Distribution And Age-related Changes Of Nerve Growth Factor In The Cochlea Of Mouse, Construction Of Eukaryotic Expression Plasmid For NGF Gene

Objective:Investigate the distribution and the age-related of NGF changes in the cochlea of mouse and construct eukaryotic expression plasmid for NGF gene to provide support to NGF gene therapy on presbycusis.Methods:Distribution in the cochlea and age-related expression of NGF protein in the cochlea spiral ganglion cell and hair cell were studied in SAMP8mice of5and7months. Normal aging senescence accelerated mouse/resistance1(SAMR1) mice served as the control groups. Taken human fetal brain (fed from human fetus five months induced by labor with water bag which from the affiliated hospital of guilin medical collage)50g, the total RNA was extracted and NGF fragment was amplified to construct eukaryotic expression vector of pcDNA3-β-NGF.Results:1Distribution and age-related changes of nerve growth factor in the cochlea of mouse:NGF expression was observed in inner and outer hair cell, spiral ganglion cell, auditory nerve fibers and stria vasculari, the NGF expression in hair cell and spiral ganglion cell is the major. The optical density of NGF immunohistochemical staining in the mouse cochlea spiral ganglion cell and hair cell:There were NGF protein expressed in the cochlea of the senescence accelerated mouse throughout the development. Compared with the SAMR1, the SAMP8at5,7months developed a significant descend(P<0.05). The optical density(OD) of NGF immunohistochemical staining in the mice cochlea spiral ganglion cell of each group was:5months SAMP8mice0.291±0.007;5months SAMR1mice0.420±0.039;7months SAMP8mice0.220±0.008;7months SAMR1mice;0.342±0.017. Compared with the SAMP8mice of5months, the optical density(OD) of NGF immunohistochemical staining in the SAMP8mice at7months showed a remarkable significant reduction (P<0.01). Compared with the SAMR1mice of7months, the optical density(OD) of NGF immunohistochemical staining in the SAMP8mice at7months showed a significant reduction (P<0.01). Compared with the SAMR1mice of5months, the optical density(OD) of NGF immunohistochemical staining in the SAMP8mice at5months showed a remarked reduction (P<0.01).The optical density(OD) of NGF immunohistochemical staining in the mice cochlea hair cell of each group was:5months SAMP8mice0.291±0.007;5months SAMR1mice0.410±0.010;7months SAMP80.213±0.009;7months SAMR1mice;0.334±0.012. Compared with the SAMP8mice of5months, the optical density(OD) of NGF immunohistochemical staining in the SAMP8mice at7months showed a remarkable reduction (P<0.01). Compared with the SAMR1mice of7months, the optical density(OD) of NGF immunohistochemical staining in the SAMP8mice at7months showed a remarked reduction (P<0.01). Compared with the SAMRl mice of5months, the optical density(OD) of NGF immunohistochemical staining in the SAMP8mice at5months showed a remarked reduction (P<0.01).2Construction of eukaryotic expression vector pcDNA3-β-NGF:The result of pcDNA3-β-NGF sequence demonstrated that β-NGF DNA was linked correctly with pcDNA3+vector plasmid by double-enzyme cleavage and gene sequence analysis.Conclusions:NGF expresses in the cochlea of mouse and the expression level changes with age and the result of pcDNA3-β-NGF sequence demonstrated that β-NGF DNA was linked correctly with pcDNA3+ vector plasmid indicate that NGF gene probably has relationship with maintaining functional status of the cochlea and provide support to the experimental research of prevention and treatment of presbyacusis from NGF gene level.