| ABSTRACT | 第11-14页 |
| ABBREVIATIONS | 第15-17页 |
| 1. INTRODUCTION | 第17-39页 |
| 1.1 General introduction | 第17-18页 |
| 1.2 Epidemiology and pathological features of ovarian cancer | 第18-19页 |
| 1.3 Clinical features of ovarian cancer | 第19页 |
| 1.4 Molecular model of ovarian cancer | 第19-25页 |
| 1.4.1 The Ovarian surface epithelium and cortical inclusion cysts (OSE-CIC) model | 第19-20页 |
| 1.4.2 Two-pathway model | 第20-21页 |
| 1.4.3 Fallopian tube as a site of origin | 第21-25页 |
| 1.5 Molecular alteration of ovarian cancer | 第25-34页 |
| 1.5.1 p53 and ovarian cancer | 第25-27页 |
| 1.5.2 K-Ras and ovarian cancer | 第27-29页 |
| 1.5.3 c-Myc and ovarian cancer | 第29-30页 |
| 1.5.4 Pax 2 and pax8 gene with ovarian cancer | 第30-34页 |
| 1.6 Introduction of lentivirus | 第34-37页 |
| 1.6.1 The evolution history of lentiviruses | 第34-35页 |
| 1.6.2 Current use of lentiviral vectors | 第35-37页 |
| 1.7 Objectives | 第37-39页 |
| 2. MATERIALS AND METHODS | 第39-74页 |
| 2.1 Cell culture | 第39-52页 |
| 2.1.1 Experimental cell lines | 第39-40页 |
| 2.1.2 Passaging cells | 第40页 |
| 2.1.3 Growth of cells in soft agar | 第40-42页 |
| 2.1.3.1 Preparation of reagents for growth of cells in soft agar | 第40-41页 |
| 2.1.3.2 Protocol of growth of cells in soft agar | 第41-42页 |
| 2.1.4 Freezing cells | 第42页 |
| 2.1.5 Reviving frozen cells | 第42页 |
| 2.1.6 Viable Cell count | 第42-43页 |
| 2.1.7 Primary oviductal epithelial cell culture and cloning | 第43-51页 |
| 2.1.7.1 Oviductal tissue was derived from wild-type p53 mice and p53~(loxP/loxP) mice | 第43-44页 |
| 2.1.7.2 Primary oviductal cells culture from wild-type p53 mice and p53~(loxP/loxP) mice | 第44-45页 |
| 2.1.7.3 Primary oviductal clonal cells derived from oviductal cells | 第45-51页 |
| 2.1.8 Cell invasion assay | 第51-52页 |
| 2.2 Genomic DNA extraction from cells | 第52-53页 |
| 2.2.1 Protocol of genomic DNA extraction from cells | 第52-53页 |
| 2.2.2 Genotyping for the p53~(flox/flox) and p53~(Δ2-10) allele | 第53页 |
| 2.3 Purification of total RNA from cells | 第53-54页 |
| 2.4 Synthesis of first-strand cDNA from total RNA | 第54-55页 |
| 2.4.1 Protocol of preparing 10 mM dNTP Mix solution | 第54-55页 |
| 2.4.2 Protocol of synthesis of first-strand cDNA from total RNA | 第55页 |
| 2.5 Amplification of open reading frames from cDNA | 第55-56页 |
| 2.6 Plasmid constructs | 第56-57页 |
| 2.7 Subcloning of ORF PCR products | 第57-59页 |
| 2.8 Subcloning of pax2 and pax8 ORF into lentiviral vectors | 第59页 |
| 2.9 Plasmid DNA Purification | 第59-61页 |
| 2.9.1 Prepare lysogeny broth (LB) solution | 第59-60页 |
| 2.9.2 Plasmid DNA Purification using the Qiagen Miniprep kit | 第60页 |
| 2.9.3 Plasmid DNA Purification Using QIAGEN Plasmid Maxi Kits | 第60-61页 |
| 2.10 Transient transfection protocol | 第61-62页 |
| 2.11 Lentiviral vector generation | 第62-66页 |
