Elucidation of fibroblast growth factor 1 and 2 effects on the adipogenesis and osteogenesis of primary human bone marrow stromal cells: = Aufklärung der Effekte von Fibroblasten-Wachstumsfaktor 1 und 2 auf die Adipogenese und Osteogenese von primären humanen Knochenmark-Stroma-Zellen
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
Würzburg
2015
|
| Schlagworte: | |
| Online-Zugang: | Volltext Inhaltsverzeichnis |
| Beschreibung: | 179, XXVIII Seiten Diagramme |
Internformat
MARC
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| 245 | 1 | 0 | |a Elucidation of fibroblast growth factor 1 and 2 effects on the adipogenesis and osteogenesis of primary human bone marrow stromal cells |b = Aufklärung der Effekte von Fibroblasten-Wachstumsfaktor 1 und 2 auf die Adipogenese und Osteogenese von primären humanen Knochenmark-Stroma-Zellen |c submitted by Meike Simann, born in Soltau |
| 246 | 1 | 1 | |a Aufklärung der Effekte von Fibroblasten-Wachstumsfaktor 1 und 2 auf die Adipogenese und Osteogenese von primären humanen Knochenmark-Stroma-Zellen |
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Datensatz im Suchindex
| _version_ | 1804176688479731712 |
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| adam_text | ELUCIDATION OF FIBROBLAST GROWTH FACTOR 1 AND 2 EFFECTS ON
THE ADIPOGENESIS AND OSTEOGENESIS OF PRIMARY HUMAN
BONE MARROW STROMAL CELLS
AUFKLAERUNG DER EFFEKTE VON FIBROBLASTEN-WACHSTUMSFAKTOR 1 UND 2
AUF DIE ADIPOGENESE UND OSTEOGENESE VON PRIMAEREN HUMANEN
KNOCHENMARK-STROMA-ZELLEN
DOCTORAL THESIS FOR A DOCTORAL DEGREE AT THE
GRADUATE SCHOOL OF LIFE SCIENCES (GSLS),
JULIUS-MAXIMILIANS-UNIVERSITAET WUERZBURG,
SECTION BIOMEDICINE
SUBMITTED BY
MEIKE SIMANN
BOM IN SOLTAU
WUERZBURG 2015
CONTENTS
ACKNOWLEDGMENTS V
ABSTRACT VII
ZUSAMMENFASSUNG IX
I INTRODUCTION 1
1 OSTEOPOROSIS AND THE ADIPOESE TISSUE ACCUMULATION IN BONE MARROW 3
1.1 BONE DISORDER OF HIGH CLINICAL RE LE V A N C E
.............................................................................
3
1.2 BONE RESORPTION AND NEW FORMATION
...................................................................................
4
1.3 PREFERENTIAL ADIPOGENESIS AND FAT ACCUM
ULATION..................................................................
5
2 HUMAN BONE MARROW STROMAL CELLS 7
2.1 DIFFERENTIATION INTO THE ADIPOCYTIC AND OSTEOBLASTIC LIN E A G E
.............................................. 7
2.2 LINEAGE PLASTICITY AND
CONVERSION.........................................................................................
7
2.3 IN VITRO MODEL FOR HBMSC DIFFERENTIATION AND CO N V ERSIO N
................................................. 8
3 FIBROBLAST GROWTH FACTORS AND THEIR RECEPTORS 11
3.1 FIBROBLAST GROWTH
FACTORS.......................................................................................................
11
3.2 FGF RE C E P TO
RS........................................................................................................................
11
3.3 POLE IN OSTEOGENESIS AND BONE FORMATION
.............................................................................
13
4 AIM OF MY DOCTORAL RESEARCH 15
II MATERIALS AND METHODS 17
5 MATERIALS 19
5.1 E QUIPM
ENT................................................................................
19
5.2 SOFTWARE AND ONLINE SO U
RCES.................................................................................................
21
5.3 CONSUMABLES
........................................................................................................................
22
5.4 CHEMICALS AND REAGENTS
.......................................................................................................
23
5.5 BUFFERS AND
SOLUTIONS.............................................................................................................
27
5.5.1 H IS TO LO G Y
..................................................................................................................
27
5.5.2 ALKALINE PHOSPHATASE (ALP) A S S A Y
..........................................................................
28
5.5.3 MINERALIZATION A S S A Y
.................................................................................................
29
5.5.4 RNA ISOLATION AND P C R
............................................................................................
29
5.5.5 PROTEIN ISOLATION
.......................................................................................................
29
5.5.6 SDS-PAGE
.................................................................
