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In vivo mr imaging and quantification of primary and metastatic cancers using biomodal lentiviral vector encoding human ferritin and green fluorescent protein
사람 페리틴과 녹색형광단백질 동시 발현 렌티바이러스 벡터를 이용한 원발성 및 전이암의 자기공명영상화 및 정량화 기법에 대한 연구

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dc.contributor.advisor문우경-
dc.contributor.author조혜림-
dc.date.accessioned2017-07-14T01:17:26Z-
dc.date.available2017-07-14T01:17:26Z-
dc.date.issued2015-02-
dc.identifier.other000000024900-
dc.identifier.urihttps://hdl.handle.net/10371/121805-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 협동과정 방사선응용생명과학전공, 2015. 2. 문우경.-
dc.description.abstractA combination of reporter genes for magnetic resonance imaging (MRI) and optical imaging can provide an additional level of noninvasive and quantitative information about biological processes occurring in deep tissues. Cellular MRI with a reporter gene offers the opportunity to track small numbers of tumor cells and to study metastatic processes in their earliest developmental stages in the target organs of interest.
I developed a bimodal lentiviral vector to monitor deep tissue events using MRI to detect myc tagged human ferritin heavy chain (myc-hFTH) expression and fluorescence imaging to detect green fluorescent protein (GFP) expression. No cellular toxicity due to overexpression of myc-hFTH and GFP was observed in MTT and trypan blue exclusion assays. Iron accumulation was observed in myc-hFTH cells and tumors by Prussian blue staining and iron binding assays.
First of all, for primary tumor imaging, the transgene construct was stably transfected into MCF-7 and F-98 cells. After transplantation of the cells expressing myc-hFTH and GFP into mice or rats, serial MRI and fluorescence imaging were performed with a human wrist coil on a 1.5T MR scanner and optical imaging analyzer for 4 weeks. The myc-hFTH cells and tumors had significantly lower signal intensities in T2 weighted MRI than mock-transfected controls (P ≤ 0.05). This is direct evidence that myc-hFTH expression can be visualized noninvasively with a 1.5T clinical MR scanner.
Second, for the noninvasive imaging and quantification of metastatic melanoma cells in the lymph nodes (LNs) of living mice, a B16F10 murine melanoma cell line expressing hFTH and GFP was constructed to allow the detection of cells by MRI and fluorescence imaging. Stable overexpression of hFTH and GFP in B16F10 murine melanoma cells was feasible and showed no cellular toxicity. In addition, hFTH cells were detectable by 9.4T MRI in vitro and in vivo, yielding significant changes in T2* relative to control cells. In BALB/c nude mice, the presence of hFTH and GFP expressing metastatic melanoma cells in deep seated axillary LNs was demonstrated as areas of low T2* on MRI, but the same LNs were not visible by fluorescence imaging because the light was unable to penetrate the tissue. Furthermore, the metastatic volume of each LN, which was assessed by cumulative histogram analysis of the T2* MRI data, correlated well with tumor burden, which was determined by histology (r = -0.8773, p = 0.0001).
This study shows that MRI and fluorescence imaging of transplanted cells or metastatic cancer cells at molecular and cellular levels can be performed simultaneously using my bimodal lentiviral vector system. In addition, this techniques can be used to monitor tumor growth, regression during cell and gene-based therapy in deep tissues.
