| Parkinson's disease is becoming common disease afflicting the elderly people, there is no effective way to stop the progress of this disease. In recent years, with the progression of the disease's reseach, the most effective treatment will count on new technologies, such as gene therapy and stem cell therapy. In particular, many studies indicated that Parkinson's disease is related to overexpression of NF-κB. Torres, etc. published the original article in Nat Cell Biol. in 2008 and found that Nanog gene could inhibit the expression of NF-κB. In order to fully apply this discovery, our team constructed the plasmid PNL-Nanog-IRES2-EGFP carrying Nanog gene with double digestion and gene recombinant. The lentiviral (LV) vectors carrying Nanog gene were constructed, then infected the mouse mesenchymal stem cells (mMSCs), the effects of Nanog overexpression on NF-κB gene expression in mMSCs and mice with Parkinsonism were observed. This facilitates our next study on neurogeneration diseases. This study consisted of three parts.Part1 : Construction, culture and identification of the bone marrow mesenchymal stem cells carrying Nanog geneMaterials and Methods1) PNL-Nanog-IRES2-EGFP or PNL-IRES2-EGFP was cotransfected along with pHELPER and pVSVG into 293T to package lentivirus particles.2) According to the enhanced green fluorescent protein (EGFP) expression, the functional titer of LV wector was determined by flow cytometry (FCM) after transduction into 293T cells. 3) The mMSCs were infected with lentiviral vectors at the same titer.4) RT-PCR analysis of Nanog mRNA, Western Blot analysis of Nanog protein and ELISA analysis of Nanog protein in the cell culture supernatant.Results1) The three plasmid PNL-Nanog-IRES2-EGFP, or PNL-IRES2-EGFP, pHELPER and pVSVG were cotransfected into and packaged in 293T cells.2) The functional titer of unconcentrated virus and concentrated virus were 6.5×104~4 TU/mL and 6. 8×104~6 TU/mL respectively.3) The resuls of RT-PCR, Western blot and ELISA analyses showed that, compared with the empty vector group, the expression of Nanog protein was singnificantly increased in Nanog group.Conclusions1) High titer lentiviral particles were produced and efficiently transduced into mMSCs.2) The results of RT-PCR analysis of Nanog mRNA, Western Blot analysis of Nanog protein and ELISA analysis of Nanog protein in the cell culture supernatant comfirmed that the constructed lentiviral vectors could increase Nanog gene expression in the mMSCs.Part2:Overexpression of Nanog gene in mouse mesenchymal stem cells and its effect on NF-κB expressionMaterials and Methods1) Lentivirus vector was packed, concentrated and then used to infect mMSCs with the method mentioned above. The lentiviral vector was added into mMSCs and cultured. The expression of green fluorescence was observed under fluorescence microscope.2) Experimental group: Nanog-mMSCs group was the mMSCs infected by lentivirus carrying Nanog gene; Mock-mMSCs group ( PNL-mMSCs group) was the mMSCs infected by lentivirus not carrying the target gene; mMSCs group was the mMSCs without being infected by lentivirus.3) Detection of the expression of Nanog and NF-κB in each group.Results1) After the constructed lentivirus infected mMSCs, the expression of green fluorescence was observed under fluorescence microscope in the experimental groups, but no green fluorescence was observed in the control group.2) The resuls of Immunofluorescence staining, RT-PCR and Western blot analyses showed that, compared with Nanog-mMSCs group, the expression of NF-κB protein was singnificantly increased in Mock-mMSCs group and mMSCs group.ConclusionsOverexpression of Nanog might inhibit NF-κB expression in cultured mMSCs.Part3:Overexpression of Nanog gene in mouse mesenchymal stem cells and its effect on NF-κB expression in mouse with ParkinsonismMaterials and Methods1) The adult male C57BL mice, 10-12 weeks old, weighing 25-30 g, PD models of C57BL mice were induced by MPTP.2) Simultaneously, Nanog-mMSCs, Mock-mMSCs were once injected into the striatum of experimental mice by stereotatic technique. PBS was only injected into control group.3) The tissue samples of brain were collected and made frozen sections, we observed the expression of NF-κB in the substantia nigra and striatum of each group under a fluorescence microscope, and counted the number of neurons in the substantia nigra.Results1) The Parkinson's disease model of mice were successfully established. 2) Frozen sections of mice brain were observed under fluorescence microscope, the results showed that the expression of NF-κB protein was singnificantly increased in Mock-mMSCs group and mMSCs group, compared with Nanog-mMSCs group. The number of dopaminergic neurons in the substantia nigra was increased in Nanog-mMSCs group, compared with the other two groups.ConclusionsOverexpression of Nanog can inhibit NF-κB expression, which may protect substantia nigra dopaminergic neurons in animal models of Parkinson's disease. |
