Dual strategies to correct abnormal ion transports in cystic fibrosis using construct therapy

Mukoviszidose (CF) wird durch Mutationen im CFTR-Gen (cystic fibrosis transmembrane conductance regulator) verursacht. Das zugrunde liegende Problem der tödlichen Erbkrankheit ist eine unausgewogene Homöostase von Ionen- und Wassertransporten in sekretorischen Epithelien, was zu Problemen in mehrere...

Volltext Mehr ...
Verfasser: Kolonko, Anna Katharina
Weitere Beteiligte: Weber, Wolf-Michael (Gutachter)
FB/Einrichtung:FB 13: Biologie
Dokumenttypen:Dissertation/Habilitation
Medientypen:Text
Erscheinungsdatum:2019
Publikation in MIAMI:10.12.2019
Datum der letzten Änderung:10.12.2019
Angaben zur Ausgabe:[Electronic ed.]
Schlagwörter:Mukoviszidose; CFTR; ENaC; Chitosan; mRNA; Antisense-Oligonukleotide; Capsaicin Cystic fibrosis; CFTR; ENaC; chitosan; mRNA; antisense oligonucleotides; capsaicin
Fachgebiet (DDC):570: Biowissenschaften; Biologie
Lizenz:CC BY 4.0
Sprache:English
Format:PDF-Dokument
URN:urn:nbn:de:hbz:6-02199695381
Permalink:https://nbn-resolving.de/urn:nbn:de:hbz:6-02199695381
Onlinezugriff:diss_kolonko.pdf
Inhaltsverzeichnis:
  • TABLE OF CONTENTS............................................................................................................................ I
  • LIST OF FIGURES .................................................................................................................................... V
  • LIST OF TABLES ................................................................................................................................... VII
  • ABBREVIATIONS .................................................................................................................................... IX
  • 1 INTRODUCTION .............................................................................................................................. 1
  • 1.1 Chitosan .................................................................................................................. 2
  • 1.2 Nanosystems .......................................................................................................... 4
  • 1.2.1 Nanocomplexes ................................................................................................................................ 6
  • 1.2.1.1 mRNA ....................................................................................................................................... 7
  • 1.2.1.2 Antisense oligonucleotides .................................................................................................... 8
  • 1.2.2 Nanocapsules .................................................................................................................................... 9
  • 1.2.2.1 Capsaicin ...............................................................................................................................10
  • 1.2.3 Characterization of nanosystems ..................................................................................................11
  • 1.3 Lung and airway epithelium .................................................................................. 13
  • 1.4 Cystic fibrosis ........................................................................................................ 16
  • 1.4.1 Cystic fibrosis transmembrane conductance regulator ................................................................19
  • 1.4.2 Epithelial sodium channel ..............................................................................................................21
  • 1.4.1 Interaction of CFTR and ENaC ........................................................................................................23
  • 1.4.2 Treatment of cystic fibrosis lung disease ......................................................................................24
  • 1.5 Aim ....................................................................................................................... 28
  • 2 MATERIALS & METHODS ........................................................................................................ 31
  • 2.1 Materials ............................................................................................................... 31
  • 2.2 Microbiological methods ...................................................................................... 31
  • 2.2.1 Plasmid isolation from CopyCutter™ EPI400™ Escherichia coli ....................................................31
  • 2.2.1.1 Cultivation of CopyCutter™ EPI400™ Escherichia coli .........................................................32
  • 2.2.1.2 Mini preparation of plasmid DNA ........................................................................................32
  • 2.2.1.3 Maxi preparation of plasmid DNA .......................................................................................33
  • 2.3 Molecular biological methods ............................................................................... 33
  • 2.3.1 Linearization of pSTI-A120/hCFTR-cDNA .......................................................................................33
  • 2.3.2 DNA gel electrophoresis .................................................................................................................34
  • 2.3.3 In vitro transcription .......................................................................................................................35
  • 2.3.4 RNA gel electrophoresis .................................................................................................................37
  • 2.3.5 RNA isolation from cells .................................................................................................................38
  • 2.3.6 Reverse transcriptase polymerase chain reaction ........................................................................ 38
  • 2.3.7 Polymerase chain reaction ............................................................................................................. 39
  • 2.3.8 Sequencing ...................................................................................................................................... 40
  • 2.4 Preparation of nanoformulations .......................................................................... 41
  • 2.4.1 Chitosan .......................................................................................................................................... 41
  • 2.4.2 Chitosan-mRNA complexes ............................................................................................................ 41
  • 2.4.3 Chitosan-ASO complexes ............................................................................................................... 42
  • 2.4.4 Chitosan-mRNA-ASO complexes .................................................................................................... 44
  • 2.4.5 Chitosan-lecithin oil-core nanocapsules ....................................................................................... 46
  • 2.5 Characterization of nanoformulations................................................................... 47
  • 2.5.1 Determination of hydrodynamic diameter and polydispersity index .......................................... 47
  • 2.5.2 Determination of zeta potential .................................................................................................... 47
  • 2.5.3 Stability measurements.................................................................................................................. 47
  • 2.5.4 Gel retardation assay ..................................................................................................................... 48
  • 2.5.5 Asymmetric flow field-flow fractionation ..................................................................................... 48
  • 2.5.6 Transmission electron microscopy ................................................................................................ 50
  • 2.6 Cell culture ............................................................................................................ 50
  • 2.6.1 CFBE41o- cells ................................................................................................................................. 50
  • 2.6.2 H441 cells ........................................................................................................................................ 52
  • 2.6.3 Primary human nasal epithelial cells ............................................................................................. 54
  • 2.6.3.1 Processing of nasal specimens ............................................................................................. 55
  • 2.6.3.2 Nasal brushing procedure .................................................................................................... 56
  • 2.6.3.3 Liquid covered cultured filters ............................................................................................. 56
  • 2.6.3.4 Submerged culture ............................................................................................................... 57
  • 2.6.3.5 Expansion Phase ................................................................................................................... 58
