| 研究課題/領域番号 |
19K23728
|
|---|
| 研究機関 | 広島大学 |
|---|
研究代表者 |
IJAZ FARYAL 広島大学, 医系科学研究科(医), 助教 (80845595)
|
|---|
| 研究期間 (年度) |
2019-08-30 – 2021-03-31
|
| キーワード | Primary Cilium / Extracellular Vesicles / Signal Transduction / Cell Migration |
|---|
| 研究実績の概要 |
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.
|
| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
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.
|
| 今後の研究の推進方策 |
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.
|
| 次年度使用額が生じた理由 |
The incurring amount will be used to purchase the antibodies to confirm the results of the proteomics analysis.
|
