UNIVERSITI PUTRA MALAYSIA NON-VIRAL TRANSFECTION OF … · 2018. 4. 9. · daripada aspirasi sumsum...
Transcript of UNIVERSITI PUTRA MALAYSIA NON-VIRAL TRANSFECTION OF … · 2018. 4. 9. · daripada aspirasi sumsum...
UNIVERSITI PUTRA MALAYSIA
NON-VIRAL TRANSFECTION OF BONE MARROW-DERIVED MESENCHYMAL STEM CELL WITH HUMAN INTERFERON-GAMMA GENE AND IN VITRO
EFFICIENCY AGAINST CHRONIC MYELOID LEUKAEMIA CELLS
LIEW LEE CHUEN
FPSK(m) 2013 22
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NON-VIRAL TRANSFECTION OF BONE MARROW-DERIVED
MESENCHYMAL STEM CELL WITH HUMAN INTERFERON-GAMMA
GENE AND IN VITRO EFFICIENCY AGAINST CHRONIC MYELOID
LEUKAEMIA CELLS
By
LIEW LEE CHUEN
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,
in Fulfilment of the Requirements for the Degree of Master of Science
June 2013
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COPYRIGHT
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Malaysia unless otherwise stated. Use may be made of any material contained within
the thesis for non-commercial purposes from the copyright holder. Commercial use
of material may only be mad with the express, prior, written permission of University
Putra Malaysia.
Copyright © University Putra Malaysia
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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment
of the requirement for the degree of Master of Science
NON-VIRAL TRANSFECTION OF BONE MARROW-DERIVED
MESENCHYMAL STEM CELL WITH HUMAN INTERFERON-GAMMA
GENE AND IN VITRO EFFICIENCY AGAINST CHRONIC MYELOID
LEUKAEMIA CELLS
By
LIEW LEE CHUEN
June 2013
Chairman : Maha Abdullah, PhD
Faculty : Medicine and Health Sciences
Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder caused by
the BCR/ABL gene rearrangement, known as the Philadelphia (Ph) chromosome. To
date, the only curative therapy for CML is allogeneic stem cell transplantation.
However, significant morbidity and mortality are associated with the procedure and
the need for a matched donor makes this option not available to the majority of the
patients. Currently, various studies have been carried out to develop an alternative
approach for CML treatment, for example targeted gene delivery of therapeutic
cytokines. In this study, the feasibility of using bone marrow-derived mesenchymal
stem cell (BM-MSC) in delivering human interferon-gamma (hIFN-γ) gene for
targeted CML therapy was explored. Mesenchymal stem cells (MSC) were
successfully isolated from human bone marrow aspirates and their biological
properties were similar to those of MSC reported. Expanded BM-MSC were
transfected with plasmid containing hIFN-γ gene (pORF-hIFN-γ) via nucleofection.
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Gene transfer efficiency was determined based on intracellular hIFN-γ expression via
flow cytometry and was found to be at 54.28±11.34%. The in vitro expression of
hIFN-γ mRNA and protein in BM-MSC were also analysed at intervals of 24 h, up to
5 days post nucleofection, via real-time PCR and ELISA, respectively. Real-Time
PCR data analysis showed significant up-regulation of hIFN-γ mRNA in
nucleofected BM-MSC when compared to non-transfected BM-MSC (P=0.043).
BM-MSC harbouring pORF-hIFN-γ could express hIFN-γ protein in vitro. This
cytokine production was achieved as high as 3.47±1.03 ng/ml after 72 hours of
nucleofection. The effect of hIFN-γ produced in nucleofected BM-MSC on the
proliferation of CML cell line (K562) in vitro was also investigated. K562 growth
was inhibited at 61.12±16.38% after seven days of co-culture with BM-MSC
expressing hIFN-γ (P=0.006). In conclusion, findings in the current study indicated
that hIFN-γ produced by genetically engineered BM-MSC successfully inhibited the
proliferation of K562 cells in vitro. Thus, MSC as cellular vehicle in hIFN-γ gene
delivery could be further explored as a promising treatment option for CML patients.
