内耳转基因表达的实验研究

目的　了解内耳转基因表达的可行性。方法　将人复制缺陷重组腺病毒基因(Adenoviruses ,Ad ,含大肠杆菌 β 半乳糖苷酶基因LacZ基因 ,Ad5 LacZ)经豚鼠耳蜗蜗窗接种到鼓阶外淋巴后 ,观察在不同时间、不同内耳组织中LacZ基因的表达 (X Gal染色 )及Ad对豚鼠声反应 (听性脑干反应 )、听毛细胞 (扫描电镜 )的影响。结果　Ad介导的LacZ基因在内耳组织中的表达至少可持续 4周 ,其中在螺旋神经节细胞表达稳定 ,Corti器、前庭囊斑、壶腹嵴的毛细胞等也有较强的表达。Ad未对豚鼠声反应 (≤ 80 0 0Hz)造成明显的损伤 ,除耳蜗底回外 ,其余各回未见明显的毛细胞缺失。结论　腺病毒载体可成功地将LacZ基因转导致豚鼠内耳组织中 ,并且未对豚鼠声反应 (低、中频 )及听毛细胞造成明显损伤 ,这对未来的内耳基因治疗研究可能具有重要的参考价值。     

二亚硝基哌嗪对转基因小鼠TNF受体相关因子2和p16的表达

目的研究二亚硝基哌嗪(N,N′-dinitrosopi- perazine,DNP)对TgN(p53mt-LMP1)/HT小鼠鼻腔和鼻咽黏膜上皮细胞增殖与TNF受体相关因子2(TNF receptor-associated Factor 2,TRAF2)、p16基因表达的影响及两者关系。方法HE染色法观察转基因小鼠组(TC)、转基因小鼠DNP诱导组(T1)和野生小鼠组(CC)、野生小鼠DNP诱导组(CI)鼻腔和鼻咽黏膜上皮细胞增殖特征;免疫组织化学染色法检测小鼠鼻腔和鼻咽黏膜上皮组织中TRAF2、p16基因的表达水平。结果TI、TC、CI和CC组小鼠鼻腔或鼻咽黏膜上皮癌前病变率分别为90%、10%、0和0。与TC、CI和CC组相比,TI组TRAF2基因表达水平显著增高(P<0.01),而p16基因表达水平显著降低(P<0.01);TRAF2和p16基因表达水平在TC和CC组之间的差异亦有统计学意义(P<0.01)。结论TgN(p53mt-LMP1)/HT小鼠鼻腔和鼻咽黏膜上皮细胞增殖活性增加,DNP可提高其增殖活性,而细胞增殖活性增加与TRAF2基因表达水平上调和p16基因表达水平下调密切相关。     

COL1A2 and COL2A1 expression in temporal bone of lethal osteogenesis imperfecta

OBJECTIVE: COL1A2 and COL2A1 genes are expressed at high levels in many cochlear cells of 16- to 23-week-old human fetuses. Given these prior observations and the rare opportunity to obtain temporal bones from a deceased neonate with osteogenesis imperfecta (OI) type II, we determined the cellular distribution and level of expression of COL1A2 mRNA in OI type II inner ear compared with the expression in second-trimester human fetal cochlea. Expression of COL2A1 mRNA was assessed for its normal role in OI type II neonatal cochlea and to address potential spatial and temporal changes along with our observations in fetal cochlea. We describe our tissue in situ hybridization protocol and document its usefulness in assessing gene expression in human temporal bone obtained at autopsy. DESIGN: RNA-RNA in situ hybridization was performed in formaldehyde-fixed, decalcified, paraffin-embedded temporal bone sections from a neonate with OI type II. Semi-quantitative assessment of gene expression was performed by visual inspection of grain densities. RESULTS AND CONCLUSIONS: COL1A2 and COL2A1 were expressed at moderate-to-high levels in many membranous cochlear cells, and no dramatic alterations in pattern or level of expression of these genes was noted compared with human fetal cochlea. Consistent with in vitro studies, expression of COL1A2 in osteoblasts lining enchondral and endosteal layers is less than that in identical cells of the fetal otic capsule undergoing osteoid deposition and mineralization. Expression of COL1A2 mRNA in osteoblasts lining the outer periosteum of otic capsule is markedly higher than osteoblasts lining enchondral and endosteal layers, suggesting that differential expression may exist between osteoblasts lining endosteal, enchondral, and periosteal surfaces of bone in OI type II.     