30
5.5.7 WESTERN BLOTTING
.......................................................................................................
31
5.6 CELL CULTURE MEDIA AND A D D ITIV E S
.........................................................................................
32
5.7 K I T S ...................................................... 34
5.8 P RIM
ERS...................................................................................................................................
35
5.9 E N ZY M
ES................................................................................................................................
36
5.10 A
NTIBODIES............................................................................................................................
37
6 METHODS 39
6.1 CELL C U LTU R E
...........................................................................................................................
39
6.1.1 ISOLATION OF HUMAN BONE MARROW STROMAL C E L LS
....................................................... 39
6.1.2 CULTIVATION OF PRIMARY HBMSCS
..............................................................................
39
6.2 MULTIPOTENCY TE S TIN G
.............................................................................................................
39
6.2.1 ADIPOGENIC DIFFERENTIATION AND CONV ERSION
............................................................
40
6.2.2 OSTEOGENIC DIFFERENTIATION AND CONVERSION
............................................................
40
6.3 INHIBITOR
EXPERIMENTS.............................................................................................................
40
6.4 HISTOLOGICAL
STAININGS.............................................................................................................
42
6.4.1 OIL RED O
STAINING.......................................................................................................
42
6.4.2 ALP STAINING
.............................................................................................................
42
6.4.3 ALIZARIN RED S S TA IN IN G
...............................................................................................
42
6.5 SPECIFIC ASSAYS QUANTIFYING DIFFERENTIATION AND CONVERSION
.............................................. 42
6.5.1 LIPID DROPLET A S S A Y
....................................................................................................
42
6.5.2 ALP ACTIVITY A S S A Y
....................................................................................................
43
6.5.3 MINERALIZATION A S S A Y
..................................................................................................
44
6.6 MRNA EXPRESSION
ANALYSIS....................................................................................................
44
6.6.1 RNA
ISOLATION.............................................................................................................
44
6.6.2 REVERSE
TRANSCRIPTION..................................................................................................
45
6.6.3 SEMIQUANTITATIVE REVERSE TRANSCRIPTASE PCR (RT-PCR)
............................
45
6.6.4 QUANTITATIVE REAL-TIME REVERSE TRANSCRIPTASE PCR (QP C R )
.....................................
46
6.6.5 PRIMER DESIGN AND ESTABLISHMENT FOR Q P C R
............................................................
47
6.7 PROTEIN EXPRESSION A N A LY S E S
..................................................................................................
47
6.7.1 PROTEIN ISOLATION
.......................................................................................................
47
6.7.2 WESTERN BLOTTING
.......................................................................................................
47
6.8 STATISTICAL
ANALYSIS...................................................................................................................
48
III RESULTS 51
7 FGF1 AND FGF2 PREVENT ADIPOGENIC DIFFERENTIATION AND CONVERSION 53
7.1 FORMATION OF LIPID DROPLETS
.................................................................................................
53
7.1.1 FGF1 COMPLETELY INHIBITS LIPID DROPLET FORMATION
................................................. 53
7.1.2 FGF2 DISTINCTLY REDUCES LIPID DROPLET FO RM A TIO N
.................................................... 56
7.2 EXPRESSION PROFILE OF ADIPOGENIC MARKER G E N E S
.................................................................. 57
7.2.1 FGF1 COMPLETELY PREVENTS ADIPOGENIC MARKER MRNA EX P RESSIO N
.......................
57
7.2.2 FGF2 MARKEDLY REDUCES ADIPOGENIC MARKER MRNA E X P RE S S IO N
..........................
60
7.3 EXPRESSION OF OSTEOGENIC MARKERS DURING AD IP O G EN
ESIS.................................................... 62
7.3.1 ADIPOGENIC CONVERSION DOWNREGULATES OSTEOGENIC KEY M A RK E RS
..........................
62
7.3.2 FGF1 UPREGULATES INHIBITORY MINERALIZATION MARKER G E N E S
..................................
65
7.3.3 FGF2 INCREASES INHIBITORY MINERALIZATION MARKER GENE EXPRESSION
.
......................
66
8 FGF1 AND FGF2 AFFECT OSTEOGENIC DIFFERENTIATION AND CONVERSION 69
8.1 REDUCTION OF MATRIX
MINERALIZATION......................................................................................
69
8.2 GENE EXPRESSION AND ENZYME ACTIVITY OF A L P
..................................................................... 73
8.2.1 FGF1 DOWNREGULATES ALPL MRNA EXPRESSION WHILE ONLY PARTLY
DECREASING ENZYME A C TIV ITY
......................................................................................