  • 2.6.3.6 Maintenance Phase .............................................................................................................. 58
  • 2.6.4 Freezing and thawing of cells ......................................................................................................... 59
  • 2.6.5 MTT assay ....................................................................................................................................... 60
  • 2.6.6 Mycoplasma test ............................................................................................................................ 60
  • 2.6.7 Determination of osmolality .......................................................................................................... 61
  • 2.7 Transfection of cells .............................................................................................. 61
  • 2.7.1 Transfection with Lipofectamine®2000 ......................................................................................... 61
  • 2.7.2 Transfection with nanoformulations ............................................................................................. 62
  • 2.7.3 Transfection efficiency ................................................................................................................... 63
  • 2.8 Fluorescence optical methods .............................................................................. 65
  • 2.8.1 Preparation of glass cover slips ..................................................................................................... 65
  • 2.8.2 Fixation of transfected cells on glass cover slips .......................................................................... 65
  • 2.9 Protein biochemical methods ............................................................................... 66
  • 2.9.1 Isolation of protein from cells ........................................................................................................ 66
  • 2.9.2 BCA assay ........................................................................................................................................ 66
  • 2.9.3 SDS-PAGE ........................................................................................................................................ 67
  • 2.9.4 Semi-dry western blot .................................................................................................................... 68
  • 2.9.5 Enhanced chemiluminescence detection ......................................................................................71
  • 2.10 Ussing chamber .................................................................................................... 71
  • 2.10.1 Functional Ussing chamber measurements .............................................................................72
  • 2.11 Statistical analysis ................................................................................................. 74
  • 3 RESULTS .......................................................................................................................................... 75
  • 3.1 Chitosan-mRNA complexes ................................................................................... 75
  • 3.1.1 Evaluation of wtCFTR-mRNA ..........................................................................................................75
  • 3.1.2 Experiments with CFBE41o- cells ...................................................................................................76
  • 3.1.3 Experiments with primary HNE cells ..............................................................................................79
  • 3.2 Chitosan-ASO complexes ...................................................................................... 83
  • 3.2.1 Characterization of chitosan-ASO complexes ...............................................................................83
  • 3.2.2 Stability of chitosan-ASO complexes .............................................................................................91
  • 3.2.3 Cell culture experiments with chitosan-ASO complexes ..............................................................95
  • 3.3 Chitosan-mRNA-ASO complexes ......................................................................... 101
  • 3.3.1 Characterization of chitosan-mRNA-ASO complexes ................................................................ 101
  • 3.4 Chitosan-lecithin oil-core nanocapsules .............................................................. 106
  • 3.4.1 Detection of TRPV1 in primary HNE cells ................................................................................... 106
  • 3.4.2 Effect of capsaicin on primary HNE cells .................................................................................... 108
  • 3.4.3 Characterization of chitosan-lecithin oil-core nanocapsules .................................................... 111
  • 3.4.4 Stability of chitosan-lecithin oil-core nanocapsules .................................................................. 119
  • 3.4.5 Cell culture experiments with chitosan-lecithin oil-core nanocapsules ................................... 121
  • 4 DISCUSSION ................................................................................................................................ 125
  • 4.1 Chitosan-mRNA complexes ................................................................................. 127
  • 4.1.1 wtCFTR-mRNA transfection leads to increased CFTR activity in human respiratory epithelial cells ...................................................................................................................................................... 127
  • 4.2 Chitosan-ASO complexes .................................................................................... 130
  • 4.2.1 Salt benefits the formation of chitosan-ASO complexes ........................................................... 130
  • 4.2.2 Supplemented transfection medium stabilizes complexes but harms human respiratory epithelial cells ............................................................................................................................................. 133
  • 4.2.3 ASO transfection successfully inhibits ENaC activity in human respiratory epithelial cells ..... 134
  • 4.3 Chitosan-mRNA-ASO complexes ......................................................................... 136
  • 4.3.1 Complexation of two nucleic acids by chitosan forms moderately monodisperse nanoparticles . ...................................................................................................................................................... 136
  • 4.4 Chitosan-lecithin oil-core nanocapsules .............................................................. 138
  • 4.4.1 Capsaicin might decrease ENaC activity by activation of TRPV1 in primary human nasal epithelial cells ............................................................................................................................................. 138
  • 4.4.2 Highly monodisperse and positively charged nanocapsules successfully adsorb wtCFTR-mRNA . ...................................................................................................................................................... 140
  • 4.4.3 Nanocapsules are highly stable in transfection medium but did not transfect primary human nasal epithelial cells ..................................................................................................................................... 142
  • 4.5 Conclusion and outlook ....................................................................................... 144
  • 5 REFERENCES ............................................................................................................................... 147
  • 6 APPENDIX...................................................................................................................................... 171
  • 6.1 List of chemicals .................................................................................................. 171
  • 6.2 List of reagents and kits ...................................................................................... 172
  • 6.3 List of devices ...................................................................................................... 174
  • 6.4 Primers................................................................................................................ 177
  • 6.5 Nucleic acid markers ........................................................................................... 179
  • 6.6 Antibodies ........................................................................................................... 180
  • 6.7 Protein markers .................................................................................................. 180
  • SCIENTIFIC CONTRIBUTIONS ..................................................................................................... 181
  • CURRICULUM VITAE ........................................................................................................................ 183
  • ACKNOWLEDGEMENTS .................................................................................................................185.

Ähnliche Einträge