细胞融合法转线粒体细胞模型的构建及鉴定

Investigation of possibility of cell fusion to establish a cybrid cell lines and its identification

目的探讨细胞融合方法建立转线粒体细胞(cybrid)可行性,并对融合后细胞内mtDNA进行鉴定。方法采用细胞融合方法将正常人血小板与mtDNA缺失人肝癌细胞(ρ°SK-Hep1)融合,建立转线粒体细胞(SK-Hep1 Cyb),恢复细胞正常线粒体功能。并用PCR、Southern杂交、Western杂交及线粒体膜电位检测进行鉴定。结果PCR、Southern杂交、Western杂交及线粒体染色证实成功建立了ρ°SK-Hep1细胞转线粒体模型SK-Hep1Cyb。结论采用细胞融合法可以建立转线粒体细胞,融合细胞恢复线粒体功能,转线粒体细胞可稳定存活30d以上。

Objective To investigate the possibility of cell fusion to establish a cybrid cell and its identification. Methods Normal human blood platelets were used as mitochondrial donors,and polyethylene glycol was used as fusion promoting reagent to establish ρ°SK-Hepl cells＇ transmitochondrial model. These cybrids were confirmed by PCR,Southern hybridization,Western blot and Mito-tracker Red. Results PCR,Southern hybridization,Mito-tracker Red and Western blot confirmed that cybrid cells had objective fragments of mtDNA and positive COX Ⅱ activity. Conclusion ρ°SK-Hepl ceils transmitochondrial model was successfully established.