Jian-He Sun

Xinqiao Hospital, Ch’ung-ch’ing-shih, Chongqing Shi, China

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Publications (18)24.21 Total impact

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    ABSTRACT: To observe the morphology and function changes of cochlear hair cells before and after math1 gene injection into the cochlea of deaf guinea pigs which were induced by kanamycin and furosemide. To explore the feasibility of Math1 gene for medicine-induced deafness therapy. Kanamycin (500 mg/kg) and furosemide (50 mg/kg) were given to the healthy adult guinea pigs intramuscularly and intravenously to establish the deafness model. The guinea pigs whose auditory brainstem response (ABR) threshold > 95 dB SPL were randomly divided into five groups. Blank control group (without any treatment, n = 3), operation control group (right ear scala tympani operation, n = 3), artificial perilymph group (right ear scala tympani injection artificial perilymph, n = 3), virus vector group [right ear scala tympani injection adenovirus which carrying enhanced green fluorescent protein (EGFP) gene (Ad. EGFP) , n = 4], Math1 gene therapy group [right ear scala tympani injection adenovirus which carrying Math1 and EGFP gene (Ad. Math1-EGFP), n = 6]. Each animal received ABR test before and after injection. The cochlear tissue was observed by scanning electronic microscopy. The ABR thresholds of tone burst( 4, 8, 16, 20 kHz ) were not statistically significant in different groups (P > 0.05). The number of hair cells increased in some of severe deaf guinea pigs after the injection of Ad. Math1-EGFP gene. However, there was no obvious difference with morphology and numbers of cochlea hair cells in other groups. The injection of Math1 gene to cochlea can regenerate or repair the hair cells of medicine-induced deaf guinea pigs, but there was no improvement on the hearing loss.
    Zhonghua er bi yan hou tou jing wai ke za zhi = Chinese journal of otorhinolaryngology head and neck surgery 07/2013; 48(7):584-8.
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    ABSTRACT: Abstract Conclusion: Mouse embryonic stem cells (ESCs) transplanted into the scala tympani are able to migrate in the cochlea of rats deafened with aminoglycoside and partly restore the structure of sensory epithelia of the inner ear. Objectives: To explore the migration and differentiation of enhanced green fluorescence protein (EGFP)-expressing ESCs by transplanting them into the scala tympani of rats with amikacin sulfate-induced hearing loss. Methods: Adult Sprague-Dawley (SD) rats were deafened with amikacin sulfate. Mouse ESCs expressing EGFP (EGFP-ESCs) were transplanted into the scala tympani. The migration and differentiation were observed at different time points. Results: EGFP-ESCs transplanted into normal cochlea did not migrate, but those in the amikacin-damaged cochlea could survive and migrate into the scala media and the vestibular cisterna. For the first time, we observed that the EGFP-ESCs migrated into the scala media, took the place of the organ of Corti, and formed a structure just like the cochlear tunnel. Some grafted stem cells even expressed myosin VIIa, the molecular marker of hair cells. Some nerve fibers reached to the bottom of the hair cell-like cells. The ESCs migrated into the vestibule and restored the sensory epithelia of the ampullary crest. The number of the transplanted ESCs reduced over the 6 week period of the study.
    Acta oto-laryngologica 10/2012; · 0.98 Impact Factor
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    ABSTRACT: To investigate the histological changes in the vestibular endorgans of Smad4 gene conditional knockout mice and to explore the influence of the Smad4 gene on vestibular development. Histological changes of periphery vestibular organs in inner ear of Smad4 conditional knockout mice were investigated by frozen sections, immunofluorescence, confocal microscopy, scanning electron microscopy and transmission electron microscopy. There was no Smad4 expression in the inner ear cartilage capsule of Smad4-/- mice. In Smad4+/- mice, Smad4 expression in the same cartilage capsule was positive, and it was strong positive in Smad4+/+ mice. Smad4 expression in vestibular sense epithelium, crista ampullaris and macula, was positive. And no difference was found among these three genotypes. Studying at scanning electron microscopy and transmission electron microscopy levels and anti-filament immunofluorescence showed that no pathological changes were observed in all the three genotype mice. Although the Smad4 gene was knockout effectively in the auricular cartilage capsule of Smad4 conditional knockout mice,the histological changes of Smad4 conditional knockout mice in vestibulum auris internae were slightly.
    Zhonghua er bi yan hou tou jing wai ke za zhi = Chinese journal of otorhinolaryngology head and neck surgery 07/2012; 47(7):575-80.
