Cuiping Zhong

Identification of new altered genes in rat cochleae with noise-induced hearing loss.

Because genes that are highly expressed in the cochlea after noise stress may have crucial regulatory roles in hearing, the identification of these genes may be useful for restoring normal auditory function. This study assessed altered gene expression at 1h following the cessation of noise exposure by using microarrays and real-time polymerase chain reaction (qPCR) in rats. In addition, the auditory threshold shifts and morphological changes of hair cells were observed. This study indicated that applied noise induced outer hair cell loss and a 40-50 dB hearing loss. Totally 239 altered genes were involved in the immune system process, response to stress, or response to stimulus. The expression of five up-regulated genes (Reg3b, Lcn2, Serpina3n, Nob1 and Hamp) was confirmed by qPCR. Future experiments will focus on several of these new candidate genes and may provide insight into the underlying auditory pathophysiology.

Up-regulation of Nob1 in the rat auditory system with noise-induced hearing loss

The Nob1 gene is assumed to be associated with transcription regulation and may play important roles in mediating some physiological and pathological functions. Here, the rats were randomized equally into experimental group and control group. In experimental group, all subjects were exposed to 4-kHz octave-band noise at 110 dB SPL, 8 h per day for 7 days consecutively. Auditory thresholds were assessed by auditory brainstem response, prior to and 1 h after the cessation of noise exposure. Then, we investigated for the first time the expression of Nob1 in noise-exposed and noise-unexposed rats by quantitative polymerase chain reaction. The distribution of Nob1 in rat cochlea was further examined by immunohistochemistry. The results indicated that the hearing threshold was significantly higher in the noise-injured group than in the uninjured group after noise exposure. Nob1 mRNA was present at higher levels in regions of the noise-injured cochlea. As for noise-exposed rats, Nob1 expression was positive in the inner and outer hair cells of the organ of Corti and spiral ganglion neurons, but it undetectable in the uninjured cochlea. Therefore, Nob1 may play an important role in auditory function following acoustic trauma and can be used as a new target for the treatment of noise-induced hearing loss.

Preparation and Characterization of a Novel Monoclonal Antibody Specific to Human NOB1 Protein

The human NOB1 gene is located at chromosome 16q22.1, encoding a protein consisting of one zinc ribbon domain. Here we aimed to efficiently generate a monoclonal antibody against NOB1 protein. We synthesized the peptide APVEHVVADAGAFLRH based on published NOB1 cDNA sequences. The peptide was chemically linked with the carrier protein keyhole limpet hemocyanin and then injected into Balb/c mice. Hybridomas were screened by indirect enzyme-linked immunosorbent assay (ELISA) using either purified 6xHis-NOB1 fusion protein or the peptide. One MAb named H11 (IgG1), effective in detecting the native NOB1 protein, was characterized by ELISA and Western immunoblotting. By using the MAb, we found NOB1 protein was expressed in human lung, liver, spleen, and kidney tissues. Taken together, the MAb H11 would be helpful for understanding the distribution and functions of NOB1.

The expression of NOB1 in spiral ganglion cells of guinea pig

OBJECTIVE NOB1 was a transcription-associated protein, consisting of one zinc ribbon domain. This study aimed to investigate the NOB1 expression in spiral ganglion cells of normal guinea pigs and deaf ones. METHODS Twelve guinea pigs were randomized equally into experimental group and control group. In experimental group, guinea pigs received a single intramuscular injection of gentamicin, while the control group was treated with physiological saline. Auditory brainstem responses (ABR) test was performed before and 10 days after injection, respectively. Guinea pigs of both groups were sacrificed and temporal bones were removed. The expression of NOB1 in the spiral ganglion was evaluated by immunohistochemistry. RESULTS NOB1 staining was found expressed in spiral ganglion cells from the basal turn to the apical turn of the cochlea. The expression of NOB1 was found significantly stronger in spiral ganglion cells of deaf guinea pigs as compared with that in normal ones. CONCLUSIONS The NOB1 was presented in spiral ganglions cells of the guinea pig and might play a role in hearing transduction.

