干细胞向雏鸡和成年豚鼠耳蜗的移植方法

目的建立干细胞向雏鸡和成年豚鼠耳蜗移植的方法。方法从绿色荧光蛋白（green fluorescent protein,GFP）转基因胚胎小鼠中分离出GFP标记的嗅球神经干细胞。雏鸡耳蜗的干细胞移植是经外耳道剥离鼓膜,暴露中耳腔的蜗窗,再经蜗窗膜造孔注入千细胞。成年豚鼠耳蜗鼓阶的干细胞移植是行耳后切口暴露听泡,在耳蜗底转近镫骨动脉处钻孔,并注入干细胞。中阶的移植是经耳蜗第三转的血管纹外侧壁注入干细胞。细胞移植1天后,分离雏鸡和豚鼠的耳蜗作冰冻切片,在荧光显微镜下观察移植细胞的分布。结果分离出GFP标记的嗅球神经干细胞。移植后的千细胞可分布于雏鸡耳蜗及豚鼠耳蜗的鼓阶、中阶。结论成功建立了干细胞向雏鸡和成年豚鼠耳蜗移植的方法,为进一步将干细胞应用于耳聋的治疗及机制研究奠定基础。     

骨髓神经组织定向干细胞移植修复听神经损伤模型大鼠的实验研究

目的 观察骨髓神经组织定向干细胞(NTCSCs)移植对听神经损伤大鼠的修复作用。方法将转染绿色荧光蛋白(EGFP)基因的骨髓NTCSCs移植至螺旋神经元特异性损伤模型大鼠左耳耳蜗蜗轴处(右耳作为自身对照,注射相同体积PBS溶液),同时设对照组(A组)、实验组(B～E组),所有组注射相同体积PBS溶液,HE染色观察耳蜗中轴切片,在荧光显微镜下观察感染EGFP的NTCSCs存活、分布部位及表达情况,注射后1周、2周、1个月时检测ABR阈值和DPOAE幅值等变化情况。结果 C组内耳耳蜗切片上发现转染EGFP基因的骨髓NTCSCs分散在鼓阶的腔隙内,D组发现转染EGFP基因的骨髓NTCSCs位置靠近基底膜和柯蒂氏器部位,E组发现转染EGFP基因的骨髓NTCSCs球团和两三个在一起的细胞聚集,并且位置靠近基底膜和柯蒂氏器部位,呈现良好的分布,类似有向基底膜和柯蒂氏器迁移行为;随着骨髓NTCSCs移植时间的延长,Nestin(+)细胞数量明显增多(P<0.05),而Myosin Ⅶa(+)细胞数量无明显变化(P>0.05);随着骨髓NTCSCs移植时间的延长,ABR阈值得到明显改善(P<0.05),DPOAE幅值明显上升(P<0.05)。结论 骨髓NTCSCs移植大鼠耳蜗后可逐渐分化成为螺旋神经节神经元(SGNs),进而起到改善听神经损伤大鼠听力的作用。     

骨髓神经组织定向干细胞移植治疗大鼠听神经损伤的实验研究

目的观察骨髓神经组织定向干细胞（nerve tissue committed stem cells, NTCSCs）移植治疗大鼠听神经损伤的作用。方法 将60只SD大鼠按数字随机法分为5组,每组各12只,A组为对照组,在平静饲养环境下培养,另外4组（B、C、D、E组）构建感音神经性耳聋动物模型。B组大鼠断头后取出听泡组织行HE常规染色,详细观察耳蜗螺旋神经节神经元（spiral ganglion neurons, SGNs）损伤和内耳毛细胞存活情况,确认造模成功。将绿色荧光蛋白（green fluore scent protein, EGFP）基因表达阳性的骨髓NTCSCs注射至C、D、E 3组大鼠左耳耳蜗蜗轴处,SD大鼠右耳及A组作为对照;采用同一方法注射相同体积的0.1%PBS溶液。HE染色观察耳蜗中轴切片,在荧光显微镜下观察转染EGFP基因的NTCSCS存活、分布部位及表达情况。结果C组（移植后1周）内耳耳蜗切片上发现转染绿色荧光蛋白（green fluore scent protein, EGFP）基因的骨髓NTCSCs分散在鼓阶的腔隙内,D组（移植后2周）发现转染EGFP基因的骨髓NTCSCs位置靠近基底膜和柯蒂氏器部位,E组（移植后4周）发现转染EGFP基因的骨髓NTCSCs球团和多个在一起的细胞聚集,并且位置靠近基底膜和柯蒂氏器部位,呈现良好的分布,类似有向基底膜和柯蒂氏器迁移行为。随着骨髓NTCSCs移植时间的延长,Nestin（＋）细胞数量明显增多（P〈0.05）,而Myosin Ⅶa（＋）细胞数量无明显变化（P〉0.05）。结论骨髓NTCSCs移植大鼠耳蜗后可在一定程度上修复损伤听神经。     

