아주대학교

목차

1. INTRODUCTION 1
2. MATERIALS AND METHODS 5
2.1 Animals 5
2.2 Human MSCs Isolation and Culture 5
2.3 Lentiviral vectors and Cell lines 6
2.4 Lentiviral titration 7
2.5 FACS 8
2.6 Surface antigen analysis 8
2.7 Western blot 9
2.8 Mesodermal differentiation assay 9
2.9 Growth kinetic assay 11
2.10 Quantitative reverse transcription (qRT)-PCR 12
2.11 Chromosomal stability 12
2.12 In vitro suicide effect of HSV-TK 12
2.13 Tumorigenicity assay of lentiviral transduced MSCs 13
2.14 In vivo suicide effect of HSV-TK 13
2.15 Statistical analysis 14
3. RESULTS 15
3.1 Development of lentiviral vector encoding three transgenes, transduction into MSCs and transduced cells characterization 15
3.1.1 Generation of lentiviral transfer plasmids encoding GFP, RFP and PuroR 15
3.1.2 Transducing reagent test in MSCs 19
3.1.3 Generation of RFP, GFP and PuroR expressing MSCs and evaluation of Stem Cell Properties 21
3.1.4 Genomic stability and tumorigenesis test of MSC-Lenti-RGP 26
3.2 Improving the safety of mesenchymal stem cell-based ex vivo therapy using Herpes Simplex virus thymidine kinase 29
3.2.1 Generation of HSV-TK expressing MSCs 29
3.2.2 Stem Cell Properties of MSC- TK A168H 34
3.2.3 Genomic stability and tumorigenesis test of MSC-TK A168H 43
3.2.4 Generation of U87-TK A168H 46
3.2.5 Effect of vGCV on U87-TK A168H tumor as a SAE model 50
4. DISCUSSION AND CONCLUSION 56
5. REFERENCES 63