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    ABSTRACT: In the current study, hair cells of vestibular terminal organs in rats were completely eliminated with trans-scala vestibuli injection of neomycin, and then the Math1 gene was transferred. It was shown that type I vestibular hair cells were regenerated and synapses were formed. The objective of this study was to identify the cell type of the regenerated vestibular hair cells and relative innervation and synaptic linkage after hair cells of vestibular terminal organs in rats were completely eliminated. Neomycin injection was used to eliminate all the vestibular terminal organs, and then the animals were treated with an injection of Ad-Math1-EGFP in the scala vestibuli of the cochlea. Math1 gene transfer into the inner ear induced type I hair cell regeneration and synaptic formation. However, neither the number nor the appearance of the hair cells was normal.
    Acta oto-laryngologica 06/2012; 132(8):819-28. · 0.98 Impact Factor
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    ABSTRACT: The hallmark of mechanosensory hair cells is the stereocilia, where mechanical stimuli are converted into electrical signals. These delicate stereocilia are susceptible to acoustic trauma and ototoxic drugs. While hair cells in lower vertebrates and the mammalian vestibular system can spontaneously regenerate lost stereocilia, mammalian cochlear hair cells no longer retain this capability. We explored the possibility of regenerating stereocilia in the noise-deafened guinea pig cochlea by cochlear inoculation of a viral vector carrying Atoh1, a gene critical for hair cell differentiation. Exposure to simulated gunfire resulted in a 60-70 dB hearing loss and extensive damage and loss of stereocilia bundles of both inner and outer hair cells along the entire cochlear length. However, most injured hair cells remained in the organ of Corti for up to 10 days after the trauma. A viral vector carrying an EGFP-labeled Atoh1 gene was inoculated into the cochlea through the round window on the seventh day after noise exposure. Auditory brainstem response measured one month after inoculation showed that hearing thresholds were substantially improved. Scanning electron microscopy revealed that the damaged/lost stereocilia bundles were repaired or regenerated after Atoh1 treatment, suggesting that Atoh1 was able to induce repair/regeneration of the damaged or lost stereocilia. Therefore, our studies revealed a new role of Atoh1 as a gene critical for promoting repair/regeneration of stereocilia and maintaining injured hair cells in the adult mammal cochlea. Atoh1-based gene therapy, therefore, has the potential to treat noise-induced hearing loss if the treatment is carried out before hair cells die.
    PLoS ONE 01/2012; 7(9):e46355. · 3.53 Impact Factor
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    ABSTRACT: Conclusion. Bone marrow mesenchymal stem cells (MSCs) have the ability to differentiate into hair cells, and this method of culturing MSCs provides a useful tool for studies on mammalian cochlear hair cell regeneration. Objective: To investigate a method to induce bone marrow MSCs to differentiate into inner ear hair cells. Methods: Rat bone marrow MSCs were isolated from healthy rats and cultured in vitro. To make sure that the cultured cells were bone marrow MSCs, the expression of MSC markers such as SH2, CD31, CD34, and CD44 genes on the cultured cells was assessed by RT-PCR. Adipogenic cells and osteogenic cells were induced by the differentiation of the cultured cells, respectively, suggesting that the cultured cells have the characteristic of pluripotent differentiation. Then they were induced to differentiate into neural stem cells and hair cell progenitor cells. Immunohistochemistry experiments were carried out to detect the expression of molecular markers. Scanning electron microscope samples were prepared for observation of the morphology of the cells. Results: Rat bone marrow MSCs were successfully isolated, purified, cultured, and identified in vitro. They were also successfully induced to differentiate into neural progenitor cells and then hair cell-like cells that expressed myosin VIIa.
    Acta oto-laryngologica 01/2012; 132(1):112. · 0.98 Impact Factor
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    International Journal of Developmental Neuroscience. 11/2011; 29(7):783.
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    ABSTRACT: Bone marrow mesenchymal stem cells (MSCs) have the ability to differentiate into hair cells, and this method of culturing MSCs provides a useful tool for studies on mammalian cochlear hair cell regeneration. To investigate a method to induce bone marrow MSCs to differentiate into inner ear hair cells. Rat bone marrow MSCs were isolated from healthy rats and cultured in vitro. To make sure that the cultured cells were bone marrow MSCs, the expression of MSC markers such as SH2, CD31, CD34, and CD44 genes on the cultured cells was assessed by RT-PCR. Adipogenic cells and osteogenic cells were induced by the differentiation of the cultured cells, respectively, suggesting that the cultured cells have the characteristic of pluripotent differentiation. Then they were induced to differentiate into neural stem cells and hair cell progenitor cells. Immunohistochemistry experiments were carried out to detect the expression of molecular markers. Scanning electron microscope samples were prepared for observation of the morphology of the cells. Rat bone marrow MSCs were successfully isolated, purified, cultured, and identified in vitro. They were also successfully induced to differentiate into neural progenitor cells and then hair cell-like cells that expressed myosin VIIa.
