Basic And Prepared Experiments Of Neurotrophins (BDNF, NT3) Gene Therapy To Neurosensory Deafness Or Hearing Loss

OBJECTIVE: Hope learn about some methods of gene therapy to do some basic experiments and preparing experiments for hearing loss, meantime hope to improve the knowledge and the practice ability of molecular biology for myself. METHODS: According to designed primers, we used RT-PCR to amplify target genes(BDNF, NTS) and clone them to the vector of pUC19, then analysised the DNA sequence of genes by DNA analysis stations and compaired with GenBank. We constructed pDH-NT3 expression vector and expressed NT-3 protein through temperature.The very important step was that we amplified NTS gene both sides of which have BamH I and EcoR I cleavaging points from the pUC19-NT3 plasmid, further directional cloned them into pCDNAS of mammalian expression vector after both BamH I and EcoR I cleavaging, and transferred pCDNA3-NT3 plasmid into Jurkat cells through liposome entrapment with G418 screening those cells several times. After those, we transplanted these genetic engineering cells to cerebrospinal fluid(CSF) of BABL/C mouse to test their DPOAEs and compaired with the animals only transplanted pCDNA3 plasmids to CSF. We also constructed pLNCX-BDNF retrovirus expression vector. RESULTS: We got completely 749bp BDNF and 777bp NT3 genes, the DNA sequence of them were exact. The recombinant pDH-NTS, pCDNA3-NT3 and pLNCX-BDNF were all certified correctly by different ways. The very important progress that we had the NTS genetic engineering cells following G418 screening several times those Jurkat cells which were transferred pCDNA3-NT3 plasmids, and we have certified genetic engineering cells by extracted general RNA from part of them to amplify NT3 gene through RT-PCR and also examined the proteinactivity of NTS genetic engineering cells by western blot. Transplant the NTS genetic engineering cells to CSF could be delay hearing loss of BABL/C mouse with age. CONCLUSION: This thesis shows us that we can make genetic engineering cells of neurotrophic factors to treat and/or prevent deafness in the future.