骨髓间充质干细胞内耳移植

干细胞内耳移植是近年来治疗感音神经性聋的研究热点,骨髓间充质干细胞具有多方向分化能力、低免疫源性和可以自体移植的优点,在内耳移植治疗上具有一定优势,本文总结骨髓间充质干细胞移植内耳后细胞分布、存活、分化情况以及应用于基因治疗等方面的进展.     

干细胞治疗耳聋研究进展

干细胞治疗耳聋是近些年耳科的研究热点之一,本文通过对干细胞耳聋治疗相关的文献进行综述,从干细胞用于内耳结构的替代或修复的基础、干细胞植入的方式、干细胞移植安全性方面,对干细胞治疗耳聋的可行性进行论述,认为干细胞的内耳移植治疗已具备一定的基础,在移植前需对细胞的分化进行更精准的控制,并且可以与传统的治疗方式(如人工耳蜗植入术)结合使用。     

脑源性神经营养因子基因修饰骨髓基质细胞对体外培养小鼠螺旋神经元的影响

目的探讨人脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)基因工程细胞的建立及其对体外培养的小鼠螺旋神经元生长活性的影响。方法参照分子克隆技术成功构建BDNF真核表达载体———pcD-NA3.1(-)-BDNF,体外证实BDNF基因转染猿猴病毒40肝脏转化细胞(COS7)后正常表达,将骨髓基质细胞分离培养,体外转染骨髓基质细胞建立BDNF基因工程细胞,观察该基因工程细胞对体外培养小鼠螺旋神经元的生长活性的影响。结果成功构建BDNF基因真核表达载体,并且建立了BDNF基因工程细胞;在体外BDNF基因工程细胞能促进螺旋神经元的生长,并保护螺旋神经元免受氧化损伤。结论建立的BDNF基因修饰的骨髓基质细胞,对体外螺旋神经元生长、生存起着重要的保护作用,为进一步研究基因修饰的骨髓基质细胞内耳移植奠定了重要的基础。     

iPSCs定向分化的内耳毛细胞与支持细胞间相互作用的研究

目的利用诱导多能性干细胞定向分化的内耳毛细胞和支持细胞探究这两种体外诱导分化细胞之间的相互作用。方法首先,利用细胞单层贴壁两步诱导法将三株i PS细胞(野生株、MYO7A缺陷株、MYO7A校正株)向内耳祖细胞及内耳毛细胞诱导分化,探究i PSCs定向分化内耳毛细胞的过程中是否有支持细胞的产生;其次,通过细胞免疫化学的方法探究体外分化的内耳毛细胞和支持细胞间的相互作用;最后,将表达绿色荧光蛋白(EGFP)的上皮样内耳祖细胞以圆窗膜穿刺的方法移植到白化荣昌猪的内耳中观察分析移植细胞在体内的迁移、分化以及在体内形成的联系。结果三株i PS细胞诱导分化为内耳毛细胞的过程中均有一部分细胞分化为支持细胞;对分化细胞进行E-cadherin、N-cadherin和ZO-1的免疫荧光检测结果显示,E-cadherin、N-cadherin和ZO-1在支持细胞-支持细胞连接和支持细胞-毛细胞连接间都有表达;移植4周后,耳蜗免疫组织化学结果显示,三株不同来源的移植细胞均有少量细胞成功迁移到了毛细胞受损部位—柯底氏器,并表达毛细胞标志性蛋白MYO7A。移植细胞之间以及移植细胞与宿主细胞之间有E-cadherin、N-cadherin和ZO-1的表达。结论 i PSCs诱导分化的内耳毛细胞和支持细胞在体内、外均能形成钙粘连接和紧密连接。这些研究结果对毛细胞取代法治疗耳聋策略的完善与发展有一定的科学意义。     