73
8.2.2 FGF2 MARKEDLY DECREASES ALPL MRNA EXPRESSION WHILE PARTLY
REDUCING ASSOCIATED ENZYME ACTIV ITY
........................................................................
75
8.3 EARLY OSTEOGENIC MARKER GENE E X P RE SSIO N
...........................................................................
78
8.3.1 FGF1 DOWNREGULATES RUNX2 AND BMP4
EXPRESSION.............................................. 78
8.3.2 FGF2 REDUCES RUNX2 AND BMP4 EX P RESSIO N
.........................................................
81
8.4 LATER MARKER GENES OF
OSTEOGENESIS......................................................................................
83
8.4.1 FGF1 STRONGLY DOWNREGULATES COL1A1 AND IBSP EXPRESSION WHILE
MILDLY AUGMENTING OC
EXPRESSION...........................................................................
83
8.4.2 FGF2 MARKEDLY DOWNREGULATES COL1A1 AND IBSP WHILE ONLY
SLIGHTLY UPREGULATING OC
.........................................................................................
87
8.5 MARKER GENES OF MINERALIZATION.................................. 91
8.5.1 FGF1 MARKEDLY UPREGULATES ANKH AND OPN EXPRESSION WHILE
DECREASING ENPP1
EXPRESSION...................................................................................
91
8.5.2 FGF2 DECREASES ENPP1 EXPRESSION AND ONLY SLIGHTLY AFFECTS ANKH
AND OPN MRNA L E V E L S
............................................................................................
95
8.5.3 OPN PROTEIN
EXPRESSION............................................................................................
98
8.5.4 ANKH INHIBITION DOES NOT ABOLISH THE ANTI-MINERALIZING EFFECT OF
FGF1
..............
99
9 FGF1 EFFECTS ARE MEDIATED VIA FGFR1 AND ERK1/2 SIGNALING 101
9.1 INHIBITION OF FGFR1 RESCUES THE FGF1
PHENOTYPE..................................................................
101
9.2 GENE EXPRESSION OF FGFR1 AND
FGFR2...................................................................................
103
9.2.1 FGFR1 IS MORE STABLY EXPRESSED THAN FGFR2 DURING
ADIPOGENIC DIFFERENTIATION AND CONVERSION
...............................................................
103
9.2.2 FGFR1 IS MORE STEADILY EXPRESSED THAN FGFR2 IN
OSTEOGENIC DIFFERENTIATION AND
CONVERSION..................................................................
106
9.3 PKC, JNK, AND P38-MAPK ARE NOT RESPONSIBLE FOR FGF1 SIGNAL
TRANSDUCTION
....................
111
9.3.1 PKC
SIGNALING................................................................................................................111
9.3.2 JNK P A TH W A Y
................................................................................................................114
9.3.3 P38-MAPK
SIGNALING............................................................................................
..
. 115
9.3.4 P38-MAPK AND MEK1/2 P A TH W A Y S
...........................................................................116
9.4 ERK1/2 IS CRUCIAL FOR FGF1 SIGNAL TRANSDUCTION IN ADIPOGENIC AND
OSTEOGENIC DIFFERENTIATION AS WELL AS OSTEOGENIC CO N V ERSIO N
..............................................117
IV DISCUSSION 121
10 EFFECT OF FGF1 AND FGF2 ON HBMSC DIFFERENTIATION AND CONVERSION 123
11 IMPACT OF FGF1 AND FGF2 ON THE ADIPOGENESIS OF HBMSCS 125
11.1 INHIBITION OF ADIPOGENIC DIFFERENTIATION BY F G F 1
.................................................................. 125
11.2 REDUCTION OF ADIPOGENIC DIFFERENTIATION BY F G F 2
.................................................................. 127
11.3 IMPACT ON ADIPOGENIC
CONVERSION............................................................................................128
11.4 SIGNALING PATHWAYS MEDIATING THE ANTI-ADIPOGENIC E F F E C
T....................................................132
11.5 SUMMARY ADIPOGENIC DIFFERENTIATION AND CONVERSION
............................................................
134
12 IMPACT OF FGF1 AND FGF2 ON THE OSTEOGENESIS OF HBMSCS 139
12.1 REDUCTION OF THE ECM MINERALIZATION
...................................................................................
139
12.2 UPREGULATION OF MARKERS INHIBITING ECM MINERALIZATION
....................................................141
12.3 DOWNREGULATION OF OSTEOGENIC MARKERS FOR ECM FO RM ATIO N
.................................................142
12.4 DOWNREGULATION OF MARKERS INITIATING OSTEOGENIC COM M ITM ENT
...........................................
