2019 Fiscal Year Research-status Report
Physiological role of primary cilium-derived extracellular vesicles in fine-tuning signal transduction in target cells.
| Project/Area Number |
19K23728
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| Research Institution | Hiroshima University |
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Principal Investigator |
IJAZ FARYAL 広島大学, 医系科学研究科(医), 助教 (80845595)
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| Project Period (FY) |
2019-08-30 – 2021-03-31
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| Keywords | Primary Cilium / Extracellular Vesicles / Signal Transduction / Cell Migration |
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| Outline of Annual Research Achievements |
NIH/3T3 knock-in cell line stably expressing Arl13b/Venus(pcEVs marker) was generated and cell line was used to check the uptake of florescent pcEVs by the acceptor cells.The results showed that the target cells were able to uptake pcEVs. Next, in order to screen the effect of pcEVs on signal transduction in acceptor cells, primary cilium-deficient cells were exposed to the pcEVs and their migration rate was measured. The rate of migration of acceptor cells was increased after 12 hours exposure to pcEVs as compared to control. To rule out the possibility that the results were truly from migration and not from cell proliferation, acceptor cells were also stained with cell proliferation marker Ki-67.Furthermore, pcEVs purification protocol was developed using size exclusion chromatography.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
The research is progressing rather smoothly because we have established the protocol for the purification of pcEVs and have generated the required stable cell lines to perform the experiments. Furthermore, we have finished the initial screening in order to determine whether their is a change in acceptor cell phenotype or gene expression in response to pcEVs through cellular assays and gene expression analysis for certain pathways.
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| Strategy for Future Research Activity |
Based on the results of our initial screening, we next consolidate the results using purified pcEVs. Moreover, proteomics analysis of the acceptor cells after exposure to pcEVs will be done to identify any changes in protein expression and also determine what pathways are changed and/or regulated by pcEVs in acceptor cells. Then the proteomics results will be confirmed through biochemical assays and florescence microscopy.
In parallel, the characterization of pcEVs will be done through NTA analysis, electron microscopy and biochemical assays in order to know how they regulate the signal transduction in acceptor cells.
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| Causes of Carryover |
The incurring amount will be used to purchase the antibodies to confirm the results of the proteomics analysis.
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