Vertical distraction osteogenesis of the alveolar process using a tooth-borne device]

Since the communication of McCarthy et al. in 1992, applications for distraction osteogenesis in the maxillofacial complex have increased enormously. The advantages as compared to conventional reconstructive procedures are missing donor-site morbidity for bone graft harvesting, reduced blood loss and also a gradual gain in the soft tissue envelope. Meanwhile, even the vertical lengthening of the alveolar process has become a clinically reliable method. The case presented illustrates the treatment of a 16-year-old patient who suffered a sporting accident at the age of 7 years, leading to ankylosis of the upper middle incisors with subsequent underdevelopment of the frontal alveolar process. After fabrication of a custom-made distraction device, an osteotomy of the tooth-bearing segment in the upper front was performed and the segment was attached to the distraction device. Bone lengthening was started on the seventh postoperative day at a daily rate of 0.5 mm. The distraction was maintained until closure of the open bite was achieved. On the 22nd day, the segment was fixed using an orthodontic arch wire and the distraction device was removed. A retention period of 6 weeks was maintained until sufficient stability was detectable. Now, 1.5 years after the treatment, the situation is stable with a good consolidation of the bone fragments and a physiologically shaped gingivobuccal sulcus. Lengthening of the alveolar process by vertical distraction osteogenesis using a tooth-borne device may be indicated in certain cases. With this technique a second operation can be avoided and there is less risk of laceration of dental roots by fixation screws.     

Behavioral investigation of some possible effects of the central olivocochlear pathways in transgenic mice

This study investigated the auditory behaviors of transgenic mice with deletions of alpha9 nicotinic acetylcholine receptor subunits. In the normal mammalian cochlea, the mechanical properties of outer hair cells are modified by the release of acetylcholine from olivocochlear efferent terminals. Electrophysiological correlates of this efferent feedback have not been demonstrated in alpha9 knockout mice, presumably because they are mediated by alpha9 receptors. Previous studies have associated lesions of olivocochlear pathways with hearing impairments in background noise. The prediction that alpha9 knockout mice would show similar deficits was tested by collecting psychophysical thresholds for tone detection and intensity discrimination from knockout mice, within-strain control subjects, and CBA/CaJ mice. Comparable performance was observed for the subject groups in quiet and in continuous background noise. The preservation of auditory function in alpha9 knockout mice suggests that central efferent pathways work in combination with the peripheral olivocochlear system to enhance hearing in noise, and may compensate for profound manipulations of peripheral feedback in highly routine testing procedures. An intriguing possibility is that these central mechanisms include the brainstem collaterals of olivocochlear neurons since their post-synaptic targets do not express alpha9 receptors and therefore are likely to maintain their effects in alpha9 knockout mice.     

干细胞移植辅助丝裂霉素C治疗翼状胬肉20例

目的探讨干细胞移植辅助丝裂霉素C(MMC)治疗翼状胬肉的效果.方法初发性胬肉20例、26眼于显微镜下彻底切除胬肉组织,干细胞移植辅助MMC治疗.结果术后20例、26眼4～6d角膜上皮修复;随访6～18个月,平均11个月;3例、4眼疑复发;手术治愈率84.7%.结论 干细胞移植辅助MMC治疗翼状胬肉既能有效地阻止复发,又能减少非邻近取材移植片造成新的创面,为可能复发再次手术者留有机会,是治疗初发性翼状胬肉安全有效的方法.     

Results of stapedectomy in osteogenesis imperfecta

Osteogenesis imperfecta is a connective tissue disease connected with improper synthesis of collagen type I. Bone fragility, blue sclera and hearing loss are the main symptoms of classic type of the disease. Early (6 months) and late (10 years) results of stapedectomy in 3 patients with osteogenesis imperfecta syndrome are presented. Six months postoperatively air-bone gap was smaller than 10 dB in 2 patients and 17 dB in one patient. In all patients, bone conduction pure tone threshold improved after the operation. Audiometric tests performed 10 years after stapedectomy were similar to those obtained 6 months after the operation. Stapedectomy in patients with osteogenesis imperfecta syndrome provides good and stable hearing results.     

