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Overexpression of Nanog Gene in Mouse Mesenchymal Stem Cells and Its Effect on NF-κB Expression in Mouse with Parkinsonism

Author: HuangZhiXin
Tutor: ZhangZhiJian
School: Fujian Medical
Course: Neurology
Keywords: Nanog NF - kappa B Lentiviral vectors Bone marrow mesenchymal stem cells
CLC: R742.5
Type: Master's thesis
Year: 2010
Downloads: 61
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Abstract

Parkinson's disease is a common disease that plagued many of the elderly, there is not an effective method to prevent the progress of the disease. In recent years, with further disease research, the final treatment will hope the new technology matures, such as gene therapy and stem cell therapy. In particular, a number of studies have found that Parkinson's disease is related to the overexpression of NF-kappa B. 2008 Torres et al in Nat Cell Biol published on the Nanog gene can inhibit NF-B expression. For the full application of this discovery, our group has been cut by restriction enzyme digestion and recombinant method successfully constructed to carry Nanog gene recombinant lentiviral vector plasmid PNL-Nanog-IRES2-EGFP. This study continue to build to carry mouse Nanog gene lentiviral vector and infected mouse bone marrow mesenchymal stem cells, observed expression of Nanog expression of NF-kappa B, to provide new ideas for the treatment of neurodegenerative diseases. This topic is divided into three parts. The first part: over-expression of quality stem cells to build the bone marrow of the Nanog gene, culture and identification methods: (1) to the the the three plasmids PNL-Nanog-IRES2-EGFP or PNL-IRES2-EGFP, pHelper and pVSVG total transfer dye 293T cells the packaging production Lentiviral; (2) viral particles collected by ultracentrifugation, 293T cells were transfected EGFP protein expression was measured by flow cytometry to determine the functional titer of lentiviral; (3) were used to carry the corresponding target genes in the same titer lentiviral to infection mMSCs; (4) using RT-PCR, Western Blot and ELISA detection the Nanog mRNA of mMSCs, protein, and cell supernatants Nanog expression levels. Results: (1) the three plasmid PNL-Nanog-IRES2-EGFP or the PNL-IRES2-EGFP, pHELPER and pVSVG co-transfected 293T cells to package lentivirus. (2) is not concentrated to the functional titer of the virus-containing cell culture supernatant and concentrated viral suspension is 6.5 × 10 ~ 4 TU / mL, respectively, and 6.8 × 10 6 TU / mL. (3) RT-PCR, Western blot and ELISA confirmed the the Nanog protein of the Nanog-mMSCs group increased, compared with the empty vector group poor heterosexual significant. The Conclusion: successfully constructed expression of Nanog gene mMSCs. Second part: mMSCs the Nanog gene over-expression of NF-kappaB expression 1, the method: (1) the packaging of the lentivirus, and concentrated, and the method of infection mMSCs ibid., lentivirus liquid Add mMSCs infection mMSCs fluorescence microscope expression. (2) experimental groups: Nanog-mMSCs group to carry a the Nanog gene lentiviral infection mMSCs group; the Mock-mMSCs group (or the the PNL-mMSCs groups) do not carry the target gene infected with lentivirus mMSCs of group; mMSCs, group without mMSCs slow virus infection group. (3) detection the groups mMSCs infection in Nanog and NF-B expression. 2 results: (1) Lentiviral the infection mMSCs, observed under fluorescence microscope, PNL-MSCs group and the Nanog-mMSCs group could be detected expression of green fluorescent the uninfected mMSCs no fluorescence expression. (2) immunofluorescence, RT-PCR and Western Blot detection the Mock-mMSCs group and mMSCs group of NF-kappa B expression levels higher than the Nanog-mMSCs group. Conclusion: in vitro culture mMSCs, overexpression the Nanog inhibit NF-kappaB expression Part III: the over expression the Nanog gene mMSCs expression of Parkinson's disease in mice NF-kappaB impact, methods: (1) selected from the group 10 to 12 weeks of age, weighing 25 to 30 g male adult C57BL brown rat Country of mice Parkinson model. (2) made models on the same day to begin single-times Nanog-mMSCs of the Mock-mMSCs stereotactic injection to the model rat striatum, and the control group injected only with PBS. (3) take the mouse brain Frozen sections under a fluorescence microscope to observe the number of each group of NF-kappa B in the substantia nigra, the striatum, the expression of, and observe the substantia nigra dopaminergic neurons. Results: (1) successfully established Parkinson's disease mouse model. (2) frozen sections of mouse brain observations the the Mock-mMSCs group and mMSCs of groups of NF-kappa B expression levels higher than the Nanog-mMSCs group. The Nanog-mMSCs group substantia nigra dopaminergic neurons in number than the other two groups. Conclusion: animal models of Parkinson's disease, Nanog inhibition of NF-kappa B expression, may protect the dopaminergic neurons of the substantia nigra.

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