Title Page
Abstract
Contents
1. Introduction 9
1.1. Natural killer (NK) cells 9
1.2. NK cell-based cancer immunotherapy 10
1.2.1. IL-2 (Interleukin-2) Based Immunotherapy 12
1.3. IL-2 Signaling Pathways 13
2. Objective 16
3. Materials and Methods 18
3.1. Cell culture and count 18
3.2. Vector construction 19
3.2.1. Mutagenesis by PCR (Polymerase Chain Reaction) Overlap Extension 19
3.2.2. Cloning 21
3.2.3. Extraction of pc DNA/JAK1VF, JAK3AV plasmid[이미지참조] 21
3.3. Transfection and Selection 21
3.4. Single clone isolation 22
3.5. PCR on NK cell line genomic DNA (gDNA) 22
3.6. Real-Time Quantitative PCR 23
3.7. Cytotoxicity assay 23
3.7.1. Calcein AM cytotoxicity assay 23
3.7.2. Immunofluorescence staining 25
3.7.3. Perforin and granzyme qPCR 26
4. Results and Discussion 27
4.1. IL-2 concentration dependent cell growth profiles 27
4.2. Comparison of mRNA Expression Levels of JAK Mutants 29
4.3. Cytotoxicity assay 31
4.3.1. Calcein am cytotoxicity assay 31
4.3.2. NK cell surface marker expression on confocal microscope 33
4.3.3. Perforin and granzyme qPCR 36
5. Conclusion 39
6. Reference 42
Table 1. Comparisons between NK, CAR-T and CAR-NK 11
Table 2. JAK-STAT signaling pathway (Created with BioRender.com) 15
Table 3. Detailed information of primers 20
Figure 1. Profiles of cell growth and viability of (a) KHYG-1 (b) JAK1VF cell line (c) JAK3AV cell line (d) JAK1VF/JAK3AV cell line in cultures addition with IL-2 concentration 0, 10, 50, 100 IU/ml.[이미지참조] 28
Figure 2. Quantitative real-time RT-PCR for JAK. WT column represents the expression of JAK1 and JAK3 in untransfected cells. The remaining columns indicate the mRNA expression levels in cells transfected with mutant... 30
Figure 3. (a) Analysis of cytotoxicity ability through co-culture of effector and tumor cells. Fluorescent microscopic images of WT and JAK1VF, JAK3AV, JAK1VF/JAK3AV cell lines. (b) A quantitative graph showing the cytotoxicity activity...[이미지참조] 32
Figure 4. Confocal microscopy analysis of NK CD 56 expression. (a) Confocal fluorescence microscopy observations, Alexa Fluor 488 (green). (b) The corresponding histograms (Mean±SEM) quantitatively represent the Mean Fluorescence... 34
Figure 5. Confocal microscopy analysis of NKG2D expression. (a) Confocal fluorescence microscopy observations, Alexa Fluor 488 (green). (b) The corresponding histograms (Mean±SEM) quantitatively represent the Mean Fluorescence... 35
Figure 6. Perforin and granzyme B protein expression. qPCR for Perforin, Granzyme B and Data are corrected for b-actin expression and expressed as fold over mRNA of the respective Perforin, Granzyme B of WT. 38