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Bidirectional transcriptome analysis of rat bone marrow-derived mesenchymal stem cells and activated microglia in an in vitro coculture system

Authors
이, 다연
Department
대학원 의생명과학과
Degree
Doctor (2020)
Table Of Contents
1. Introduction 1
2. Materials and methods 5
2.1. Isolation and maintenance of rat bone marrow-derived mesenchymal stem cells (rBM-MSCs) 5
2.2. Rat microglia primary cultures 7
2.3. Coculture of LPS-stimulated or non-stimulated microglia and rBM-MSCs 9
2.4. Total RNA isolation and microarray analysis 9
2.5. Transcriptomic analysis using Ingenuity Pathway Analysis 10
2.6. Quantitative real-time PCR (qPCR) 14
2.7. Migration assay 17
2.8. Statistical analysis 17
3. Results 18
3.1. Cellular movement-related transcriptomic changes in rBM-MSCs cocultured with LPS-stimulated microglia 18
3.2. Functional prediction of transcriptomic networks in rBM-MSCs cocultured with LPS-stimulated microglia 23
3.3. Increased migratory activity in rBM-MSCs cocultured with LPS-stimulated microglia 28
3.4. Transcriptomic analysis in LPS-stimulated microglia cocultured with rBM-MSCs 30
3.5. Functional prediction of transcriptomic networks and reduced inflammatory response in LPS-stimulated microglia cocultured with rBM-MSCs 34
4. Discussion 41
CONCLUSION 44
REFERENCES 45
Abstract
Microglia contribute to the pathogenesis of brain diseases by regulating of neuroinflammation. Thus, targeting of neuroinflammation triggered by activated microglia in brain diseases has become a promising curative strategy. Bone marrow-derived mesenchymal stem cells (BM-MSCs) have been shown to have therapeutic effects, resulting from the regulation of inflammatory conditions in the brain. In this study, Gene expression pattern in rat BM-MSCs (rBM-MSCs) cocultured with lipopolysaccharide- (LPS-) stimulated primary rat microglia were investigated using microarray analysis and the functional relationships were evaluated through Ingenuity Pathway Analysis (IPA). The effects of rBM-MSC on LPS-stimulated microglia were also assessed using a reverse coculture system and the same transcriptomic analysis. In rBM-MSCs, 67 genes were differentially expressed, which were highly related with migration of cells, compared to control. The gene network was predicted using IPA and LPS-stimulated primary rat microglia increase the migration of rBM-MSCs was validated by experiments. Reversely, expression patterns of the transcriptome in LPS-stimulated primary rat microglia cocultured with rBM-MSCs were changed. Results showed that 64 genes were altered, which were highly related with inflammatory response, compared to absence of rBM-MSCs. In the same procedure with the aforementioned, the prediction of the gene network and experimental validation showed that rBM-MSCs decrease the inflammatory response of LPS-stimulated primary rat microglia. These indicate that LPS-stimulated microglia increase the migration of rBM-MSCs and that rBM-MSCs reduce the inflammatory activity in LPS-stimulated microglia. The results of this study show complex mechanisms underlying the interaction between rBM-MSCs and activated microglia and may be supportive for the advance of stem cell therapy for brain diseases. This study is based on a previously published report
Keywords
bone marrow-derived mesenchymal stem cellsinflammationmicrogliamigrationtranscriptome analysis
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Theses > Graduate School of Biomedical Sciences > Doctor
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이, 다연
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