重组腺病毒构建及转染培养耳蜗Corti器和螺旋神经节细胞的实验研究

目的构建携带绿色荧光蛋白（GFP）基因的重组腺病毒,观察GFP基因在体外培养的新生大鼠耳蜗Corti器和螺旋神经节细胞的表达。方法通过细菌内同源重组方法构建携带有绿色荧光蛋白基因的重组腺病毒（Ad-GFP）,观察重组腺病毒转染培养的新生大鼠耳蜗Corti器和螺旋神经节细胞后,不同时间内绿色荧光蛋白基因的表达情况、表达部位及病毒对培养耳蜗Corti器和螺旋神经节细胞的影响。结果构建的重组腺病毒经酶切电泳鉴定正确;荧光显微镜下观察腺病毒转染体外培养的新生大鼠耳蜗Corti器和螺旋神经节细胞后,12 h即开始表达绿色荧光蛋白基因,24 h达到高峰,表达时间可持续1周;倒置显微镜观察转染后的Corti器和螺旋神经节细胞形态结构及生长无明显改变。结论本实验成功构建了携带绿色荧光蛋白（GFP）基因的重组腺病毒,通过该方法构建的腺病毒可以介导绿色荧光蛋白（GFP）基因在体外培养的新生大鼠耳蜗Corti器和螺旋神经节细胞中表达,但腺病毒本身对体外培养的耳蜗Corti器和螺旋神经节细胞生长无明显影响,为通过腺病毒进行内耳基因治疗提供了理论依据。     

Overview of the radiology,histology, and bone morphogenetic protein expression during distraction osteogenesis of the mandible

INTRODUCTION: Distraction osteogenesis (DO) is a form of in vivo tissue engineering during which an osteotomy and controlled distraction are used to lengthen bone. The molecular signals that govern distraction-induced bone formation have not been fully elucidated. Specifically, the role of bone morphogenetic proteins (BMPs) in DO of the mandible remains unclear. OBJECTIVE: To characterize the radiologic and histologic evolution of newly formed bone during DO of the mandible and to relate these changes to the expression of BMPs. METHODS: Fourteen skeletally mature male rabbits were used. A distractor device was surgically applied to one side of the mandible following osteotomy. After 1 week (latency period), distraction was started at a rate of 0.25 mm every 12 hours for 3 weeks (distraction period) and was followed by a 3-week consolidation period. Two animals were sacrificed each week after surgery (weeks 1 to 7). The mandible was resected and the new bone assessed by radiography and histology. The expression of BMPs was also analyzed using immunohistochemistry. RESULTS: There was radiographic and histologic evidence of bone formation during the distraction period. By week 6, there was mature woven bone within the distraction zone. Bone morphogenetic proteins 2 and 4 were strongly expressed in osteoblasts during distraction and in chondrocytes during consolidation. The expression of BMP-7 was relatively minor. CONCLUSION: The temporal and spatial pattern of BMP expression suggests that these proteins are important mediators of mandibular DO. Understanding the expression of BMPs may facilitate the use of recombinant proteins to enhance the rate and quality of bone generation during craniofacial DO.     

Histopathology of the stapes in osteogenesis imperfecta

Conventional histological studies of stapes footplates from patients with osteogenesis imperfecta revealed, in nine out of 15 stapes examined, an otospongiotic-like lesion. Although the morphology of the stapedial lesion was comparable to the early otosclerotic focus, a greater structural disorganization and larger resorption spaces in osteogenesis imperfecta distinguished the two conditions. The histopathology of the stapes was related to the morphology of cortical and trabecular bone from the iliac crest. Various degrees of immature, osteogenic bony tissue were found in the iliac crest, showing no resemblance to the otospongiotic-like focus in the footplate. Even though the histologic appearance of the stapes footplates differed from the findings in peripheral bone, the present study indicates that the stapedial lesion in osteogenesis imperfecta is most likely a manifestation of the generalized bone and connective tissue disorder. The study further supports the view that osteogenesis imperfecta and otosclerosis are of different aetiology.     

Localization of prestin and expression in the early period after radiation in mice

This study aimed to examine expression and microstructural distribution of prestin in outer hair cells, and the effect of dose and time of radiation on prestin expression in the BALB/c mouse. We also investigated the molecular biological characteristics of prestin and possible mechanisms of sensorineural hearing loss caused by radiation. Seventy 4-week-old mice were randomly divided into four groups, including one control group and three experimental groups. Each experimental group was randomly divided into two groups, which were killed to collect specimens of the cochlea on the 3rd and 7th days after exposure to different doses of 8, 12, and 16 Gy radiation. These cochleas were embedded in paraffin, and then cut into sections. The sections were immunostained with anti-prestin antibodies. The distribution of prestin was observed under optical microscopy and the density of prestin-positive expression was quantitatively calculated by Image-Pro Plus. Prestin had high expression in the lateral membrane and low expression in the cytoplasm of outer hair cells above the nucleus. The density of prestin protein expression of the basal turn was not significantly different after exposure to the different doses of radiation compared with the control group, but up-regulation occurred after radiation in the apex turn. We conclude that prestin protein is mainly expressed in the lateral membrane above the nucleus. Prestin protein may be responsible for the mechanism of injury to the inner ear caused by radiation.     