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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai
memenuhi keperluan untuk ijazah Master Sains
TRANSFEKSI GEN INTERFERON-GAMMA KE DALAM SEL STEM
MESENKIMIA DARIPADA SUMSUM TULANG SECARA BUKAN VIRUS
DAN KEBERKESANAN MENENTANG SEL LEUKEMIA MIELOGENUS
KRONIK IN VITRO
Oleh
LIEW LEE CHUEN
Jun 2013
Pengerusi : Maha Abdullah, PhD
Fakulti : Perubatan dan Sains Kesihatan
Leukemia mielogenus kronik (CML) merupakan sejenis leukemia kronik yang
dicirikan oleh peningkatan penghasilan klon sel-sel mieloid secara tidak terkawal
akibat daripada translokasi gen ABL dan gen BCR, umumnya dikenali sebagai
kromosom Philadelphia. Sehingga kini, transplantasi sumsum tulang merupakan
satu-satunya terapi kuratif yang berpotensi untuk CML. Walau bagaimanapun,
morbiditi and mortaliti yang dikaitkan dengan prosedur transplantasi and keperluan
penderma yang sesuai telah menyebabkan terapi tersebut tidak dapat disediakan
kepada kebanyakan pesakit. Oleh yang demikian, pelbagai penyelidikan telah
dijalankan pada masa kini dengan tujuan untuk memperkembangkan satu rawatan
novel untuk CML, contohnya penghantaran sitokin terapeutik ke sasaran. Dalam
kajian ini, penggunaan sel stem mesenkima yang diperoleh daripada sumsum tulang
dalam penghantaran gen interferon-gamma manusia (hIFN-γ) sebagai terapi sasaran
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bagi CML telah dikaji. Sel stem mesenkima (MSC) telah berjaya dipencilkan
daripada aspirasi sumsum tulang manusia dan sifat-sifat biologi sel ini adalah serupa
dengan MSC yang dilaporkan. MSC sumsum tulang manusia (BM-MSC) ini
kemudian ditransfeksi dengan plasmid mengekod gen hIFN-γ dengan menggunakan
teknologi nukleofeksi. Keberkesanan transfeksi telah ditentu berdasarkan ekspresi
hIFN-γ intraselular dengan menggunakan sitometri aliran. Keberkesan transfeksi
sebanyak 54.28±11.34% telah tercapai selepas 24 jam nukleofeksi. Ekspresi mRNA
dan protein hIFN-γ di dalam BM-MSC secara in vitro juga dikaji setiap 24 jam
selepas nukleofeksi, selama 5 hari dengan menggunakan tindak balas rantai
polimerase masa nyata (real-time PCR) dan ELISA masing-masing. Keputusan
tindak balas rantai polimerase masa nyata menunjukan peningkatan ekspresi mRNA
hIFN-γ yang ketara dalam sampel nukleofeksi BM-MSC berbanding dengan BM-
MSC yang tidak dinukleofeks (P=0.043). Di samping itu, BM-MSC yang telah
dinukleofeks dengan plasmid pORF-hIFN-γ juga berupaya menghasilkan protein
hIFN-γ secara in vitro. Produksi protein hIFN-γ yang paling tinggi ialah 72 jam
selepas nukleofeksi, dimana sebanyak 3.47±1.03 ng/ml protein hIFN-γ telah dikesan.
Selain itu, kesan hIFN-γ yang dihasilkan oleh nukleofeksi BM-MSC terhadap
proliferasi sel CML (K562) juga turut dikaji. Proliferasi sel K562 telah ditindas
sebanyak 61.12±16.38% apabila kultur bersama nukleofeksi BM-MSC selepas 7 hari
(P=0.006). Secara kesimpulannya, penemuan dalam kajian semasa menunjukkan
bahawa hIFN-γ yang dihasilkan secara modifikasi genetik BM-MSC telah berjaya
menindas proliferasi sel K562 in vitro. Oleh itu, MSC sebagai pengangkutan selular
dalam penghantaran gen hIFN-γ boleh terus diterokai sebagai rawatan alternatif yang
berkesan untuk pesakit CML.
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ACKNOWLEDGEMENTS
First and foremost, I would like to express utmost gratitude to my supervisor, Assoc.
Prof. Dr. Maha Abdullah, for her patience to guide me along the research with her
immense knowledge and motivation. Her valuable guidance has helped me
throughout the research and completion of this thesis.
It gives me great pleasure in acknowledging my co-superviors, Emeritus Prof. Dr.
Cheong Soon Keng, Assoc. Prof. Dr. Leong Chooi Fun and Assoc. Prof. Dr. Chong
Pei Pei, for their timely support and insightful comments. My sincere thanks also go
to Dato’ Mohd Farid Ariffin, the Founder and President of National Cancer Council
Malaysia (MAKNA), for his moral support in research activities and generosity in
providing grant to enable the research to be carried out. I would also like to share the
credit of my work with my seniors and fellow labmates – Wei Yi, Chee Yin, Erica,
Yuen Fen and especially Hoon Koon and Rachel, for their guidance and discussion
that has polished up both my research and thesis writing.
Last but not least, I would like to thank my family and friends for their love and
unconditional support, letting me focus on my study and help me to make it through
hurdles that I faced throughout the study.
This dissertation would not have been possible without the help and guidance of all
the individuals mentioned above, and to whom that I did not mention but
nevertheless contributed in one way or another to the success of this thesis, a big
THANK YOU to you all.