144
12.5 IMPACT ON OSTEOGENIC C O N V ERSIO N
............................................................................................145
12.6 SIGNALING PATHWAYS MEDIATING THE ANTI-OSTEOGENIC EFFECT
....................................................147
12.7 SUMMARY OSTEOGENIC DIFFERENTIATION AND CONVERSION
............................................................
150
13 CONCLUSIONS 153
14 PERSPECTIVES 155
BIBLIOGRAPHY 179
V APPENDIX XI
LIST OF FIGURES XV
LIST OF TABLES XVII
LIST OF ABBREVIATIONS XIX
CURRICULUM VITAE XXV
LIST OF PUBLICATION XXVII
|
| any_adam_object | 1 |
| author | Simann, Meike 1984- |
| author_GND | (DE-588)1116296160 |
| author_facet | Simann, Meike 1984- |
| author_role | aut |
| author_sort | Simann, Meike 1984- |
| author_variant | m s ms |
| building | Verbundindex |
| bvnumber | BV043828180 |
| collection | ebook |
| ctrlnum | (OCoLC)962085097 (DE-599)BVBBV043828180 |
| dewey-full | 610 |
| dewey-hundreds | 600 - Technology (Applied sciences) |
| dewey-ones | 610 - Medicine and health |
| dewey-raw | 610 |
| dewey-search | 610 |
| dewey-sort | 3610 |
| dewey-tens | 610 - Medicine and health |
| discipline | Medizin |
| format | Thesis Book |
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| owner | DE-384 DE-473 DE-BY-UBG DE-703 DE-1051 DE-824 DE-29 DE-12 DE-91 DE-BY-TUM DE-19 DE-BY-UBM DE-1049 DE-92 DE-739 DE-898 DE-BY-UBR DE-355 DE-BY-UBR DE-706 DE-20 DE-1102 DE-860 DE-2174 |
| owner_facet | DE-384 DE-473 DE-BY-UBG DE-703 DE-1051 DE-824 DE-29 DE-12 DE-91 DE-BY-TUM DE-19 DE-BY-UBM DE-1049 DE-92 DE-739 DE-898 DE-BY-UBR DE-355 DE-BY-UBR DE-706 DE-20 DE-1102 DE-860 DE-2174 |
| physical | 179, XXVIII Seiten Diagramme |
| psigel | ebook |
| publishDate | 2015 |
| publishDateSearch | 2015 |
| publishDateSort | 2015 |
| record_format | marc |
| spelling | Simann, Meike 1984- Verfasser (DE-588)1116296160 aut Elucidation of fibroblast growth factor 1 and 2 effects on the adipogenesis and osteogenesis of primary human bone marrow stromal cells = Aufklärung der Effekte von Fibroblasten-Wachstumsfaktor 1 und 2 auf die Adipogenese und Osteogenese von primären humanen Knochenmark-Stroma-Zellen submitted by Meike Simann, born in Soltau Aufklärung der Effekte von Fibroblasten-Wachstumsfaktor 1 und 2 auf die Adipogenese und Osteogenese von primären humanen Knochenmark-Stroma-Zellen Würzburg 2015 179, XXVIII Seiten Diagramme txt rdacontent n rdamedia nc rdacarrier Dissertation Julius-Maximilians-Universität Würzburg 2015 Zusammenfassung in deutscher und englischer Sprache Genexpression (DE-588)4020136-3 gnd rswk-swf Fibroblastenwachstumsfaktor (DE-588)4310092-2 gnd rswk-swf Mesenchymzelle (DE-588)4278308-2 gnd rswk-swf Osteoporose (DE-588)4044021-7 gnd rswk-swf Fettzelle (DE-588)4154243-5 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Fibroblastenwachstumsfaktor (DE-588)4310092-2 s Genexpression (DE-588)4020136-3 s Mesenchymzelle (DE-588)4278308-2 s Osteoporose (DE-588)4044021-7 s Fettzelle (DE-588)4154243-5 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:20-opus-119322 https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-119322 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=029239028&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Simann, Meike 1984- Elucidation of fibroblast growth factor 1 and 2 effects on the adipogenesis and osteogenesis of primary human bone marrow stromal cells = Aufklärung der Effekte von Fibroblasten-Wachstumsfaktor 1 und 2 auf die Adipogenese und Osteogenese von primären humanen Knochenmark-Stroma-Zellen Genexpression (DE-588)4020136-3 gnd Fibroblastenwachstumsfaktor (DE-588)4310092-2 gnd Mesenchymzelle (DE-588)4278308-2 gnd Osteoporose (DE-588)4044021-7 gnd Fettzelle (DE-588)4154243-5 gnd |
