Objective To explore the changes of superoxide dismutase(SOD),malondialdehyde(MDA) and apoptosis in the auditory cortex,the cochlear nuclei and the cochlea of the rats with age-related hearing loss,and to understand the relationship between oxidative damage and apoptosis with presbyacusis.Methods Three-month-old rats(ABR 20?5 dB SPL) and eighteen-month-old rats(ABR 55?10 dB SPL) were used in the study.T-SOD activity was measured by xanthine oxidase(XO) method.The MDA level was examined by the thibabituric acid(TBA) method.Apoptosis was examined by Annexin V-FITC/PI staining with flow cytometry(FCM).Results The SOD activities decreased and the MDA levels increased significantly in the auditory cortex,the cochlear nuclei and the cochlea of the eighteen-month-old rats(P

Objective To investigate the function of bone-marrow mesenchymal stem cells(BMSCs) differentiating into neuron-like cells in vitro after transfected by human brain-derived neurotrophic factor (BDNF) gene.Methods Human BDNF genes were cloned and recombinant pcDNA3.1(-)-BDNF plasmids were constructed. BMSCs from five guinea pigs were isolated and cultured while their morphologies were observed by microscope. The surface antigen was detected by flowcytometry. BDNF genes were transfected into BMSCs with electroporation,and the transfected BMSCs were induced by ratinoic acid(RA)after bolted by Geneticin-418(G418),then the differentiated BMSCs were identified by immunocytochemistry. Results The culture cells demonstroted the typical morphology and surface antigen of BMSCs. The transfected cells expressed neuron-specific enolase(NSE),Nestin and glial fibrillary acid protein(GFAP) also secreted BDNF.Conclusion BMSCs transfected by BDNF genes can differentiate into neuron-like cells in vitro,electroporation can enhame the transfection efficiency,RA can promote cell induction.

Objective Galactosialidosis(GS) is an autosomal recessive lysosomal storage disease caused by a combined deficiency of lysosomal ?-galactosidase and neuraminidase as a result of a primary defect in the protective protein/cathepsin A(PPCA).Mouse model of GS has been generated by targeted deletion of PPCA gene and closely resembled the phenotypes in human conditions.However,it remains to be determined whether hearing loss observed in human also occurs in the mouse model.In this study,we observed their alterations of the auditory function and morphology of the ear,and explored pathophysiological mechanisms of hearing impairment.Methods PPCA homozygous(PPCA-/-) mice at 1 and 2 months of age,and their wildtype littermates(PPCA+/+) were examined for auditory thresholds through auditory brainstem responses(ABR) to click,tone pips 8,16,and 32 kHz stimuli.Morphological analyses in ears were performed by series temporal bone section and light microscopy.Results PPCA-/-mice at 1 month of age showed a normal threshold and the morphology of ears.Up to 2months of age,their thresholds were elevated 40～45 dB SPL above those of PPCA+/+ mice.There were distinct pathological changes of middle and inner ear in PPCA-/-mice of 2 months old.The severe otitis media and the vacuolation associated with lysosomal storage were observed within ossicles and cochlear bone cells,stria vascularis cells,spiral ganglion neurons,spiral limbus,Reissner's membrane cells,and the mesothelial cells of the perilymphatic scala and basilar membrane,but not within the organ of Corti.Vestibular organ did not show vacuolation.Conclusion The deficiency of lysosomal protective protein/cathepsin A may result in hearing loss and morphological alterations of ear.The otitis media and ossicle changes,and the defects in lysosomal storage of neurons,stria vascularis,spiral limbus,Reissner's membrane and basilar membrane cells may contribute to the conductive and sensorineural hearing loss respectively.