Effect of c-myc on the ultrastructural structure of cochleae in guinea pigs with noise induced hearing loss

Noise over-stimulation may induce hair cells loss and hearing deficit. The c-myc oncogene is a major regulator for cell proliferation, growth, and apoptosis. However, the role of this gene in the mammalian cochlea is still unclear. The study was designed to firstly investigate its function under noise condition, from the aspect of cochlear ultrastructural changes. We had established the adenoviral vector of c-myc gene and delivered the adenovirus suspension into the scala tympani of guinea pigs 4 days before noise exposure. The empty adenoviral vectors were injected as control. Then, all subjects were exposed to 4-kHz octave-band noise at 110dB SPL for 8h/day, 3 days consecutively. Auditory thresholds were assessed by auditory brainstem response, prior to and 7 days following noise exposure. On the seventh days after noise exposure, the cochlear sensory epithelia surface was observed microscopically and the cochleae were taken to study the ultrastructural changes. The results indicated that auditory threshold shift after noise exposure was higher in the ears treated with Ad.EGFP than that treated with Ad.c-myc-EGFP. Stereocilia loss and the disarrangement of outer hair cells were observed, with greater changes found in the Ad.EGFP group. Also, the ultrastructure changes were severe in the Ad.EGFP group, but not obvious in the Ad.c-myc-EGFP group. Therefore, c-myc gene might play an unexpected role in hearing functional and morphological protection from acoustic trauma.

A comparison of the proliferative capacity and ultrastructure of proliferative cells from the cochleae of newborn rats of different ages

OBJECTIVE Recent reports have shown that multipotent stem cells/progenitor cells that are capable of proliferation and regeneration are present in mammalian cochleae. However, progenitor cells have not been isolated from the adult cochlea. We examined the proliferative potential of cells derived from neonatal rats of various ages. The determination of the differences between the proliferative cells from rats of different ages may provide clues to the mechanisms controlling the destiny of these cells. METHODS Proliferative cells were isolated from the cochleae of 1-, 7-, and 14-day-old rats, and the proliferative capacity and ultrastructure of the cells from each age group were assessed using flow cytometry and transmission electron microscopy, respectively. RESULTS During the first two postnatal weeks, the number of proliferative cells gradually fell to zero. This decrease occurred in parallel with the impairment of the proliferative capacity of the cells and the accumulation of proliferative cells in G0/G1. In addition, some of the cells exited the cell cycle by means of gradual maturity and apoptosis. CONCLUSIONS Our study suggests that cochlear proliferative cells are remnants of the progenitor cells that originally gave rise to the sensory epithelium. The disappearance of the cochlear proliferative cells in adult mammalian cochleae may result from their differentiation and/or apoptosis.

Decreased level of cyclin A2 in rat cochlea development and cochlear stem cell differentiation.

Cyclin A2 plays a major role in cell cycle modulation. Cyclin A2 level has not been determined in the inner ear yet. RT-PCR, Western blotting, immunohistochemistry and immunocytochemistry were used to measure cyclin A2 expression in rat cochlea tissues of different ages, isolated cochlear stem cells and stem cell-derived differentiated cells. The results indicated that cyclin A2 level in cochlea tissues decreased gradually from newborn to adult. Furthermore, cyclin A2 level fell down after differentiation of cochlear stem cells. It was suggested that cyclin A2 might be involved in the modulation of rat cochlea development and cochlear stem cell differentiation.