| subject_GND | (DE-588)4020136-3 (DE-588)4310092-2 (DE-588)4278308-2 (DE-588)4044021-7 (DE-588)4154243-5 (DE-588)4113937-9 |
| title | Elucidation of fibroblast growth factor 1 and 2 effects on the adipogenesis and osteogenesis of primary human bone marrow stromal cells = Aufklärung der Effekte von Fibroblasten-Wachstumsfaktor 1 und 2 auf die Adipogenese und Osteogenese von primären humanen Knochenmark-Stroma-Zellen |
| title_alt | Aufklärung der Effekte von Fibroblasten-Wachstumsfaktor 1 und 2 auf die Adipogenese und Osteogenese von primären humanen Knochenmark-Stroma-Zellen |
| title_auth | Elucidation of fibroblast growth factor 1 and 2 effects on the adipogenesis and osteogenesis of primary human bone marrow stromal cells = Aufklärung der Effekte von Fibroblasten-Wachstumsfaktor 1 und 2 auf die Adipogenese und Osteogenese von primären humanen Knochenmark-Stroma-Zellen |
| title_exact_search | Elucidation of fibroblast growth factor 1 and 2 effects on the adipogenesis and osteogenesis of primary human bone marrow stromal cells = Aufklärung der Effekte von Fibroblasten-Wachstumsfaktor 1 und 2 auf die Adipogenese und Osteogenese von primären humanen Knochenmark-Stroma-Zellen |
| title_full | Elucidation of fibroblast growth factor 1 and 2 effects on the adipogenesis and osteogenesis of primary human bone marrow stromal cells = Aufklärung der Effekte von Fibroblasten-Wachstumsfaktor 1 und 2 auf die Adipogenese und Osteogenese von primären humanen Knochenmark-Stroma-Zellen submitted by Meike Simann, born in Soltau |
| title_fullStr | Elucidation of fibroblast growth factor 1 and 2 effects on the adipogenesis and osteogenesis of primary human bone marrow stromal cells = Aufklärung der Effekte von Fibroblasten-Wachstumsfaktor 1 und 2 auf die Adipogenese und Osteogenese von primären humanen Knochenmark-Stroma-Zellen submitted by Meike Simann, born in Soltau |
| title_full_unstemmed | Elucidation of fibroblast growth factor 1 and 2 effects on the adipogenesis and osteogenesis of primary human bone marrow stromal cells = Aufklärung der Effekte von Fibroblasten-Wachstumsfaktor 1 und 2 auf die Adipogenese und Osteogenese von primären humanen Knochenmark-Stroma-Zellen submitted by Meike Simann, born in Soltau |
| title_short | Elucidation of fibroblast growth factor 1 and 2 effects on the adipogenesis and osteogenesis of primary human bone marrow stromal cells |
| title_sort | elucidation of fibroblast growth factor 1 and 2 effects on the adipogenesis and osteogenesis of primary human bone marrow stromal cells aufklarung der effekte von fibroblasten wachstumsfaktor 1 und 2 auf die adipogenese und osteogenese von primaren humanen knochenmark stroma zellen |
| title_sub | = Aufklärung der Effekte von Fibroblasten-Wachstumsfaktor 1 und 2 auf die Adipogenese und Osteogenese von primären humanen Knochenmark-Stroma-Zellen |
| topic | Genexpression (DE-588)4020136-3 gnd Fibroblastenwachstumsfaktor (DE-588)4310092-2 gnd Mesenchymzelle (DE-588)4278308-2 gnd Osteoporose (DE-588)4044021-7 gnd Fettzelle (DE-588)4154243-5 gnd |
| topic_facet | Genexpression Fibroblastenwachstumsfaktor Mesenchymzelle Osteoporose Fettzelle Hochschulschrift |
| url | https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-119322 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029239028&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT simannmeike elucidationoffibroblastgrowthfactor1and2effectsontheadipogenesisandosteogenesisofprimaryhumanbonemarrowstromalcellsaufklarungdereffektevonfibroblastenwachstumsfaktor1und2aufdieadipogeneseundosteogenesevonprimarenhumanenknochenmarkstromazellen AT simannmeike aufklarungdereffektevonfibroblastenwachstumsfaktor1und2aufdieadipogeneseundosteogenesevonprimarenhumanenknochenmarkstromazellen |