| 2.11.1 Preparation of polyethyleneimine (PEI) stocks | 第62页 |
| 2.11.2 PEI-based transfections were performed to generate lentiviral vectors | 第62页 |
| 2.11.3 CaPO_4-based transfections were performed to generate lentiviral vectors | 第62-63页 |
| 2.11.3.1 Preparation of 2×HBS stock solutions | 第63页 |
| 2.11.3.2 Preparation of 0.5M CaPO_4 stock solutions | 第63页 |
| 2.11.4 Infection protocol | 第63-64页 |
| 2.11.5 The optimal HBS solution pH was determined for production of lentiviral vectors in HEK 293T cells | 第64-65页 |
| 2.11.6 Titration of lentiviral vector stocks | 第65-66页 |
| 2.11.7 Titer formula | 第66页 |
| 2.12 Protein extraction protocol | 第66-67页 |
| 2.12.1 Protein extraction from cells | 第66-67页 |
| 2.12.2 Protein extraction from tissues | 第67页 |
| 2.13 Western Blot Protocol | 第67-69页 |
| 2.13.1 Protein quantification | 第67页 |
| 2.13.2 Preparing the gel | 第67-68页 |
| 2.13.3 Loading and running the gel | 第68页 |
| 2.13.4 Transferring | 第68页 |
| 2.13.5 Protein transfer confirmed | 第68-69页 |
| 2.13.6 Probing | 第69页 |
| 2.13.7 ECL step and imaging | 第69页 |
| 2.14 Flow Cytometry | 第69-70页 |
| 2.15 Immunofluorescence Staining | 第70-71页 |
| 2.15.1 Preparation of a 4% paraformaldehyde fixative solution | 第70页 |
| 2.15.2 Immunofluorescence Staining Protocol | 第70-71页 |
| 2.16 Immunohistochemistry protocol | 第71-72页 |
| 2.17 Q-PCR (Relative Quantification) Protocol | 第72页 |
| 2.18 Tumorigenesis of MOSE-RM cells expressing pax2 in SCID mice | 第72-73页 |
| 2.18.1 Animal experiment groups | 第72-73页 |
| 2.18.2 Mice survive assay | 第73页 |
| 2.19 Statistical analyses | 第73-74页 |
| 3. RESULTS | 第74-184页 |
| 3.1 Development of an oviductal epithelial cell line (OECL)-model for fallopian tube cancer | 第74-96页 |
| 3.1.1 Screen out oviductal epithelial cell lines | 第74-90页 |
| 3.1.1.1 Primary oviductal epithelial cells obtained from transgenic mice | 第74页 |
| 3.1.1.2 Selection of primary oviduct clonal cells | 第74-81页 |
| 3.1.1.3 Screening of primary oviduct clonal cell lines for epithelial cell markers | 第81-90页 |
| 3.1.2 Characteristic of the oviductal epithelial cells | 第90-96页 |
| 3.1.2.1 Evaluation of the genotyping in the oviductal epithelial cells | 第90页 |
| 3.1.2.2 Evaluation pax2 and pax8 expression in oviduct epithelial cells | 第90-92页 |
| 3.1.2.3 Oviductal epithelial cell line (OECL) growth assay | 第92-96页 |
| 3.2 Cloning of the pax2 and pax8 cDNAs into lentiviral plasmids pWPI and WPI-RFP | 第96-112页 |
| 3.2.1 Amplification of the pax2 and pax8 ORF cDNAs from oviductal tissue | 第96-97页 |
| 3.2.2 Evaluation of expression of the pax2 and pax8 genes in oviductal and ovarian tissues | 第97页 |
| 3.2.3 Gel extraction cDNA of pax2 and pax8 from oviductal tissues | 第97页 |
| 3.2.4 Cloning of the pax2 and pax8 ORF cDNAs | 第97-100页 |
