxic condition (E) hEBs... 49