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The two parts of this thesis, including literature and experimental studies. Literature through a lot of literature, a systematic review of the Schwann cells cultured in vitro, and progress summary and analysis of recombinant adenovirus expression vector and brain-derived neurotrophic factor. Through the analysis of the main problems in the past Schwann cells in vitro and gene transfection method, it is envisaged that the encoding neurotrophic factor gene fragment Schwann cells by adenoviral vector import that can achieve sustained, stable expression of the target gene, to explore gene therapy the new way of the outer periphery of nerve injury. The repair and regeneration of peripheral nerve injury is a multi-factor, multi-factor involved in physiological processes, supplement the very limited role of a single factor on nerve regeneration. Peripheral nerve injury symptoms belonging to the Weizheng and arthralgia, Chinese medicine and traditional Chinese medicine is a multi-factor complex, may provide more the ratio closer neurophysiological demand growth environment of the active factor has its unique advantages, this The success of the experiment provides a good foundation for the future combined with the overall concept of the traditional Chinese medicine to promote the recovery of nerve injury. Objective: To study the joint use of a variety of methods to be obtained in a short period of time a number of high purity of Schwann cells and Schwann cells transfected with the recombinant adenovirus vector carrying brain-derived neurotrophic factor gene. Explore the application of recombinant adenovirus vector carrying brain-derived neurotrophic factor gene transfection feasibility of Schwann cells and effective way to promote the recovery of peripheral nerve injury. Method: take 6-7 d SD neonatal rat sciatic nerve, using enzyme digestion, differential adherence method isolated Schwann cells, Ara-C G418 joint application to remove fibroblasts, bovine pituitary extract (BPE) to promote the proliferation of Schwann cells, as well as a series of steps with low concentrations of trypsin digestion passaged access to a large number of high-purity Schwann cells. Explore the BPE on cultured Schwann cell proliferation and to determine the optimal concentration of BPE applications, the second generation of the Schwann cells are divided into five groups, with different concentrations of BPE (50μg/ml, 100μg/ml, 150μg / ml, 200μg/ml), and without the BPE as a control. Joined the BPE after the second, four, six, eight days in each group, respectively, for S-100 protein antigen staining of Schwann cells, fibroblasts were counted for statistical analysis. The adenovirus vector infection of HEK293 cells, until CPE phenomenon obviously used when the virus harvest of repeated freezing and thawing method, and then again the harvested virus infection of HEK293 cells, harvesting the virus, was repeated three times. The last harvest of the virus titer was determined by plaque assay (plaque assay). BDNF gene recombinant adenovirus vector containing a multiplicity of infection (multiplicity of infection MOI) virus infection in vitro culture of rat derived SC. RT-PCR and Western blot analysis detected 72 h after infection, the infection efficiency, further MOI virus infection with high infection efficiency SC ELISA quantitative analysis after infection 3d, 6d, 9d, 12d, 15d, 18d and 21d infection SC culture supernatant the expression of BDNF. Results: the state of the Schwann cell morphology and S-100 protein-associated antigen immune cells staining verified, high purity, and up to 90%. BPE significant effect on the proliferation of Schwann cells; BPE concentration of 100μg/ml less affected fibroblast division maintained the effect on the proliferation of Schwann cells. After the amplification of the three virus Ad-BDNF titer: 1.78 × 109pfu/ml. Adenovirus SC 72 h after infection MOI of 200 infected group most efficient. Further at a MOI of 200 the amount of virus infection SC, ELISA results showed that at each time point after infection the SC of BDNF expression levels in the culture supernatant was significantly higher than the normal SC. Conclusion: by enzymatic digestion, differential adherence method, Ara-C G418-BPE and low concentrations of trypsin digestion combined use of high purity can be obtained in a short period of time, the state of good primary cultured Schwann cells. The appropriate concentration of bovine pituitary extract (BPE) can effectively promote the proliferation of Schwann cells. The adenovirus vector was amplified using HEK293 cells can be obtained in a short period of high titers of adenoviral vectors. Ad-BDNF infection SC capable of recombinant adenovirus-mediated exogenous BDNF gene transcription and translation, the ultimate expression of BDNF amount higher than normal SC, and the ability to maintain a longer period of time. This is the application of recombinant adenovirus vector carrying brain-derived neurotrophic factor gene transfection of Schwann cells to promote the damage nerve repair an experimental basis, to explore new ways of gene therapy for peripheral nerve injury.
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