| 3.2.5 Subcloning of the pax2 and pax8 ORF cDNAs into the lentiviral expression vectors pWPI and pWPI-RFP | 第100页 |
| 3.2.6 Identification of the pax2 and pax8 lentiviral clones | 第100-108页 |
| 3.2.7 Functional Verification of recombinant plasmid | 第108-112页 |
| 3.3 Optimization of lentivirus production | 第112-136页 |
| 3.3.1.C omparison of different titration method of LvV stocks | 第112-114页 |
| 3.3.2 Influence of different culture media and culture duration on efficiency of PEI-mediated transfection | 第114-119页 |
| 3.3.3 Influence of cell density on the efficiency of PEI-mediated transfections | 第119-124页 |
| 3.3.4 Optimization of DNA amounts for PEI-mediated lentiviral vector production | 第124-125页 |
| 3.3.5 Comparison of lentiviral vector production using PEI- and CaPO_4-mediated transient transfection | 第125-136页 |
| 3.3.5.1 Optimization of the pH for LvV production using CaPO_4 | 第125-133页 |
| 3.3.5.2 Comparison of lentiviral vector production using PEI- and CaPO_4-mediated transient transfection | 第133-136页 |
| 3.4 Transduction of the pax2, pax8 and k-Ras genes to MOSE cell lines and OECLs | 第136-142页 |
| 3.5 The function of pax2 and pax8 genes in MOSE-RM cells | 第142-170页 |
| 3.5.1 Preliminary experiments for cell growth assay | 第142-147页 |
| 3.5.2 Effect of pax2 and pax8 on growth of MOSE-RM derived cell lines in soft agar | 第147-151页 |
| 3.5.3 k-Ras, c-Myc and pax2 gene expression assay | 第151-152页 |
| 3.5.4 Tumorigenesis of MOSE-RM cells expressing pax2 | 第152-170页 |
| 3.5.4.1 Survival assay of mice | 第152-153页 |
| 3.5.4.2 The pathological features of mice | 第153-166页 |
| 3.5.4.3 Immunohistochemical staining of mice’s tumor tissues | 第166-170页 |
| 3.6 In vitro transformation of p53~(loxP/loxP) MOSE cell, p53~(Δ2-10) MOSE cell and OECLs | 第170-184页 |
| 3.6.1 In vitro transformation of p53~(loxP/loxP) and p53~(Δ2-10) MOSE cells | 第170-176页 |
| 3.6.1.1 Transgenic p53~(loxP/loxP) and p53~(Δ2-10) MOSE cells | 第170-172页 |
| 3.6.1.2 Growth assays of transgenic p53~(loxP/loxP0 MOSE cells and p53~(Δ2-10) MOSE cells | 第172-173页 |
| 3.6.1.3 Growth of transgenic p53~(loxP/loxP) MOSE cells and p53~(Δ2-10) MOSE cells in soft agar | 第173页 |
| 3.6.1.4 Ability of transformed MOSE to invade through Transwell~(?) Membrane | 第173-176页 |
| 3.6.2 In vitro transformation of OECLs | 第176-184页 |
| 3.6.2.1 Establishment of transgenic oviductal epithelial cell lines | 第176页 |
| 3.6.2.2 Transgenic OECLs growth assay | 第176-177页 |
| 3.6.2.3 Transgenic OECLs invasion assay | 第177页 |
| 3.6.2.4 Transgenic OECLs growth assay in soft agar | 第177-184页 |
| 4. DISCUSSION | 第184-202页 |
| 4.1 LvV production | 第184-189页 |
| 4.2 Derivation of oviductal epithelial cell lines | 第189-194页 |
| 4.3 Pax gene in the derivation and development of ovarian cancer | 第194-202页 |
| Reference | 第202-229页 |
| ACKNOWLEDGEMENTS | 第229-230页 |