    Acta oto-laryngologica 08/2011; 131(11):1136-41. · 0.98 Impact Factor
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    ABSTRACT: In mammals, hair cells do not undergo spontaneous regeneration when they are damaged and result in permanent hearing loss. Previous studies in cultured Organ of Corti dissected from neonatal animals have shown that both DAPT (r-secretase inhibitor in the Notch signal pathway) treatment and Atoh1 overexpression can induce supernumerary hair cells. The effects of simultaneous DAPT treatment and Atoh1 over expression in the cells of cultured Organ of Corti from neonatal rats are still obscure. In this study, we set out to investigate the interaction of DAPT treatment and Atoh1 overexpression as well as culture time and the location of basilar fragment isolated form neonatal rat inner ear. Our results showed that DAPT treatment induced more hair cells in the apical turn, while Atoh1 overexpression induced more extra hair cells in the middle turn of the cultured Organ of Corti. When used together, their effects are additive but not synergistic. In addition, the induction of supernumerary hair cells by both DAPT and Atoh1 overexpression is dependent on the treatment time and the location of the cochlear tissue. Moreover, DAPT treatment causes dramatic changes in the orientation of the stereociliary bundles of hair cells, whereas Atoh1 overexpression didn't induce drastic change of the polarity of stereociliary bundles. Taken together, these results suggest that DAPT treatment are much more potent in inducing supernumerary hair cells than Atoh1 overexpression and that the new hair cells mainly come from the trans-differentiation of supporting cells around hair cells. The orientation change of stereociliary bundle of hair cells may be attributed to the insertion of the newly formed hair cells. The immature hair bundles on the newly formed hair cells may also contribute to the overall chaos of the stereociliary bundle of the sensory epithelia.
    PLoS ONE 01/2011; 6(10):e23729. · 3.53 Impact Factor
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    ABSTRACT: Mammalian cochlear hair cells don't regenerate naturally after injury, which usually leave permanent hearing loss. Math1 gene is a positive regulator of hair cell differentiation during cochlear development and was proved to be very critical in hair cell regeneration in deaf animals. Generating new cochlear hair cells by forced Math1 expression may be a cure for hearing loss. However, satisfying gene delivering vectors in gene therapy are not available. We combined quaternized chitosan (QCS) with Na-carboxymethyl-beta-cyclodextrin (CM-beta-CD) as novel non-viral vector, which adsorbs pRK5-Math1-EGFP perfectly at the mass ratio of 4:1. In vitro cell transfection can reach a 40% transfect efficiency and relatively low cytotoxity than liposomes. These results suggest that QCS/CM-beta-CD nanoparticle complexes could be a novel non-viral gene carrier in further clinical application.
    Journal of Nanoscience and Nanotechnology 11/2010; 10(11):7262-5. · 1.15 Impact Factor
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    ABSTRACT: The regulation of the bone morphogenetic protein (BMP) signal transduction pathway is important in the development of the inner ear and vestibular system. We reported previously that small mothers against decapentaplegic homolog-4 (Smad4) is required for inner ear cochlear development and normal auditory function in mammals; however, the distribution and functional mechanisms of Smad4 at various stages of vestibular development remained unclear. To investigate the relationship between the Smad4 gene and vestibular organ development, we measured changes in the expression of BMP4 and Smad4 during vestibular development in C57BL/6 mice. In addition, vestibular structures, pathologic changes, and the vestibular function of chondrocyte-specific Smad4 knockout mice were compared to those of the control group. We found that the expression of Smad4 in the inner ear was delayed compared with that of BMP4. Moreover, chondrocyte-specific Smad4 knockout homozygous mice showed stunted growth and partial vestibular deformities, but it showed less histologic changes in the vestibular end-organs and saccule dysfunction. These results suggest that Smad4 participates in late-stage shaping of the configuration of the vestibule and development of vestibular functional, but a Smad4-independent pathway for the inner ear vestibular BMP4 signal transduction could not be rule out.
