Delivering DNA to Mammalian Cells Using Magnetic Nanoparticles
- 주제(키워드) nanoparticle , DNA delivery , gene therapy , magnetic nanoparticle
- 발행기관 고려대학교 대학원
- 지도교수 김익환
- 발행년도 2014
- 학위수여년월 2014. 8
- 학위구분 석사
- 학과 일반대학원 생명공학과
- 세부전공 생물법제학
- 원문페이지 46 p
- 실제URI http://www.dcollection.net/handler/korea/000000051965
- 본문언어 영어
- 제출원본 000045808871
초록/요약
Gene therapy has received considerable interest as a potential method for the treatment of numerous inherited and acquired diseases. For developing the safe and efficient non-viral gene delivery system, we used magnetic stirring system. The main objective of this study was to investigate the delivery of the N-terminal Aequorea coerulescens green fluorescent protein plasmid (pAcGFP1-N1) attached to amine groups coated silica nanoparticles into the Chinese Hamster Ovary (CHO-K1) and the Human lung fibroblast (MRC-5) adherent cells. The nanoparticle was designed a core-shell structure and a rhodamine B isothiocyanate (RITC) flurescent dye which is incorporated in a silica shell. By modifying (coated with amine groups) surface of nanoparticles, DNA can be attached with nanoparticles enhanced biocompatibility. The results presented that DNA coding GFP gene with nanoparticles could be delivered to cells. Which result has efficiency when the DNA-nanoparticle complexes treated in optimized condition (pH 7.4 with buffer and the distance between cells and magnetic field around 3 mm). The transfection efficiency was confirmed via GFP expression. Magnetic nanoparticle attached DNA significantly improved the delivery efficiency to cells relaive to DNA only. The evidence obtained in the present study suggests that this approach of intracellular delivery technology can be used to delivering gene for gene therapy.
more목차
List of Figures……………………………………………………...i
List of Tables………………………………………………….......... ii
List of Abbreviations…………………………………………….... iii
Abstract………………………………………………………............iv
Introduction………………………………………………………….1
Materials and Methods……………………………………………..4
1. Cell culture …………………………………………………….…4
2. Measurement of viable cell density and cytotoxicity……….…..5
3. Nanoparticles………………….……..………………………….6
4. DNA………………………..............................................……... 8
5. Nanoparticles complexed with DNA: Electrostatic coupling.....10
6. Magnetic field system and delivery nanoparticles to cells……..11
7. Endogenous gene expression: GFP protein.…………………..13
Results…………………………………………………………….14
1. DNA binding assay………………….......………………14
2. Cytotoxicity of DNA-nanoparticles complexes…...…16
3. Deliverying of nanoparicles to cells……………… 18
3.1. Confocal microscopy image of deliveried nanoparticles to target cells ……….…………………………………………...18
3.2. Flow cytometry analysis……………………………...22
4. Transfection efficiency of delivering DNA with nanoparticles to cells…………….……………………24
Discussion…….……………………………………………………..29
References……………………….…………………………..………32
Abstract in Korean……………………….………………..………36
