Functional analysis of angiogenic factors in tumor cells and endothelia:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | German |
| Veröffentlicht: |
2014
|
| Schlagworte: | |
| Online-Zugang: | kostenfrei Inhaltsverzeichnis |
| Beschreibung: | Zsfassung in dt. und engl. Sprache |
| Beschreibung: | IV, 104 Bl. Ill., graph. Darst. |
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Datensatz im Suchindex
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| adam_text | FUNCTIONAL ANALYSIS OF ANGIOGENIC FACTORS
IN TUMOR CELLS AND ENDOTHELIA
FUNKTIONELLE ANALYSE ANGIOGENER FAKTOREN
IN TUMORZELLEN UND ENDOTHELIEN
DOCTORAL THESIS FOR A DOCTORAL DEGREE
AT THE GRADUATE SCHOOL OF LIFE SCIENCES,
JULIUS-MAXIMILIANS-UNIVERSITAT WURZBURG,
SECTION BIOMEDICINE
SUBMITTED BY
MELANIE HEIN
FROM
MAINZ
WURZBURG 2014
HTTP://D-NB.INFO/1052956300
TABLE OF CONTENTS
TABLE OF CONTENTS
TABLE OF CONTENTS I
1 SUMMARY 1
2 ZUSAMMENFASSUNG 3
3 INTRODUCTION 5
3.1 ANTI-ANGIOGENIC THERAPY OF HUMAN TUMOR CELLS 5
3.1.1 TUMOR ANGIOGENESIS 5
3.1.2 ANGIOGENIC PATHWAY MOLECULES 6
3.1.3 BIOLOGICAL FUNCTION OF VEGF-A 8
3.1.4 THE COMPLEXITY OF CANCER 9
3.1.5 BEVACIZUMAB - A MONOCLONAL ANTIBODY FOR ANTI-ANGIOGENIC TREATMENT
AND
CANCER THERAPY 10
3.1.6 TUMOR CELL LINES FOR THE EXPERIMENTAL RESEARCH OF CANCER 11
3.1.7 AIM OF THE THESIS
- PART 1 12
3.2 THE ROLE OF HEY GENES DURING VASCULAR DEVELOPMENT 13
3.2.1 BLOOD VESSEL DEVELOPMENT AND FUNCTION OF THE ENDOTHELIUM 13
3.2.2 THE CANONICAL NOTCH SIGNALING PATHWAY 13
3.2.3 HEY BASIC HELIX-LOOP-HELIX TRANSCRIPTION FACTORS 15
3.2.4 EXPRESSION AND FUNCTION OF HEY GENES DURING CARDIOVASCULAR
DEVELOPMENT ....16
3.2.5 THE ROLE OF NOTCH SIGNALING AND HEY GENES IN ENDOTHELIAL CELL
SPECIFICATION ....17
3.2.6 NOTCH SIGNALING AND HEY GENE FUNCTIONS IN EMBRYONIC STEM CELLS AND
ENDOTHELIAL CELLS 18
3.2.7 AIM OF THE THESIS-PART II 19
4 MATERIAL 20
4.1 CHEMICALS 20
4.2 PLASMIDS 20
4.3 CELL CULTURE MATERIAL 21
4.3.1 CELL CULTURE MEDIA AND SUPPLEMENTS 21
4.3.2 EUKARYOTIC CELL LINES 22
4.3.2.1 HUMAN TUMOR CELL LINES 22
4.3.2.2 HUMAN ENDOTHELIAL CELL LINES 22
4.3.2.3 MOUSE EMBRYONIC STEM CELL LINES 22
4.3.2.4 MOUSE ENDOTHELIAL CELL LINES 22
4.4 BUFFERS AND SOLUTIONS 23
4.5 OLIGONUCLEOTIDES 23
4.5.1 HUMAN OLIGONUCLEOTIDES FOR QUANTITATIVE REAL-TIME PCR 24
I
TABLE OF CONTENTS
4.5.2 MURINE OLIGONUCLEOTIDES FOR QUANTITATIVE REAL-TIME PCR 24