Characterization of fungi in chronic rhinosinusitis using polymerase chain reaction and sequencing

The role of fungi in chronic rhinosinusitis (CRS) remains unknown. Fungi were also determined as one of the responsible agents in the etio-pathogenesis, while several studies found fungi in 6–93% of the cases. The aim of this study is to test the presence of fungi in samples taken from the middle meatus of patients with CRS, using traditional culture methods and polymerase chain reaction (PCR), and to compare the efficacy of these methods. Thirty patients diagnosed with CRS, with or without nasal polyposis, undergoing an operation in the Otorhinolaryngology Clinic, were prospectively included in the study. Nasal mucosa samples from ten patients, who were operated for pathologic evaluation, and without CRS, were used as controls. Nasal samples were taken from each patient by swabbing with a cytology brush. Middle meatus culture samples were taken by using nasal cotton swab, and the polyp and/or sinus mucosa samples were taken during endoscopic sinus surgery. Fungal specific PCR, using 18S rRNA primers and standard cultures, were performed on every sample. All amplicons were sequenced. There was no fungal growth in the Sabouraud dextrose agar (SDA) medium from middle meatus samples and tissue parts. Of 30 tissue and brush samples, 3 and 2 were positive for fungal DNA, respectively. Sequence analysis showed that four amplicons were homologus to Cladosporium herbarum and one to Aspergillus amstelodami. We concluded that fungal etiology is overestimated and fungi rarely play a role in patients with CRS. Large-scale studies should be done using molecular methods.     

Histological investigation of skin biopsies in otosclerosis and osteogenesis imperfecta

Summary The hypothesis that homeostatic control mechanisms control mucociliary function in ciliated mucous membrane was tested. Nasal mucociliary transport rates were recorded in chickens in vivo at successive intervals during exposure to SO₂ or after inoculation with Newcastle disease virus (NDV), or both. Either agent alone caused deceleration of the turbinate clearance. However, when SO₂ exposure was limited to one nasal fossa and turbinate mucociliary rates were determined in the unexposed and infected side, the two acted antagonistically and yielded approximately normal rates. Exposure of the nasal mucosae to SO₂ caused decreased rates of sinus clearance, while NDV infection of nasal membranes induced increased rates of sinus clearance. Exposure of nasal mucosae to both agents acted antagonistically to effect rates of sinus clearance in normal ranges. These data support the idea of homeostasis.Research support from NIH grant no. 5 RO1 ES-00741     

Sustained cadherin 23 expression in young and adult cochlea of normal and hearing-impaired mice

Cadherin 23 encodes a single-pass transmembrane protein with 27 extracellular cadherin-domains and localizes to stereocilia where it functions as an inter-stereocilia link. Cadherin 23-deficient mice show congenital deafness in combination with circling behavior as a result of organizational defects in the stereocilia hair bundle; common inbred mouse strains carrying the hypomorphic Cdh23(753A) allele are highly susceptible to sensorineural hearing loss. Here, we show that an antibody (N1086) directed against the intracellular carboxyterminus reacts specifically with cadherin 23 and detects with high sensitivity the isoform devoid of the peptide encoded by exon 68 (CDH23Delta68). Cochlea, vestibule, eye, brain and testis produce the CDH23Delta68 isoform in abundance and form moieties with different molecular weight due to variations in glycosylation content. In the cochlea, CDH23Delta68 expression is highest at postnatal day 1 (P1) and P7; expression is down regulated through P14 and P21 and persists at a low steady-state level throughout adulthood (P160). Furthermore, CDH23Delta68 expression levels in young and adult cochlea are similar among normal and hearing deficient strains (C3HeB/FeJ, C57BL/6J and BUB/BnJ). Finally, by immunofluorescence using an antibody (Pb240) specific for ectodomain 14, we show that cadherin 23 localizes to stereocilia during hair bundle development in late gestation and early postnatal days. Cadherin 23-specific labeling becomes weaker as the hair bundle matures but faint labeling concentrated near the top of stereocilia is still detectable at P35. No labeling of cochlea stereocilia was observed with N1086. In conclusion, our data describe a cadherin 23-specific antibody with high affinity to the CDH23Delta68 isoform, reveal a dynamic cochlea expression and localization profile and show sustained cadherin 23 levels in adult cochlea of normal and hearing-impaired mice.