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세포투과성 항-DNA 단클론 항체의 가변부위 도메인을 이용한 효율적인 세포내 물질 전달 도구에 관한 연구
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 장영주 | - |
| dc.contributor.author | Im sunwoo | - |
| dc.date.accessioned | 2019-10-21T07:28:19Z | - |
| dc.date.available | 2019-10-21T07:28:19Z | - |
| dc.date.issued | 2016-08 | - |
| dc.identifier.other | 23101 | - |
| dc.identifier.uri | https://dspace.ajou.ac.kr/handle/2018.oak/18893 | - |
| dc.description | 학위논문(석사)--아주대학교 일반대학원 :의생명과학과,2016. 8 | - |
| dc.description.tableofcontents | I. INTORDUCTION 1 II. MATERIALS AND METHODS 3 1. Cell lines 3 2. Purification of recombinant 2C10 VH domain 3 3. Conjugation of 2C10 VH domain with FITC or si-RNA 4 4. Western blotting 4 5. Analysis of cell penetration by flow cytometry 5 6. Analysis of cell penetration by confocal microscopy 5 7. Inhibition of cell penetration by endocytosis inhibitors 6 8. Gene transcription analysis 6 9. Cell proliferation assay 6 10. Cell cycle assay 7 III. RESULTS 1. Expression and purification of recombinant 2C10 VH domain 8 2. The purified 2C10 VH domain penetrated into various cell lines in a dose-dependent manner 8 3. The penetrated 2C10 VH domain was localized in cytoplasm and nucleus of living cells 13 4. Internalization of 2C10 VH domain into living HeLa cells occurred mainly via clathrin-mediated endocytosis 13 5. 2C10 VH domain did not affect cell proliferation in the various mammalian cell lines and change the cell cycle 19 6. 2C10 VH domain transferred siRNA into mammalian cell lines 19 7. Expression of tNASP was decreased in Caki cells by the tNASP-siRNA delivered by 2C10 VH domain 23 8. The tNASP-siRNA delivered by 2C10 VH domain effectively inhibited cell proliferation and caused G1 phase 24 IV. DISCUSSION 28 V. REFERENCES 31 VI. 국문요약 37 | - |
| dc.language.iso | eng | - |
| dc.publisher | The Graduate School, Ajou University | - |
| dc.rights | 아주대학교 논문은 저작권에 의해 보호받습니다. | - |
| dc.title | 세포투과성 항-DNA 단클론 항체의 가변부위 도메인을 이용한 효율적인 세포내 물질 전달 도구에 관한 연구 | - |
| dc.title.alternative | Studies on the efficient intracellular delivery vehicle using variable region domain of a cell-penetrating anti-DNA monoclonal antibody | - |
| dc.type | Thesis | - |
| dc.contributor.affiliation | 아주대학교 일반대학원 | - |
| dc.contributor.alternativeName | Sun-Woo Im | - |
| dc.contributor.department | 일반대학원 의생명과학과 | - |
| dc.date.awarded | 2016. 8 | - |
| dc.description.degree | Master | - |
| dc.identifier.localId | 758765 | - |
| dc.identifier.url | http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000023101 | - |
| dc.subject.keyword | Cell-penetrating antibody | - |
| dc.subject.keyword | Single domain antibody | - |
| dc.subject.keyword | Cellular delivery vehicle | - |
| dc.description.alternativeAbstract | The cell-penetrating antibodies and antibody fragments can be utilized as a tool for the medical diagnosis and treatment. In this study, we have produced the protein of recombinant VH domain of a monoclonal anti-DNA antibody 2C10, IgG of which has been previously shown to have cell-penetrating activities for studying the applicability of the domain as a cellular delivery vehicle. To evaluate the cell-penetrating property of 2C10 VH domain in various mammalian cell lines, the flow cytometric and confocal microscopic analysis and cell proliferation assay were used. The VH domain penetrated into all cell lines we tested in time- and dose-dependent manner, although the internalization efficiency was varied. We also found that the VH domain was localized in nuclei as well as cytoplasm of living cells and it was mainly internalized by the clathrin-mediated endocytosis pathway. The VH domain did not change the cell cycle and significantly affect the cell proliferation in various mammalian cell lines at the concentration that we used for functional assays. We tested further the delivering activity of a valuable molecule by the VH domain using the conjugates of VH and siRNA for testicular Nuclear Auto-antigenic Sperm Protein (tNASP). It was found that the siRNA was successfully transferred by 2C10 VH domain into Caki and HeLa cancer cell lines, and the knockdown effects of tNASP-siRNA were observed in Caki cells, in where the level of tNASP expression is known to be high; the levels of RNA transcripts and proteins of tNASP were decreased by the delivered tNASP-siRNA; the down-regulated tNASP effectively inhibited cell proliferation and caused G1 phase arrest of cell cycle. These results indicate that the recombinant 2C10 VH domain could be applied as a valuable vehicle which can deliver specific biomolecule(s) into the cytoplasm or cell nuclei for therapy and diagnosis of diseases. | - |
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