BACKGROUND:Gene transfection of cells includes virus and non-virus vector.As virus vector has some issues,such as safety and immunological rejection,the present study explored lipofectamine and electroporation transfection methods.OBJECTIVE:To establish genetic engineering cells using human brain-derived neurotrophic factor(hBDNF)gene transfected bone marrow mesenchymal stem cells(BMMSCs)by lipofectamine or electroporation,and explore its characteristics and expression in vitro.METHODS:Lipofectamine method:The BMMSCs were obtained from the tibias and femurs of the guinea pigs.The third passage BMMSCs were cultured with plasmid-lipofectamine mixture for 6 hours,followed by fetal bovine medium for 48 hours.Immunohistochemistry was performed for transient expression.G418 was added after 48 hours.Electroporation method:BMMSCs were trypsinized and resuspended with serum-free medium.Cell suspension was added into electrotransformation pool,and plasmid was added.The electrotransformation pool was moved between electrodes.After transfection for 48 hours,gene transient expression was detected.G418 was added after 48 hours.Brain-derived neurotrophic factor expression was detected by immunohistochemistry and RT-PCR.RESULTS AND CONCLUSION:Immunohistochemistry showed that BDNF transient expression was 5.80% by lipofectamine and 24.29% by electroporation.Cells almost died at 14 days following lipofectamine transfection.Stable expression cell lines of BDNF engineered BMMSC were successfully established by electroporation,with 90% expressive rate by immunohistochemistry and expression of BDNF mRNA by RT-PCR.Genetic engineering cells using BDNF transected BMMSC were established by electroporation whereas failed by lipofectamine,and the expressed BDNF was confirmed by immunohistochemistry and RT-PCR in vitro.

TMPRSS3 (transmembrane protease, serine 3) is a member of Ⅱ transmembrane serine proteases (TTSPs), and like the other members of this family, it contains typical domains including a serine protease domain, a transmembrane domain, a LDL receptor-like domain (LDLRA), and a scavenger receptor cysteine-rich domain (SRCR). Four alternative protein isoforms have been described, and isoform A is thought to be primary isoform which is expressed in many tissues, especially in the cochlea. TMPRSS3 protein is primarily localized in the endoplasmic reticulum membranes where it may be anchored by its transmembrane domain. TMPRSS3 is mutated in non-syndromic autosomal recessive deafness (DFNB8/10). Therefore TMPRSS3 is thought to be involved in the development and maintenance of the inner ear, and isoform D may be proposed as a novel diagnostic marker in ovarian carcinoma. TMPRSS3 protein is the first protease which mutation could lead to deafness. These data indicate that important signaling pathways in the inner ear are controlled by proteolytic cleavage. However, it is not clear about TMPRSS3 substrates and its function. The epithelial amiloride-sensitive sodium channel (ENaC) which is regulated by membrane-bound channel activating serine proteases (CAPs), a member of TTSPs, may be a potential substrate of TMPRSS3, but this hypothesis is still to be verified in vivo. With the development of protease research and the application of protease proteomics, substrate degradomes of a protease may therefore represent an important tool for the research of TMPRSS3 function and its molecular mechanism.

Objective To study the expression of brain-derived neurotrophic factor(BDNF) and nerve growth factor(NGF) in the guinea pig cochlea of cisplatin-induced ototoxicity, and to understand its protective role for the inner ear.Methods The animals were divided into normal group and experimental group with cisplatin. Sodium chloride was used for the control. All animals were sacrificed on the day 3, 5 and 7, and the cochlea were used for BDNF and NGF staining.Results The weak expression of BDNF and the moderate expression of NGF were observed in the cochlea in the normal group. The strongest expression of BDNF and NGF was observed in the animals with cisplatin.Conclusion The existence of NGF in the normal cochlea indicates that NGF plays an important role to maintain auditory physiology.BDNF and NGF may be involved in self-protection mechanism for the cochlea and auditory nerves from cisplatin-induced ototoxicity.

Objective To establish human brain-derived neurotrophic factor (BDNF) genetical modified marrow stromal cells and to evaluate the influence on the viability of the cultured spiral ganglion cells from Kunming mice.Methods Eukaryotic expression plasmid——pcDNA3.1(-)-BDNF was constructed according to the molecular clone approach. Marrow stromal cells(MSC) were separated and cultured. BDNF genetical modified marrow stromal cells were established and verified by biochemical analysis.The bio-effect to spiral ganglion cells, especially with the oxidative damage under the different concentration of H_2O_2 was observed.Results PcDNA3.1(-)-BDNF and BDNF genetical engineering cells were successfully established. The supernatant from BDNF-MSCs remarkably improved the survival rate of the spiral ganglion cells.Conclusion BDNF genetical modified marrow stromal cells are successfully established, and the genetical engineering cell plays an important role in the protection the spiral ganglion cells from the oxidative damage. This study provide a strong basis for genetical engineering cells transplantation into the inner ear.

