Establishment and identification of a mitochondrial tracking system
10.3867/j.issn.1000-3002.2023.12.006
- VernacularTitle:线粒体示踪体系的建立及验证
- Author:
Lin LYU
1
;
Sihan WANG
;
Quan ZENG
;
Han DUAN
;
Zhuang MAO
;
Changyao WANG
;
Xuetao PEI
;
Hua WANG
;
Yanhua LI
Author Information
1. 军事科学院军事医学研究院辐射医学研究所,北京 100850
- Keywords:
mitochondrial transfer;
mitochondrial fluorescent reporting system;
mesenchymal stem cell
- From:
Chinese Journal of Pharmacology and Toxicology
2023;37(12):928-935
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE To observe whether mitochondria can be transferred from mesenchymal stem cells(MSCs)to irradiated cells by establishing a mitochondrial fluorescent reporting system.METHODS The lentiviral vector pSIN-EF1α-COX8A-DsRed2(named COX8A-DsRed2)that might guide the expres-sion of red fluorescence protein in the membrane of mitochondria was constructed.A lentivirus(named Lv-COX8A-DsRed2)was prepared in 293T cell line.Dental pulp stem cells(DPSCs)(named DPSC-COX8A-DsRed2)was infected with Lv-COX8A-DsRed2.The intracellular expression of the red fluores-cence protein in DPSC was observed under fluorescence microcopy.The mitochondrial localization of the expressed red fluorescent probe in DPSC-COX8A-DsRed2 was confirmed according to TOMM20 immunostaining and MitoTracker Green staining results,which could specifically label mitochondria.The IEC-6 cells that received 10 Gy X-ray radiation were used as an injured cell model.The co-culture system was established by supplementing DPSC-COX8A-DsRed2 into the culture plate with the irradi-ated IEC-6 labelled by CFSE for 24 h.RESULTS The imaging results of fluorescent microcopy obser-vation showed that DPSC-COX8A-DsRed2 expressed the mitochondrial fluorescent reporting system,which was co-located with TOMM20 protein and Mito Tracker Green.The imaging results of confocal fluorescence microcopy showed that the mitochondria with red fluorescent protein were transferred from DPSC-COX8A-DsRed2 to the irradiated IEC-6 cells,suggesting that the established mitochondrial fluorescent reporting system could indicate mitochondrial transfer from donor cells to injured ones.CONCLUSION DPSC-COX8A-DsRed2 stably expressing the mitochondrial fluorescent reporting system is established,which can be used to track mitochondrial transfer.