    International journal of developmental neuroscience: the official journal of the International Society for Developmental Neuroscience 10/2010; 29(1):15-23. · 2.03 Impact Factor
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    ABSTRACT: Our previous studies have shown that both apoptosis and necrosis are involved in hair cell (HC) pathogenesis in aging cochleae. To better understand the biological mechanisms responsible for the regulation of HC death, we examined the activity of succinate dehydrogenase (SDH), a mitochondrial bioenergetic enzyme, in the HCs of aging cochleae. The auditory brainstem response thresholds elicited by tone bursts at 4, 10 and 20 kHz were measured in both young (2-3 months) and aging (22-23 months) Wistar rats. SDH activity was evaluated with a colorimetric assay using nitroblue tetrazolium monosodium salt. The SDH-labeled organs of Corti were double stained with propidium iodide, a DNA intercalating fluorescent probe for illustration of HC nuclei. All the specimens were examined with fluorescence microscopy and confocal microscopy. Aging rats exhibited a significant elevation of ABR thresholds with threshold shifts being 34 dB at 20 kHz, 28 dB at 10 kHz, and 25 dB at 4 kHz. Consistent with the reduction in the cochlear function, aging cochleae exhibited the reduction of SDH staining intensity in the apical and the basal ends of the cochleae, where a large number of apoptotic, necrotic, and missing HCs were evident. The reduction in SDH staining appeared in a cell-death-mode dependent fashion. Specifically, SDH labeling remained in apoptotic HCs. In contrast, SDH staining was markedly reduced or absent in necrotic HCs. In the aging cochlea, SDH activity is preserved in HCs undergoing apoptosis, but is substantially reduced in necrosis. These results suggest that mitochondrial energetic function is involved in the regulation of cell death pathways in the pathogenesis of aging cochleae.
    Chinese medical journal 07/2010; 123(13):1633-8. · 0.90 Impact Factor
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    ABSTRACT: Smad4 is the central intracellular mediator of transforming growth factor-beta (TGF-beta) signaling, which plays crucial roles in tissue regeneration, cell differentiation, embryonic development, and regulation of the immune system. Conventional Smad4 gene knockout results in embryonic lethality, precluding its use in studies of the role of Smad4 in inner ear development. We used chondrocyte-specific Smad4 knockout mice (Smad4Co/Co) to investigate the function of Smad4 in inner ear development. Smad4Co/Co mice were characterized by a smaller cochlear volume, bone malformation, and abnormalities of the osseous spiral lamina and basilar membrane. The development of the hair cells was also abnormal, as evidenced by the disorganized stereocilia and reduced density of the neuronal processes beneath the hair cells. Auditory function tests revealed the homozygous Smad4Co/Co mice suffered from severe sensorineural hearing loss. Our results suggest that Smad4 is required for inner ear development and normal auditory function in mammals.
    Developmental Dynamics 09/2009; 238(8):1897-908. · 2.59 Impact Factor
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    ABSTRACT: The Smads are a group of related intracellular proteins critical for transmitting the signals to the nucleus from the transforming growth factor-beta superfamily at the cell surface. Knockout of the Smad5 is embryonic lethal. However, the Smad5 knockout of single allele (+/-) could survive. We used Smad5 heterozygous knockout (+/-) to determine the role of Smad5 in the development of inner ear morphology and function. In situ hybridization showed that Smad5 was expressed predominantly in hair cells, spiral ganglion, and supporting cells. Measurements of hearing thresholds using auditory brainstem response showed that Smad5 defect resulted in progressive hearing loss between 4 and 24 weeks after birth. Morphological examination revealed apoptosis in the inner ear, with significant loss of outer hair cells in adult Smad5 mutant mice. Our results indicated that deficiency in the Smad5-mediated signaling resulted in apoptosis of hair cells, suggesting Smad5 is a gene that may be related with presbycusis.
    Developmental Neurobiology 01/2009; 69(2-3):153-61. · 4.42 Impact Factor
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    ABSTRACT: The purpose of this study was to observe the ultrastructure of the fibroblasts, collagen and elastic fibers in vocal fold polyps. Ten vocal fold polyps and 3 normal vocal fold specimens obtained from total laryngectomy were studied by means of transmission electron microscope and scanning electron microscope. The result showed that in vocal fold polyps, the quantity of fibroblasts increased and there were abundant organelles, suggesting that the fibroblast were in the status of activation. As the main cell to produce lamina propria extracellular matrix, the representation suggested that the extracellular matrix metabolism was active. Leucocytes soakage was observed, suggesting that the inflammation may play a role in the lesion. It was found by scanning electron microscopy that in case of lesions, collagen fibers and elastic fibers arrayed irregularly. Under pathologic circumstance, fibroblasts, collagen and elastic fibers altered in morphology, which possibly induced the functional alteration.
    Zhonghua er bi yan hou tou jing wai ke za zhi = Chinese journal of otorhinolaryngology head and neck surgery 05/2008; 43(4):287-90.
