Huang Z.,Fujian Medical University |
Huang Z.,Fujian Institute of Neuroloscience |
Zhang Z.,Fujian Medical University |
Zhang Z.,Fujian Institute of Neuroloscience |
And 5 more authors.
Shengwu Gongcheng Xuebao/Chinese Journal of Biotechnology | Year: 2010
The expression of NF-κB is considered to be involved in the progress of neurodegeneration. It has been reported that Nanog can suppress the expression of NF-κB. To inspect and verify this finding, we constructed lentivirus (LV) vector that overexpressed the Nanog gene, infected mouse mesenchymal stem cells (mMSCs), and examined the influence of Nanog overexpression on NF-κB gene expression. The plasmid pNL-Nanog-IRES 2-EGFP was constructed by double digestion and genetic recombination. Sequencing results confirmed that our cloned Nanog gene in the PNL-Nanog-IRES 2-EGFP plasmid was consistent with the sequence reported in the GenBank. The three plasmids: pNL-Nanog-IRES 2-EGFP, HELPER, and VSVG were cotransfected into 293T cells to produce LV particles. After co-transfection of the three lentiviral plasmids, green fluorescence was observed confirming successful transfection. The mMSCs were infected by the LV and the expression of Nanog was then also verified by the presence of green fluorescence. Nanog expression levels in the mMSCs were examined using Western blotting. Expression of NF-κB was also examined using RT-PCR and Western blotting, and in addition with fluorescent microscope after immunocytochemical staining. The levels of Nanog protein expression in Nanog-mMSCs were significantly increased, and the levels of NF-κB mRNA and protein expression in Nanog-infected mMSCs were significantly lower than those of Mock-mMSCs and the mMSCs control groups. Our findings suggest that mMSCs genetically modified to overexpress Nanog can lead to the suppression of NF-κB expression. This suppression of NF-κB could have important implications for the treatment of neurodegeneration, and hence further scientific investigations of these interactions will have significant impact on future clinical attempts to attenuate disease progression. © 2010 CJB, All rights reserved.