|
Background: Diabetic Neurogenic bladder is a common complication of diabetes, bladder dysfunction, the current lack of effective treatment. Some studies suggest that the lack of diabetic neurogenic bladder with local neurotrophic factor, local supplement neurotrophic factors will help improve diabetes bladder function. Nerve growth factor (nerve growth factor, NGF) is the most important neurotrophic factor, one of the β subunit is constituted only NGF three subunits of biologically active subunits, also known as of β-NGF, maintaining sympathetic growth and development of nerve and sensory nerve function is essential. Studies have shown that using a suitable carrier gene into the lesion of the nerve growth factor expression is expected to obtain good therapeutic effect. Lentivirus vector can infect dividing cells and non-dividing cells, transfer the gene fragment of the larger capacity, the target gene expression for a long time, difficult to induce a host immune response, the two different genes can be introduced to the same cell, or infection of the same cells repeatedly, etc. advantages, is an ideal vector for gene therapy. Therefore, this study intends to construct a recombinant lentiviral vector containing the human nerve growth factor β subunit (β-NGF), and lay the foundation for future in vivo gene therapy. Objective: To construct a lentiviral vector expressing nerve growth factor β subunit (β-NGF) gene, build β-NGF gene modified rat bone marrow mesenchymal stem cell transplantation in the treatment of diabetic neurogenic bladder experimental basis . Methods: PCR technology from the plasmid pGC-E1-NGFB containing β-NGF gene amplification target gene of β-NGF and β-NGF gene was cloned into the lentiviral vector plasmid pGC-FU, construct recombinant lentiviral vector plasmid pGC -FU-β-NGF. Β-NGF genes verified by restriction enzyme digestion, sequencing, plasmid and packaging pGC-FU-β-NGF plasmid pHelper1.0, envelope plasmid pHelper2.0 co-transfected human embryonic kidney epithelial cell line 293T cells, carrying the β- NGF gene recombinant lentivirus GC-FU-β-NGF was. Real time quantitative PCR to detect recombinant lentiviral GC-FU-β-NGF was titer. Recombinant lentivirus GC-FU-β-NGF transfected human embryonic kidney epithelial cell line 293T cells, RT-PCR was used to detect the expression of the target gene β-NGF, Wstern blot to detect the expression of the target protein β-NGF. Results: β-NGF gene obtained by sequencing reported in GeneBank sequence exactly correct β-NGF gene of pGC-FU-β-NGF was carrying. the pGC-FU-β-NGF plasmid and packaging plasmid pHelper1.0, envelope plasmid pHelper2.0 has transfected packaging cells 293T can produce recombinant lentivirus GC-FU-β-NGF was. Real-time quantitative PCR to high titer recombinant lentivirus (2.43E 9 TU / ml). Β-NGF target genes can be efficiently transfected with recombinant lentivirus 293T, RT-PCR detected the expression of the target gene β-NGF in target cells, Western blot detected expression of β-NGF protein in the target cells. Conclusion: We successfully constructed a high-titer recombinant lentiviral vector carrying the β-NGF gene can correct expression.
|