지원사업
학술연구/단체지원/교육 등 연구자 활동을 지속하도록 DBpia가 지원하고 있어요.
커뮤니티
연구자들이 자신의 연구와 전문성을 널리 알리고, 새로운 협력의 기회를 만들 수 있는 네트워킹 공간이에요.
논문 기본 정보
- 자료유형
- 학위논문
- 저자정보
- 지도교수
- 나재운
- 발행연도
- 2015
- 저작권
- 순천대학교 논문은 저작권에 의해 보호받습니다.
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Ternary complexes are designed to deliver psiRNA-bcl2 and (KLA)4 peptide into tumor cells for cancer therapy. In this study, complexes containing psiRNA-bcl2 and (KLA)4 peptide with transferrin ligand-conjugated LMWSC (LMWSC-PEG-TfP) were prepared by electrostatic interaction. The delivered psiRNA-bcl2 induced gene-silencing in a nucleus of cancer cells, while (KLA)4 peptide inhibited tumor growth via mitochondrial apoptosis,
indicating that ternary complexes exert very strong synergistic effects on tumor growth suppression by acting on psiRNA-bcl2 and (KLA)4 peptide at the same time. In this study, ternary complexes having a targeting-ligand, transferrin (TfP), were found to be especially effective at binding to the TfP receptor rich cancer cells, HCT119.
The plasmid DNA (pDNA) in ternary complexes was completely condensed at various content of LMWSC-PEG-TfP (32?64 times more than pDNA) and released into cells. pDNA in the complexes was protected from DNase present on the exterior of cells. The size (165 nm?248 nm) of ternary complexes with LMWSC-PEG-TfP was increased, but surface charges (3 mV?4.5 mV) were decreased. These results likely occurred because the free amine-group of LMWSC decreased in response to conjugated transferrin. Moreover, transfected ternary complexes with LMWSC-PEG-TfP were not expressed in the normal cells (HEK293), but were over expressed in HCT119 cells. These findings indicate that the ternary complexes can be specifically targeted to HCT119 cancer cells. The ternary complexes had a low cytotoxicity in L929 cells at up to 4 μg of pDNA and high antitumor activities via a synergistic effect due to co-action of psiRNA and (KLA)4 peptide in HCT119 cells. Thus,
the ternary system is useful as a gene and peptide delivery system with high gene transfection, low toxicity, and high antitumor activities.
indicating that ternary complexes exert very strong synergistic effects on tumor growth suppression by acting on psiRNA-bcl2 and (KLA)4 peptide at the same time. In this study, ternary complexes having a targeting-ligand, transferrin (TfP), were found to be especially effective at binding to the TfP receptor rich cancer cells, HCT119.
The plasmid DNA (pDNA) in ternary complexes was completely condensed at various content of LMWSC-PEG-TfP (32?64 times more than pDNA) and released into cells. pDNA in the complexes was protected from DNase present on the exterior of cells. The size (165 nm?248 nm) of ternary complexes with LMWSC-PEG-TfP was increased, but surface charges (3 mV?4.5 mV) were decreased. These results likely occurred because the free amine-group of LMWSC decreased in response to conjugated transferrin. Moreover, transfected ternary complexes with LMWSC-PEG-TfP were not expressed in the normal cells (HEK293), but were over expressed in HCT119 cells. These findings indicate that the ternary complexes can be specifically targeted to HCT119 cancer cells. The ternary complexes had a low cytotoxicity in L929 cells at up to 4 μg of pDNA and high antitumor activities via a synergistic effect due to co-action of psiRNA and (KLA)4 peptide in HCT119 cells. Thus,
the ternary system is useful as a gene and peptide delivery system with high gene transfection, low toxicity, and high antitumor activities.
목차
Ⅰ. Introduction ?????????????????????????????????????????????????????????????????????????????????????1 Ⅱ. Materials and Methods ???????????????????????????????????????????????????????????????????52.1. Materials ???????????????????????????????????????????????????????????????????????????????????????52.2. Method ??????????????????????????????????????????????????????????????????????????????????????????62.2.1. Cell culture ????????????????????????????????????????????????????????????????????????????????62.2.2. Extraction of plasmid DNA ???????????????????????????????????????????????????????72.2.3. Synthesis of Synthesis of PEG-TfP-Fmoc?????????????????????????????????7 2.2.4. Synthesis of LMWSC-PEG-TfP-Fmoc??????????????????????????????????????72.2.5. Fmoc deprotection of LMWSC-PEG-TfP-Fmoc???????????????????????8 2.2.6. 1H-NMR spectra of LMWSC-PEG-TfP?????????????????????????????????????8 2.2.7. Preparation of ternary complexes ??????????????????????????????????????????????92.2.8. The size and zeta potential of ternary complexes??????????????????????92.2.9. Gel retardation assay of ternary complexes ?????????????????????????????102.2.10. pDNA protection and release assay of ternary complexes?????????????????????????????????102.2.11. The transfection of ternary complexes???????????????????????????????????112.2.12. The Cytotoxicity and antitumor activity of ternary complexes?????????????????????????????????????112.2.13. Gene silencing of ternary complexes with psiRNA-bc l2Ⅲ. Results & Discussion ?????????????????????????????????????????????????????????????????????143.1. The scheme and action of ternary complexes ?????????????????????????????14 3.2. 1H-NMR spectra of LMWSC-PEG-TfP ????????????????????????????????????163.3. The gel retardation assay of ternary complexes ??????????????????????????193.4. The size and zeta potential of ternary complexes????????????????????????223.5. In-vitro transfection of ternary complexes ??????????????????????????????????283.6. In vitro cytotoxicity of ternary complexes???????????????????????????????????353.7. In vitro antitumor activities of ternary complexes???????????????????????393.8. In vitro western blot analysis of inhibited bcl-2 protein??????????????47 Ⅳ.Conclusion???????????????????????????????????????????????????????????????????????????????????????49 References????????????????????????????????????????????????????????????????????????????????????????????50 Korean Abstract??????????????????????????????????????????????????????????????????????????????????56 List of Publications ??????????????????????????????????????????????????????????????????????????????59 감사의 글 ???????????????????????????????????????????????????????????????????????????????????????????64
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