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dc.description.tableofcontentsContents
Overall Introduction………………………………………………………….......……………....1
Chapter 1. In vivo imaging of Tumor Transduced with Bimodal Lentiviral Vector Encoding Human Ferritin and Green Fluorescent Protein on a 1.5T Clinical Magnetic Resonance Scanner …...3
1.1 Introduction ……………………………………………………………………………....3
1.2 Material and Methods ……………………………………………………………………5
1.2.1 Construction of dual promoter lentiviral vectors encoding hFTH and GFP…………..5
1.2.2 Lentivirus production ………………………………………………………………… 5
1.2.3 Generation of MCF-7 and F-98 cells stably expressing myc-hFTH and GFP ………...5
1.2.4 Fluorescence-activated cell sorting analysis ………………………....………………..6
1.2.5 RT-PCR ………………………………………………………………………………...6
1.2.6 Western blot ……………………………………………………………………………6
1.2.7 Assessment of iron accumulation ……………………………………………………...7
1.2.8 Immunofluorescence …………………………………………………………………..7
1.2.9 Cell toxicity assay …………………...………………………………………………...8
1.2.10 Determination of the mitochondrial membrane potential ……………………………8
1.2.11 Tumor model ……………………………………………………………………….…8
1.2.12 Fluorescence imaging ………………………………………………………………...8
1.2.13 MRI….………………………………………………………………………………..9
1.2.14 Statistical analysis …...……………………………………………………………….9
1.3 Results …………………………………………………………………………………...11
1.3.1 Expression of myc-hFTH and GFP in cancer cells …………………………………..11
iv
1.3.2 Increased iron accumulation in cells expressing myc-hFTH …………....…………....11
1.3.3 In vitro MRI and fluorescence imaging of myc-hFTH cells ………………………....12
1.3.4 In vivo MRI and fluorescence imaging of myc-hFTH tumors …………………….....16
1.3.5 Histologic analysis of myc-hFTH tumors …………....………………………………16
1.4 Discussion …………………………………………………………………………........19
1.5 References ………………………………………………………………………...........23
Chapter 2. Imaging and Quantification of Metastatic Melanoma Cells in Lymph Nodes with a Ferritin MR Reporter in Living Mice ………..27
2.1 Introduction ………………………………………………………………………….….27
2.2 Material and Methods ……………………………………………………………….….29
2.2.1 myc-hFTH and GFP expression in B16F10 melanoma cell line ………………...…...29
2.2.2 Fluorescence-activated cell sorting analysis …....………………………………..…..29
2.2.3 Western blot ………………………………………………………………………......29
2.2.4 Immunocytochemistry ……………………………………………………………..…30
2.2.5 Cell viability assay ……………………………………………………………….…..30
2.2.6 Determination of in vitro iron concentration …………………………………………30
2.2.7 Preparation of cell pellets for in vitro imaging …………………………………….…31
2.2.8 Metastatic LN model …………………………………………………………………31
2.2.9 MRI …………………………………………………………………………………...31
2.2.10 Fluorescence imaging ……………………………………………………………….32
2.2.11 Analysis of MRI data ……………...………………………………………………...32
2.2.12 Histological analysis ………………………………………………………………...34
2.2.13 Statistical analysis ……………………………………………………………….…..35
2.3 Results …………………………………………………………………………………...36
v
2.3.1 In vitro analysis of control and myc-hFTH cells …………………………………......36
2.3.2 In vivo MRI, in vivo and ex vivo fluorescence imaging and hitological analysis …….36
2.4 Discussion …………………………………………………………………………........44
2.5 References ………………………………………………………………………...........48
Overall Conclusions……...…………......…………..…………………………………………..52
국문 초록 (Abstract in Korean)………………….……....53
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dc.formatapplication/pdf-
dc.format.extent1658284 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjecthuman ferritin heavy chain (hFTH)-
dc.subjectgreen fluorescent protein (GFP)-
dc.subjectMR reporter gene-
dc.subjectMagnetic resonance (MR)-
dc.subject.ddc616-
dc.titleIn vivo mr imaging and quantification of primary and metastatic cancers using biomodal lentiviral vector encoding human ferritin and green fluorescent protein-
dc.title.alternative사람 페리틴과 녹색형광단백질 동시 발현 렌티바이러스 벡터를 이용한 원발성 및 전이암의 자기공명영상화 및 정량화 기법에 대한 연구-
dc.typeThesis-
dc.description.degreeDoctor-
dc.citation.pages54-
dc.contributor.affiliation의과대학 협동과정 방사선응용생명과학전공-
dc.date.awarded2015-02-
Appears in Collections:
College of Medicine/School of Medicine (의과대학/대학원)Dept. of Radiation Applied Life Science (대학원 협동과정 방사선응용생명과학전공)Theses (Ph.D. / Sc.D._협동과정 방사선응용생명과학전공)
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