`교장선출보직제` 제안의 논리적 구조 분석

학위논문(석사)--서울대학교 대학원 :교육학과 교육행정전공,2004.Maste

Cytosine deaminase를 포함한 사람의 중간엽 줄기세포를 이용한 유전자 치료 연구

학위논문(박사)--아주대학교 일반대학원 :분자과학기술학과,2009. 8ABSTRACT Background and Aim: Stem cell transplantation offers potential gene therapy for brain tumors. However, this approach has received little attention as a treatment for gastrointestinal tumors. In the present study, we explored the possibility of human bone marrow derived mesenchymal stem cells (hMSC) producing cytosine deaminase (CD), followed by systemic 5-fluorocytosine (5-FC) administration, showing an antitumor effect on a mouse gastric cancer xenograft. Methods: We first explored the ability of hMSC, coated with fluorescent dye, to migrate to human gastric cancer MKN45 cells in vitro and in vivo. We then used hMSC in which a gene expressed the prodrug-activating enzyme CD, which can convert the prodrug 5-FC into the cytotoxic agent 5-fluorouracil (5-FU), and further investigated the potential of these cells to deliver the CD gene and to reduce tumor growth in nude mice. The migratory capacity of hMSC was confirmed by an in vitro migration assay, as well as in an in vivo model of nude mice bearing subcutaneous tumors of MKN45 cells when hMSC were injected. Results: The migration ability of hMSC towards MKN45 cells was confirmed by migration assay. Effective conversion of 5-FC to 5-FU by hMSC transfected with the CD gene (CD-hMSC) showed therapeutic anticancer potential in a MKN45 cell co-culture system, as confirmed by thin layer chromatography, MTT assay in vitro. Conclusion: The CD-hMSC system showed anticancer therapeutic potential, and minimized the side-effects of 5-FU.CONTENTS ABSTRACT ………………………………………………...………..……………………… ⅰ CONTENTS ……………………………....…………………...………………………….…. ⅲ LIST OF FIGURES ……………………………………………………………………...... ⅴ ABBREVIATION ………………………………………………………………………….. ⅶ Ⅰ. INTRODUCTION ……………...………………………………………………..… 1 Ⅱ. MATERIALS AND METHODS …………………...……………………………... 3 1. Culture of human bone marrow-derived mesenchymal stem cells .……………………..….. 3 2. hMSC labeling with fluorescent dye, CM-Dil …………………………………………… 3 3. In vitro migraitnion assay ………………………………………………..……………….. 4 4. In vivo migration study …..……………….………………………………………………… 5 5. Tissue processing and imaging studies ……………………………………..………………. 5 6. Apoptosis assay ……………............…………………………………..….………………… 6 7. Transfection to MSC ……………………………………………………………………….. 7 8. PCR of cytosine deaminase ………………………………..……………………………….. 7 9. The design of primer for PCR ...……………………………………………………………. 7 10. Construction of plasmids CD and transfection …………...………………………………. 8 11. Cytosine deaminase conversion assay in vitro ……………………………………………… 8 12. Microtitration (MTT) assay ………………………………................……………..……….. 9 13. Statistical analysis ….……………………………………..……..………………………….. 9 Ⅲ. RESULTS ………………………………………………………………………..……… 11 1. The migration ability of hMSC toward MKN45 cells in vitro .………………………….. 11 2. Transplanted hMSCs migrated to MKN45 tumor regions ………….………..……...…… 11 3. CD gene transfected hMSCs showed strong CD conversion activity in vitro ….………… 12 Ⅳ. DISCUSSION …………………………………………………………………………… 25 Ⅴ. REFERENCES ………………………...……………………………………………….. 29 LIST OF FIGURES Fig. 1 Migraion assay in vitro ………............................................…………………………….… 14 Fig.2 Time dependent migraion of MSC toward tumor cell MKN45 …………………………... 15 Fig. 3 Migration assay using CM-Dil labeled MSCs in vitro ……………………..……….… 16 Fig. 4 Nude mice injected with MKN45, then i.v injected with MSC/CM-Dil ……………...….. 17 Fig. 5 Intravenously injected human bone marrow-derived mesenchymal stem cells (hMSCs) migrate to tumor regions of athymic mice harboring MKN45 tumors ………………….…...… 18 Fig. 6 The ligation alignment of Cytosine deaminase(CD) with pcDNA3.1(-) …………………. 19 Fig. 7 The digestion was confirmed by restriction enzyme digestion using EcoRI, XhoI ………. 20 Fig. 8 Efficeint transfection of MSC with GFP using lipofectamine 2000 …………………….. 21 Fig. 9 Cytosine deaminase (CD) activity in vitro ..…………………………………………….. 22 Fig. 10 Cell sensitivity to 5-FU and selective cytotoxicity effects were mediated by CD-hMSCs in the presence of 5-FC on MKN45 cells …………………………………………………………. 23 Fig. 11 MTT assay ……………………………………………………………………………… 24 ABBREVIATION BM ………………………………………............................................. Bone marrow CD ………………………………………………………….…………………. Cytosine deaminase CM-Dil …………………………………….………...… chloromethybenzamido-1,19-dioctadecyl-3,3,39,39-tetramethylindocarbocyanine perchlorate DNA ……………………………………………………………………… Deoxyribonucleic acid FBS ………………………………………………………….………. Fetal bovine serume 5-FC ………………………………………………………………...................... 5-fluorocytosine 5-FU ……………………………………………………………………….……… 5-fluorouracil 5-FdUMP ………………………………………………… 5-fluoro-deoxyuridine-monophosphate GFP............................................................................................................. Green fluorescent protein HSC ………………………………………..……………………………. Hematopoeitic stem cell hMSC …………………………...………………………..…. Human mesenchymal stem cell MTT assay ……………………………………………………………..…. Microtitration assay RNA ……………………………………………………………….. Ribonucleic acid RT-PCR …………………...……….………… Reverse transcriptase-polymerase chain reaction TLC ………………………………………………………….…… Thin layer chromatography TS …………………………………………………………………………… Thymidylate synthaseMaste