4.5.3 ADDITIONAL OLIGONUCLEOTIDES 27
4.6 ANTIBODIES 27
4.6.1 ANTIBODIES FOR WESTERN BLOT ANALYSIS 27
4.6.2 ANTIBODIES FOR IMMUNOFLUORESCENCE 27
4.6.3 ANTIBODIES FOR FLOW CYTOMETRY 28
4.6.4 HUMANIZED MONOCLONAL ANTIBODIES 28
4.7 ENZYMES AND KITS 28
4.8 TECHNICAL DEVICES 28
5 METHODS 29
5.1 CELL CULTURE 29
5.1.1 CELL CULTURE OF HUMAN TUMOR CELL LINES AND HUMAN ENDOTHELIAL CELLS
29
5.1.2 CELL CULTURE UNDER HYPOXIC CONDITIONS AND BEVACIZUMAB TREATMENT 29
5.1.3 CELL CULTURE OF MOUSE EMBRYONIC STEM CELLS 29
5.1.4 ESTABLISHMENT OF STABLE MOUSE EMBRYONIC STEM CELL LINES 29
5.1.4.1 ELECTROPORATION 29
5.1.4.2 TRANSFECTION 30
5.1.4.3 SELECTION OF STABLE CLONES 30
5.1.5 DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS INTO ENDOTHELIAL
CELLS 30
5.2 CELL BIOLOGICAL METHODS 31
5.2.1 CELL PROLIFERATION ASSAY 31
5.2.2 CELL MIGRATION ASSAY 31
5.2.3 TUBE FORMATION ASSAY 31
5.2.4 IMMUNOFLUORESCENCE 31
5.2.5 ALKALINE PHOSPHATASE STAINING 32
5.3 MOLECULAR BIOLOGICAL METHODS 32
5.3.1 MOLECULAR CLONING 32
5.3.2 REVERSE TRANSCRIPTION AND QUANTITATIVE REAL-TIME PCR (QRT-PCR) 32
5.3.3 ISOLATION OF GENOMIC DNA AND PCR ANALYSIS 33
5.3.4 AGAROSE GEL ELECTROPHORESIS 33
5.3.5 RNA-SEQUENCING 33
5.4 PROTEIN BIOCHEMICAL METHODS 34
5.4.1 WESTERN BLOT ANALYSIS 34
5.4.2 FLOW CYTOMETRY 34
6 RESULTS 35
6.1 IMPACT OF BEVACIZUMAB TREATMENT ON HUMAN TUMOR CELL LINES IN VITRO
35
II
TABLE OF CONTENTS
6.1.1 TUMOR CELL AND CELL SURFACE EXPRESSION OF VEGF RECEPTORS 35
6.1.2 HYPOXIC INDUCTION OF VEGF 38
6.1.3 REGULATION OF ANGIOGENIC PATHWAY MOLECULES UPON BEVACIZUMAB
TREATMENT ...38
6.1.4 EFFECTS OF BEVACIZUMAB ON BIOLOGICAL FUNCTIONS 40
6.1.4.1 EFFECTS OF BEVACIZUMAB ON TUMOR CELL SURVIVAL 40
6.1.4.2 EFFECTS OF BEVACIZUMAB ON TUMOR CELL PROLIFERATION 41
6.1.4.3 EFFECTS OF BEVACIZUMAB ON TUMOR CELL MIGRATION 43
6.2 IN VITRO DIFFERENTIATION OF HEY DEFICIENT AND HEY RE-EXPRESSING
ENDOTHELIA TO DELINEATE
VASCULAR HEY GENE FUNCTIONS 45
6.2.1 GENERATION OF STABLE EMBRYONIC STEM CELL LINES FOR ENDOTHELIAL
CELL SELECTION ...45
6.2.1.1 ESTABLISHMENT OF A DIRECTED ESC-EC DIFFERENTIATION METHOD 46
6.2.1.2 CHARACTERIZATION OF EMBRYONIC STEM CELL DERIVED ENDOTHELIAL
CELLS 49
6.2.2 GENERATION OF HEY INDUCIBLE EMBRYONIC STEM CELL LINES 53
6.2.2.1 DIFFERENTIATION AND CHARACTERIZATION OF HEY RE-EXPRESSING
ENDOTHELIAL CELLS..
