中国药理学与毒理学杂志2023，Vol.37Issue(12)：928-935,8.DOI:10.3867/j.issn.1000-3002.2023.12.006
线粒体示踪体系的建立及验证
Establishment and identification of a mitochondrial tracking system
摘要
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.关键词
线粒体转移/线粒体荧光探针/间充质干细胞Key words
mitochondrial transfer/mitochondrial fluorescent reporting system/mesenchymal stem cell分类
医药卫生引用本文复制引用
吕琳,王思涵,曾泉,段晗,毛壮,王唱垚,裴雪涛,王华,李艳华..线粒体示踪体系的建立及验证[J].中国药理学与毒理学杂志,2023,37(12):928-935,8.基金项目
国家自然科学基金(82270132) （82270132）
国家自然科学基金(81872553) （81872553）
国家自然科学基金(82200122) Nature Science Foundation of China(82270132) （82200122）
Nature Science Foundation of China(81872553) （81872553）
and Nature Science Foundation of China(82200122) （82200122）