Alginate microcapsules co-embedded with MSCs and anti-EGF mAb for the induction of hair cell-like cells in guinea pigs by taking advantage of host EGF

As the irreversibility of hair cell loss in mammals is among the main reasons giving rise to permanent hearing loss, studies have been focused on the development of biological technologies to generate new hair cells as a means of replacing lost hair cells. Bone marrow-derived mesenchymal stem cells (BMSCs) possess the capacity to differentiate into hair cell-like cells, and may find applications in the regeneration of mammalian cochlear hair cells. In order to efficiently induce BMSCs into hair cells, alginate microcapsules co-delivering rat bone marrow-derived mesenchymal stem cells (rBMSCs) and anti-EGF monoclonal antibody (mAb) were developed to examine the feasibility of differentiating rBMSC into hair cell-likes cells in vitro and in vivo by taking advantage of epidermal growth factor (EGF) ligands. In vitro analysis showed that anti-EGF mAbs bonded with exogenous EGF ligands activated the EGF receptors on rBMSCs, enhancing the expression of myosin 7a (a hair cell marker) and Notch1 (supporting cell marker) via the EGFR/Ras/Raf/ERK1/2 signal pathway. In in vivo experiments, alginate microcapsules loaded with rBMSCs (2 × 106 cells per microcapsule) together with Iso mAbs or anti-EGF mAbs were grafted into guinea pig cochlea. After 6 weeks of treatment, immunofluorescence analyses indicated that the rBMSCs embedded into anti-EGF microcapsules were more efficiently transformed into hair cells compared with the group with Iso mAb microcapsules and displayed an ordered arrangement in Reissners membranes. The results highlighted the significance of engineering the microenvironment of stem cells for hair cell differentiation, and particularly the advantage of hair cell differentiation of rBMSCs by recruiting host EGF ligands via tethered mAbs. In conclusion, this strategy of co-embedding rBMSCs and anti-EGF mAb in alginate microcapsules is a promising modality for the regeneration of hair cell-like cells.

Construction of recombinant adenoviral vector carrying cyclinA2 gene

Cell cycle related molecules in mammalian cochleae could provide a new avenue to restore hearing loss caused by a variety of genetic and environmental insults. CyclinA2 is one of the most important regulators of cell cycle, but its role in the mammalian cochlea is still unknown. So, it is necessary to construct an adenovirus vector carrying cyclinA2 gene for clarifying its function in the cochlea. In this study, the cyclinA2 genes were cloned into the shuttle plasmid pDC316-mCMV-EGFP to construct pDC316-CyclinA2-mCMV-EGFP, which was co-transfected with the rescue plasmid pBHGlox∆E1,3Cre into 293 cells to obtain the recombinant adenovirus Ad.CyclinA2-EGFP. Then, the plasmid pDC316-CyclinA2-mCMV-EGFP and recombinant adenovirus Ad.CyclinA2-EGFP were identified by restriction enzymes and reverse transcription-polymerase chain reaction (RT-PCR). The recombinant adenovirus vector was purified by CsCl banding, and was titrated. Finally, the recombinant adenovirus vector carrying cyclinA2 gene was constructed and confirmed by restriction enzyme analysis and RT-PCR. The titer of the recombinant adenovirus vectors reached 2.5 × 10(‒11) v.p/mL. Thus, we had successfully established the Ad.CyclinA2-EGFP vector, and it could express efficiently in various cells of cochlea. This study established the foundation for the further research of cyclinA2 genes function in the cochlea.

Viral-mediated expression of c-Myc and cyclin A2 induces cochlear progenitor cell proliferation

Cochlear progenitor cells have a limited proliferative capability, which prevents their application in treating sensorineural hearing loss. In this study, we showed that the expression of c-Myc and cyclin A2 was down-regulated during the development of cochlear tissue and CPC differentiation. Over-expression of these two genes using adenovirus transduction, significantly affected the CPC cell cycle and promoted the CPC proliferation. We further demonstrated that this promotion involves the classic CKI-cyclin-CDK pathway. Our study suggests that genetically modified CPCs may be a promising cell source for cochlear stem cell transplantation that improves the efficacy of cell therapy.