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I certify that a Thesis Examination Committee has met on 7 June 2013 to conduct the
final examination of Liew Lee Chuen on her thesis entitled “Non-viral Transfection
of Bone Marrow-Derived Mesenchymal Stem Cell woth Human Interferon-gamma
Gene and In Vitro Efficiency Against Chronic Myeloid Leukaemia Cells” in
accordance with the Universities and University Colleges Act 1971 and the
Constitution of the University Putra Malaysia [P.U.(A) 106] 15 March 1998. The
Committee recommends that the student be awarded the Master of Science.
Members of Thesis Examination Committee were as follows:
Mohamad Aziz bin Dollah, PhD
Associate Professor
Faculty of Medicine and Health Sciences
Universiti Putra Malaysia
(Chairman)
Sharmili Vidyadaran, PhD
Associate Professor
Faculty of Medicine and Health Sciences
Universiti Putra Malaysia
(Internal Examiner)
Noorjahan Banu binti Mohamad Alitheen, PhD
Associate Professor
Faculty of Biotechnology and Biomolecular
Universiti Putra Malaysia
(Internal Examiner)
Ainoon Othman
Professor
Faculty of Medicine and Health Sciences
Universiti Sains Islam Malaysia
(External Examiner)
____________________________
NORITAH OMAR, PhD
Assoc. Professor and Deputy Dean
School of Graduate Studies
Universiti Putra Malaysia
Date: 2 August 2013
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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been
accepted as fulfilment of the requirement for the degree of Master of Science. The
members of the Supervisory Committee were as follows:
Maha Abdullah, PhD
Associate Professor
Faculty of Medicine and Health Sciences
Universiti Putra Malaysia
(Chairman)
Chong Pei Pei, PhD
Associate Professor
Faculty of Medicine and Health Sciences
Universiti Putra Malaysia
(Member)
Cheong Soon Keng
Professor and Dean
Faculty of Medicine and Health Sciences
Universiti Tunku Abdul Rahman
(Member)
Leong Chooi Fun
Associate Professor
Department of Pathology
Universiti Kebangsaan Malaysia Medical Centre
(Member)
____________________________
BUJANG BIN KIM HUAT, PhD
Professor and Dean
School of Graduate Studies
Universiti Putra Malaysia
Date:
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DECLARATION
I declare that the thesis is my original work except for the quotations and citations
which have been duly acknowledged. I also declare that it has not been previously,
and is not concurrently, submitted for any other degree at Universiti Putra Malaysia
or at any other institution.
__________________
LIEW LEE CHUEN
Date: 7th June 2013
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TABLE OF CONTENTS
Page
ABSTRACT ii
ABSTRACK iv
ACKNOWLEDGEMENTS vi
APPROVAL vii
DECLARATION ix
LIST OF TABLES xiv
LIST OF FIGURES xv
LIST OF ABBREVIATIONS xvii
CHAPTER
1 INTRODUCTION 1
2 LITERATURE REVIEW 6
2.1 Chronic Myeloid Leukaemia
2.1.1 Introduction
2.1.2 Treatment Outcome
6
6
7
2.2 An Overview of Stem Cell 9
2.3 Mesenchymal Stem Cell (MSC) 11
2.3.1 Historical Overview and Nomenclature of
MSC
11
2.3.2 Properties of MSC 14
2.3.2.1 Morphology and Immunophenotype 14
2.3.2.2 Differentiation Capacity 16
2.3.2.3 Immunimodulatory Properties
2.3.2.4 Homing Ability
2.3.3 Clinical Potential of Mesenchymal Stem Cells
2.3.3.1 MSC for the Treatment of Graft-
Versus-Host Disease
2.3.3.2 The Use of MSC in Regenerative
Medicine
2.3.3.3 MSC as Potential Vehicle for
Therapeutic Genes Delivery
17
19
21
22
26
29
2.4 Gene Delivery Systems 33
2.4.1 Viral-based Gene Delivery System
2.4.1.1 Integrating Viral Vector
2.4.1.2 Non-integrating Viral Vector
34
35
39
2.4.2 Non-viral Gene Delivery System
2.4.2.1 Chemical Methods
2.4.2.2 Physical Methods
40
41
44
2.4.3 Nucleofection 47
2.5 The Interferons (IFNs) 49
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3 MATERIALS AND METHODS 54
3.1 Experimental Design 54
3.2 Isolation of Mononuclear Cells From Bone Marrow