Objective To explore the deafness-causing underlying mechanisms of CX26 gene recessive mutations through functional analyzing nine missense mutations (p.S19T,p.R32H,p.E47K,p.V84L,p.V95M,p.R143W,p.R165W,p.S199F,p.L214P) in exogenous expression system Hela cells.Methods The nine recessive missense mutations of CX26,which are in the different domains of CX26 protein,and the wild type CX26 were subcloned into pEGFP-N1 vector directively,following to transfect into HeLa cells by the liposome complex method.The expressions of the mutated proteins were analyzed using western blot method.The localizations of the mutated proteins and whether there were gap junction-plaques formation were observed under confocal microscopy with immunofluorescence technique.Results The nine constructs were all expressed in HeLa cells.In which,the mutated proteins of p.S19T,p.E47K,p.V84L,p.V95M and p.R165W localized at the cytoplasmic membrane of HeLa cells and formed gap junction-plaques at contact points between two cells,and the mutated proteins of p.R32H,p.R143W,p.S199F and p.L214P accumulated and localized only intracellularly and did not form gap junction-plaques on cell membrenes.Conclusion The mutations of p.S19T,p.E47K,p.V84L,p.V95M and p.R165W do not interfere the mutated connxins trafficking and inserting into the plasma membrane.The mutations of p.R32H,p.R143W,p.S199F and p.L214P impared the proteins trafficking to the cell surface.The deafness-causing mechanisms of different missense mutations might not be identical and no correlation could be observed between the mutation and the topological domain of the mutated protein.

Objective To investigate the function of bone-marrow mesenehymal stem eells(BMSCs) differ-entiating into neuron-like cells in vitro after transfected by human brain-derived neurotrophie factor (BDNF) gene. Methods Human BDNF genes were cloned and recombinant pcDNA3. 1(-)-BDNF plasmids were construc-ted. BMSCs from five guinea pigs were isolated and cultured while their morphologies were observed by microscope. The surface antigen was detected by flowcytometry. BDNF genes were transfected into BMSCs with electroporation, and the transfected BMSCs were induced by ratinoie acid(RA)after bolted by Geneticin-418 (G418), then the dif-ferentiated BMSCs were identified by immunocytochemistry. Results The culture ceils demonstroted the typical mor-phology and surface antigen of BMSCs. The transfected cells expressed neuron- specific enolase(NSE), Nestin and glial fi-brillary acid protein(GFAP) also secreted BDNF. Conclusion BMSCs transfected by BDNF genes can differentiate into neu-ron- like cells in vitro, electroporation can enhame the transfection efficiency, RA can promote cell induction.

Objectives To establish the two-dimensional electrophoresis(2-DE)profile of cell. Secreted proteins.Difierential expression profiling of fibroblast cell secreted proteins between nasopharyngeal carcinoma and normal nasopharyngeal tissue was analyzed.Methods Five tissue specimens each from patients with nasopharyngeal carcinoma and nasal polyp were collected individually.Fibroblast eells from above-mentioned tissue were cultured in serum-free medium,and cell-secreted proteins from the cultured medium were harvested by uhrafihration concentration and desalination.Samples were analyzed by 2-DE,and the differentially expressed proteins were analyzed and identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry.Galectin-1 wa8 analyzed by EUSA test.Results 2-DE diagram of fibroblast cell-secreted proteins Was constructed.1 8 protein spots displayed quantitative changes in expression,and 11 protein spots among them were identified by mass speetrometrv.3 proteins including cystatin C,complement subcomponent C1S precursor,heterogeneous nuclear ribonueleoprotein A1 were down-regulated in the cultured medium of nasopharyngeal carcinoma associated fibroblast cells(CAFs). Nevertheless,the rest cell-secreted proteins including galectin-1,14-3-3 protein sigma,eathepsin L and etc,were up-regulated.Meanwhile,the expression of galectin-1 in the cultured medium was also analyzed and Its results were compared between CAFs and the normal fibroblast cells by ELISA.There Was statistical significance difference between them,and galectin-1 was up-regulated in the cIlltured medium of CAFs.Conclusions The changes of fibroblast cell-secreted proteins during nasopharyngeal carcinogenesi8 are analyzed by 2-DE analysis.The variation of pattern of secreted proteins is involved in signal transduction,protein synthesis,degradation and other pathways.CAFs may regulate tumor microenvironment by the abeve-mentioned pathways,and influence nasopharyngeal carcinogenesis,progress,invasion and metastasis.This study provided experimental basis for the eell secreted proteomics studv in future.