The Study Of NT-3 Gene Transfection In Prevation Ototoxicity Induced By Gentamicin

The neurotrophin-3 is composed with 774 amino acids, plays an important role in neuronal differentiation ,survival , while it is the basic survival factor during the development of inner ear.. Indeed, because of its profound trophic effects on the nervous system, neurotrophin-3, NT-3 has being used to treat all kinds of sensorial hearing loss . The NT-3 cDNA gene was cloned by RT-PCR from human liver tissue, its eukaryotic expression vector (pIRES2-EGFP-NT-3) with the reporter gene -EGFP was constructed and identified by PCR and double-enzyme digestion. The pIRES2-EGFP-NT-3 was transfected into different kinds of cells of guinea pig cochlea tissue by using lipofectamine. The expression of NT-3 in the transfected cells was observed. Then the liposome -plasmid complex was injected into the guinea pig cochlea, and the expression of the reporter gene -EGF and NT-3 were observed respectively after 1 d, 2w and 4w, while the audition of the guinea pig were measured. The results are following: 1. Cloning human neurotrophin-3 (NT-3) cDNA and constructing itseukaryon vector and testing the expression of NT-3 ptotein 1) Cloning human neurotrophin-3 (NT-3) cDNA and constructing itseukaryon vectorThe total RNA was isolated by single-step (AGPC) from human liver tissue and its cDNA was gained by PT-PCR. The purified large fragment of pUC19 was digested with double-enzyme and the purified RT-PCR products were ligated and transformed into XL 10 Ecoli bacteria. The white clones were selected andthe plasmid was purified, which was further identified by double-enzyme digestion and sequenced. pUC19-NT-3 and pIRES2-EGFP were digested by double-enzyme respectively. The purified 774bp NT-3 DNA fragment of the former and the purified large fragment of the latter were ligated and transferred into XL 10 Ecoli bacteria.. The clones were randomly selected and recombinant plasmid was purified, which was identified by enzyme digestion and PCR reaction again. The human NT-3-cDNA was successfully cloned and itseukaryotic expression vector (pIRES2-EGFP-NT-3) was constructed.2) NIH3T3 cell line expressing human NT-3 was constructed and identifiedThe NT-3 cDNA gene was transfected into NIH3T3 cell line by using cationic liposome (LP2000). Then the NIH3T3 cells transfected with NT-3-cDNA were examined by RT-PCR, Western-blot and immunoreactivity methods. RESULTS: cell clones with G-418 resistance was obtained, the size of RT-PCR product of the positive cell clones was about 744bp, and the positive signals of protein were obtained.2. The influence of NT-3 tranfection in vitro on the growth of differentguinea pig cochlea tissue by using cationic liposome 1) The influence of NT-3 tranfection in vitro on the growth of guinea pigcochlea fibroblastThe pIRES2-EGFP-NT-3 was transfected into guinea pig cochlea fibroblast by using lipofectamine. After transfection 48h, the fibroblasts of transfection pIRES2-EGFP-NT-3 were observed by fluorescence microscope, and immunohistochemical methods. Results: The fibroblasts transfected with pIRES2-EGFP-NT-3 were observed by fluorescence microscope. The immunohistochemical staining showed that the NT-3 immunoreactivity was observed in the fibroblasts of transfection pIRES2-EGFP-NT-3, the NT-3 expression was not observed in the fibroblasts of transfection pIRES2-EGFP.2) The influence of NT-3 tranfection in vitro on the growth of guinea pig cochlea Schwann cellsWe optimize culture condition of SCs in vitro to eliminate fibroblast and promote proliferation of SCs in order to obtain more population and purification of SCs. The pIRES2-EGFP-NT-3 was transfected into SCs by using lipofectamine. The SCs with green fluorescence were observed by fluorescence microscope. The expression of NT-3 in transfected cells was determined by immunohistochemical method and grey measure. Results: The immunohistochemical staining showed that the NT-3 immunoreactivity was both observed in the SCs of transfection pIRES2-EGFP-NT-3 and pIRES2-EGFP, but the later one was weaker.