Aspirate
56
3.3 Cell Count 57
3.4 Cell Culture 58
3.4.1 Culture of Bone Marrow-Derived Adherent
Cells
58
3.4.2 Culture of Chronic Myeloid Leukaemia Cells
Line (K562)
59
3.5 Characterisation of Bone Marrow-Derived Adherent
Cells
59
3.5.1 Morphology Study under Microscopic
Observation
59
3.5.2 Immunophenotyping 60
3.5.3 Differentiation Assays
3.5.3.1 Adipogenic Differentiation
3.5.3.2 Osteogenic Differentiation
3.5.3.3 Chondrogenic Differentiation
61
61
62
64
3.6 Preparation of Plasmid pORF-hIFN-γ for Transfection
3.6.1 Plasmid pORF-hIFN-γ
3.6.2 E. coli Culture Media and Antibiotic
3.6.2.1 Liquid Media
3.6.2.2 Solid Media
3.6.2.3 Antibiotic
3.6.3 Growth and Storage of pORF-transformed
Bacteria
3.6.4 Verification of Plasmid pORF-hIFN-γ from
Transformed E. coli Clones
3.6.4.1 Colony PCR (Polymerase Chain
Reaction)
3.6.4.2 Gel Electrophoresis
3.6.4.3 Plasmid Extraction and Sequencing
65
65
67
67
68
68
69
69
69
71
72
3.7 Nucleofection of Bone Marrow-Derived
Mesenchymal Stem Cells
3.7.1 Nucleofection Protocol
3.7.2 Gene Transfection Efficiency and Viability
of Cells Post Nucleofection
3.7.2.1 Transfection Efficiency of pmaxGFP
into BM-MSC
3.7.2.2 Transfection Efficiency of pORF-
hIFN-γ into BM-MSC
3.7.2.3 Viability of Cells after
Nucleofection
3.7.3 Morphology, Immunophenotyping and
Differentiation Potential of Nucleofected
BM-MSC
74
74
75
75
76
77
78
3.8 Functional Status of Human Interferon Gamma Gene
in Nucleofected BM-MSC
3.8.1 Quantitative Real-Time PCR Analysis of
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hIFN-γ mRNA Expression in Nucleofected
BM-MSC
3.8.1.1 Total Ribonucleic Acid (RNA)
Extraction
3.8.1.2 DNase I Treatment of RNA
3.8.1.3 Determination of RNA Integrity
3.8.1.4 Reverse Transcription of RNA to
cDNA
3.8.1.5 Quantitative Real-Time PCR
Quantification
3.8.2 Protein Expression of hIFN-γ in
Nucleofected BM-MSC via ELISA
79
80
81
82
83
85
3.9 K562 Cell Proliferation Assay
3.9.1 Establishment of Growth Profile for K562
cells
3.9.2 Inhibition Effect of Nucleofected BM-MSC
on K562 Cell Proliferation
86
86
87
3.10 Statistical Analysis
89
4 RESULTS 90
4.1 Characterisation of Isolated Bone Marrow-Derived
Adherent Cells
90
4.1.1 Morphology Study under Microscopic
Observation
90
4.1.2 Immunophenotyping 92
4.1.3 Differentiation Assays
4.1.3.1 Adipogenic Differentiation
4.1.3.2 Osteogenic Differentiation
4.1.3.3 Chondrogenic Differentiation
94
94
96
98
4.2 Verification of Plasmid pORF-hIFN-γ from
Transfected E. coli Colonies
4.2.1 Colony PCR (Polymerase Chain Reaction)
4.2.2 Plasmid Purification and Verification
100
100
102
4.3 Transfection Efficiency and Viability of Cell Post
Nucleofection
103
4.3.1 Nucleofection of BM-MSC with plasmid
pmaxGFP
4.3.2 Nucleofection of BM-MSC with plasmid
pORF-hIFN-γ
103
106
4.4 Morphology, Immunophenotypes and Differentiation
Potential of Nucleofected BM-MSC
108
4.5 mRNA Expression of hIFN-γ in Nucleofected BM-
MSC
110
4.5.1 RNA Purity and Integrity
4.5.2 Real-Time PCR Quantification
110
111
4.5.2.1 Validation Experiment 111
4.5.2.2 mRNA Expression of hIFN-γ in
nucleofected BM-MSC
115
4.6 Protein Expression of hIFN-γ in Nucleofected BM-
MSC
117
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4.7 K562 Cell Proliferation Assay 119
4.7.1 Establishment of Growth Profile for K562
Cells
4.7.2 Inhibition Effect of Nucleofection BM-MSC
on K562 Cell Proliferation
119
120
5 DISCUSSION 122
5.1 Characterisation of Isolated Bone Marrow-Derived
Adherent Cells
123
5.2 Transfection Efficiency and Viability of BM-MSC
Post Nucleofection
126
5.3 In vitro Expression of Human Inteferon-gamma in
Nucleofected BM-MSC and Their Inhibition Effect on
K562 Cell Proliferation
129
6 CONCLUSION AND FUTURE RECOMMENDATIONS
135
6.1 Conclusion 135
6.2 Future recommendations
137
REFERENCES 139
APPENDIX A – Information Sheets 163
APPENDIX B – Data Sets 169
BIODATA OF STUDENT 186
PUBLICATION 187