NGF Gene Overexpressed Lentiviral Vectors Transfect Human Umbilical Cord-derived Mensenchymal Stem Cells And Its Differentiation Of Neural Stem Cell In Vitro

Aim: Diabetic neurogenic bladder(DNB) is a common complication of urinary system caused by diabetes. Due to a lack of pertinence in the clinical treatment, we can only temporarily relieve symptoms,but not suspend the progress of the DNB and restore bladder function. A lower expression level of NGF is a important pathogenic factor for DNB. Clinical trials confirm the added exogenous NGF can effectively improve DNB. But there is also a problem NGF has a short half-life and low bioavailability. Umbilical cord mesenchymal stem cells have a abilitry of Multi-directional differentiation potential, is a good vectors for gene therapy. After construction of lentiviral vector carrying NGF gene and differentiation of netural stem cells from h UCMSCs, using the technology of transfection, combined the function of gene therapy and cell therapy.At last, verify the mechanism on animal models.Method:1) Evaluation of the basic biological characteristics of h UCMSCs. The growth curve of h UCMSCs was counted by blood cell counting plate method. h UCMSCs cell surface was demonstrated by flow cytometry. Nestin protein expression was confirmed by immunofluorescence.2) Construction of lentiviral vector carrying NGF gene and its functional inspection.3) Induce h UCMSCs into osteogenic differentiation and adipogenic differentiation. The osteogenic differentiation ability was evaluated by collagen expression, ALP detection, Alizarin Red staining for three weeks and the adipogenic differentiation was detected by Oil Red Ostaining for three weeks.4) Through the inductive medium, h UCMSCs were induced into netural stem cells. The results were evaluated by morphological observation, RT-PCR, immunofluorescence.Results:1) Cell morphology of h UCMSCs was spindle long and swirling growth. Flow cytometry analysis showed that Men SCs are positive for CD105, CD29, CD13 and CD90 and negative for CD45, CD14, CD106, CD34 and HLA-DR. The expression of HLA-DR demonstrated that cells had low immunogenicity. Immunofluorescence showed that h UCMSCs expressed nestin.2) Recombinant lentiviral vector carrying NGF gene was constructed successfully.3) HUCMSCs had osteoblasts and adipogenic differentiation potential.4)Through the inductive medium, evaluated by morphological observation, RT-PCR, immunofluorescence, h UCMSCs were induced into netural stem cells.Conclusion:1) All the results showed that h UCMSCs could be obtained from a healthyhumam umbilical cord. HUCMSCs maintained the basic biological properties of stem cells and high amplification capacity. In addition, h UCMSCs showed low immunogenicity and pluripotent ability. The experiments further demonstrated that HUCMSCs had the potential to differentiate into netural stem cells, Our findings provided some theoretical basis for the clinical application for the treatment of diabetes and also demonstrated that HUCMSCs were ideal seed cells for cell therapy.