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    ABSTRACT: To evaluate the relationship between labeling index (LI) Ki-67, proliferating cell nuclear antigen (PCNA) and transforming growth factor-beta1 (TGF-beta1) with the clinical behavior of acoustic neuroma. Expression of Ki-67, PCNA and TGF-beta1 was detected by immunohistochemistry in 53 specimens of acoustic neuromas. The relationship among tumor proliferation, histological representation, size of tumor, clinical proliferation index of tumor and tumor proliferation activity were analyzed. In all 53 cases, the positive rate of Ki-67 was 77.4% (41/53) but the positive rate of PCNA was 84.9% (45/53). There was significant difference between the proliferate index, clinic growth rate and course of disease (t = 2.14, t = 2.70; P < 0.05). The positive rate of TGF-beta1 was 83.0% (44/53). The correlation of TGF-beta1 with LI (Ki-67) was significant difference (r = 0.36, P < 0.05). Cystic degeneration often occurred in large-size tumor (Z = 4.44, P < 0.05). There was no significant relationship between the expression of LI (Ki-67), LI (PCNA) and TGF-beta1 and the course of disease as well as between the cystic degeneration and the non-cystic degeneration. Although clinic growth rate of cystic degeneration was bigger than that of non-cystic degeneration, there was not statistically significant. Ki-67 and PCNA are reflected proliferation activities of tumor cells in acoustic neuromas. Cell proliferation-labeling index LI (PCNA) was related with clinical growth rates. TGF-beta1 might participate in the biological behavior of acoustic neuroma. Cystic degeneration was one of special pattern of acoustic neuroma, however, tumor enlargement might due to the volume of the cystic but unrelated to fast proliferation of parenchyma cell.
    Zhonghua er bi yan hou tou jing wai ke za zhi = Chinese journal of otorhinolaryngology head and neck surgery 03/2008; 43(2):125-9.
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    ABSTRACT: To establish in vitro culture systems of greater epithelial ridge (GER) cells from rat cochlear and to investigate the characterization, growth pattern and ultrastructure of GER cells. Using a combinatorial approach of enzymatic digestion and mechanical separation to allow isolation and culture of GER cells from P1 rat cochleae. The dissociated GER cells were cultured in serum-free or 10% fetal bovine serum DMEM respectively. BrdU, phalloidin, ZO1, calretinin and myosin VIIa immunostaining and scanning electron microscope observation were performed in GER cell cultures. The dissociated GER cell cultures showed positive to ZO1, phalloidin and BrdU staining, but negative to myosin VIIa and calretinin. They assumed a polygonal morphology which was similar to epithelial cells and grew in islands-like patches in medium containing 10% fetal bovine serum while forming spheres in serum-free medium. The GER cells presented significant ability to proliferate in both conditions. Scanning electron microscope showed that there was microvillus and centre bodies but not hair cell specific stereociliary bundles on the surface of GER cultures. The GER cell cultures showed significant ability to proliferate and grew in islands-like patches in medium containing 10% fetal bovine serum while forming spheres in serum-free medium. The dissociated GER cells expressed epithelial cell specific marker but not marker of hair cells.
    Zhonghua er bi yan hou tou jing wai ke za zhi = Chinese journal of otorhinolaryngology head and neck surgery 11/2007; 42(10):760-4.
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    ABSTRACT: Mammalian cochlear hair cell loss is irreversible and leads to permanent hearing loss. To restore hearing physiologically, it is necessary to generate new functional hair cells either from endogenous cells or from exogenously transplanted hair cells/progenitors. Previous studies suggest that cochlear greater epithelial ridge (GER) and lesser epithelial ridge (LER) cells are capable of differentiating into hair cells. While it was recently possible to obtain and culture pure LER progenitors, isolation of pure GER progenitors has not been reported. Here we describe a method that allows isolation of pure GER cells from neonatal rat cochleae. The cochlear epithelial sheet (CES) containing GER progenitor cells was mechanically separated from the underlying mesenchymal tissue after digestion with thermolysin. The GER area could then be dissected following mechanical removal of organ of Corti as well as all the lateral area. The isolated GER cells showed significant proliferation and expressed markers for GER cells but not markers for hair cells or LER. When the GER cells were cultured in serum-free medium containing epidermal growth factor, spheres were formed where they continued to proliferate. Furthermore, when GER cells were induced to express Hath1 or co-cultured with mesenchymal cells prepared from neonate rat cochleae, they showed the potential to differentiate into hair cell-like cells. Successful isolation, culture and differentiation of GER hair cell progenitors will shed additional light on the mechanism of hair cell differentiation and potential hair cell replacement.
    Journal of Neuroscience Methods 09/2007; 164(2):271-9. · 2.11 Impact Factor