54
6.2.3 RNA-SEQUENCING OF HEY INDUCIBLE CELL LINES 57
6.2.3.1 RNA-SEQUENCING OF HEY INDUCIBLE EMBRYONIC STEM CELLS AND
ENDOTHELIAL
CELLS AFTER HEY
1/2 OVEREXPRESSION 57
6.2.3.2 COMPARISON OF EMBRYONIC STEM CELLS AND ENDOTHELIAL CELLS AFTER
HEY
OVEREXPRESSION BY RNA-SEQUENCING 59
6.2.3.3 TARGET GENE VALIDATION IN EMBRYONIC STEM CELLS AND ENDOTHELIAL
CELLS 60
DISCUSSION 63
7.1 IMPACT OF BEVACIZUMAB TREATMENT ON HUMAN TUMOR CELL LINES IN VITRO
63
7.1.1 TUMOR CELL EXPRESSION OF VEGF AND VEGF RECEPTORS UNDER HYPOXIA AND
BEVACIZUMAB TREATMENT 63
7.1.2 ANGIOGENIC GENE EXPRESSION ANALYSIS UNDER HYPOXIA AND BEVACIZUMAB
TREATMENT 64
7.1.3 EFFECTS OF BEVACIZUMAB TREATMENT ON TUMOR CELL SURVIVAL 65
7.1.4 PROLIFERATION AND MIGRATION ANALYSIS OF HUMAN TUMOR CELL LINES
AFTER
BEVACIZUMAB TREATMENT 66
7.1.5 CONCLUSION 67
7.2 IN VITRO DIFFERENTIATION OF HEY DEFICIENT AND HEY RE-EXPRESSING
ENDOTHELIA TO DELINEATE
VASCULAR HEY GENE FUNCTIONS 69
7.2.1 HEY DEFICIENT ENDOTHELIA EFFICIENTLY DIFFERENTIATE IN VITRO 69
7.2.2 ESC-DERIVED ENDOTHELIAL CELLS SHOW TYPICAL ENDOTHELIAL
CHARACTERISTICS 70
7.2.3 RE-EXPRESSION OF HEY PROTEINS IN HEY 1/2 DOUBLE KNOCKOUT CELLS 71
7.2.4 NO SIGNIFICANT CHANGE IN ARTERIAL VERSUS VENOUS MARKER GENE
EXPRESSION UPON
HEY OVEREXPRESSION IN ESC-DERIVED ENDOTHELIAL CELLS 73
III
TABLE OF CONTENTS
7.2.5 WHOLE TRANSCRIPTOME ANALYSIS REVEALED MORE HEY DOWNSTREAM TARGET
GENES IN
EMBRYONIC STEM CELLS THAN IN ENDOTHELIAL CELLS 74
7.2.6 CONCLUSION 76
8 REFERENCES 77
9 APPENDIX 85
9.1 ABBREVIATIONS 85
9.2 RNA-SEQDATA 88
9.3 PUBLICATIONS 101
9.4 ORAL PRESENTATIONS AND POSTERS 101
9.5 CURRICULUM VITAE 102
9.6 ACKNOWLEDGEMENTS 103
9.7 AFFIDAVIT 104
IV
|
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| spelling | Hein, Melanie Verfasser (DE-588)1049995422 aut Functional analysis of angiogenic factors in tumor cells and endothelia von Melanie Hein Funktionelle Analyse angiogener Faktoren in Tumorzellen und Endothelien 2014 IV, 104 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Zsfassung in dt. und engl. Sprache Würzburg, Univ., Diss., 2014 Angiogenese (DE-588)4142449-9 gnd rswk-swf Antiangiogenese (DE-588)1025111168 gnd rswk-swf Krebs Medizin (DE-588)4073781-0 gnd rswk-swf Monoklonaler Antikörper (DE-588)4040094-3 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Krebs Medizin (DE-588)4073781-0 s Angiogenese (DE-588)4142449-9 s Monoklonaler Antikörper (DE-588)4040094-3 s Antiangiogenese (DE-588)1025111168 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:20-opus-93863 https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-93863 Resolving-System kostenfrei Volltext DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027250763&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Hein, Melanie Functional analysis of angiogenic factors in tumor cells and endothelia Angiogenese (DE-588)4142449-9 gnd Antiangiogenese (DE-588)1025111168 gnd Krebs Medizin (DE-588)4073781-0 gnd Monoklonaler Antikörper (DE-588)4040094-3 gnd |
| subject_GND | (DE-588)4142449-9 (DE-588)1025111168 (DE-588)4073781-0 (DE-588)4040094-3 (DE-588)4113937-9 |
| title | Functional analysis of angiogenic factors in tumor cells and endothelia |
| title_alt | Funktionelle Analyse angiogener Faktoren in Tumorzellen und Endothelien |
| title_auth | Functional analysis of angiogenic factors in tumor cells and endothelia |
| title_exact_search | Functional analysis of angiogenic factors in tumor cells and endothelia |
| title_full | Functional analysis of angiogenic factors in tumor cells and endothelia von Melanie Hein |
| title_fullStr | Functional analysis of angiogenic factors in tumor cells and endothelia von Melanie Hein |
| title_full_unstemmed | Functional analysis of angiogenic factors in tumor cells and endothelia von Melanie Hein |
| title_short | Functional analysis of angiogenic factors in tumor cells and endothelia |
| title_sort | functional analysis of angiogenic factors in tumor cells and endothelia |
| topic | Angiogenese (DE-588)4142449-9 gnd Antiangiogenese (DE-588)1025111168 gnd Krebs Medizin (DE-588)4073781-0 gnd Monoklonaler Antikörper (DE-588)4040094-3 gnd |
| topic_facet | Angiogenese Antiangiogenese Krebs Medizin Monoklonaler Antikörper Hochschulschrift |
| url | https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-93863 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027250763&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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