脑源性神经营养因子基因转染骨髓间充质干细胞在受损耳蜗内的生存和分化

目的探讨脑源性神经营养因子(brain-derived neurotrophic factor gene,BDNF)基因转染的骨髓间充质干细胞(bone-marrow mesenchymal stem cells,BMSC)在受损耳蜗内的生存和分化情况。方法将转染了BDNF基因并在体外诱导分化的BMSC(BDNF-BMSC)标记后,通过鼓阶注射植入阿米卡星致聋的豚鼠耳蜗内(BDNF-BMSC组,18只),并设注射未诱导分化的BMSC的豚鼠为对照组(BMSC组,18只)。在注射后1、2、4w取耳蜗组织石蜡切片,HE染色和荧光染色观察注射细胞的分布;神经元特异性烯醇化酶(neuron-specificenolase,NSE)、胶质纤维酸性蛋白(glial fibrillary acid protien,GFAP)抗体免疫组化染色观察注射细胞在耳蜗内的分化。结果 BDNF-BMSC和BMSC在耳蜗内均能成活并分布到一定的区域,鼓阶和前庭阶较多,中阶和蜗轴较少。NSE和GFAP免疫化学可见BDNF-BMSC部分细胞阳性染色,而BMSC阳性细胞较少。BDNF-BMSC在1、2w时平均光密度(AOD)值较高,在4w时显著下降(P<0.05);在各时间点,BDNF-BMSC组AOD值均显著高于BMSC组(P<0.01)。结论诱导分化前后的BMSC耳蜗移植后均能成活并分布到一定的区域,在耳蜗内BDNF-BMSC分化能力明显强于BMSC,但随时间延长分化能力下降。     

脑源性神经营养因子基因修饰的骨髓间充质干细胞在药物致聋豚鼠内耳的表达及保护作用

目的 探讨脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)基因修饰的骨髓间充质干细胞(mesenchymal stem cell,MSC)在药物致聋豚鼠内耳的表达及对螺旋神经节细胞(spiral ganglion cell,SGC)的保护作用.方法 阿米卡星致聋的豚鼠随机数字表法分为两组,治疗组经鼓阶开窗注射BDNF基因修饰的MSC,对照组注射外淋巴液.各组分别在术后7 d及28 d处死动物,荧光定量反转录聚合酶链反应(RT-PCR)检测耳蜗组织中BDNF mRNA的表达,耳蜗切片计算SGC细胞密度,末端脱氧核苷酸转移酶介导dUTP缺口末端标记法(terminal deoxynucleotidyl transferase mediated dUTP nick end labeling,TUNEL)检测SGC凋亡情况.结果 治疗组在术后7 d和28 d的BDNF mRNA相对表达量均明显高于对照组,差异具有统计学意义(P值均＜0.01).治疗组术后7 d及28 d的SGC密度均高于对照组,并且治疗组术后7 d及28 d的SGC凋亡指数也比对照组明显下降,差异具有统计学意义(P值均＜0.01).结论 BDNF基因修饰的MSC在致聋豚鼠内耳中的表达时间超过28 d,对SGC具有保护作用.     

支持细胞在内耳发育及耳蜗毛细胞损伤后的作用

耳蜗毛细胞损伤后如何再生及恢复其听功能是现今耳科学研究的热点问题之一。目前国内外的研究热点为调控参与内耳毛细胞调控的基因、干细胞移植、生长因子诱导、内耳前体细胞向毛细胞分化等。对于螺旋器中支持细胞在内耳发育、耳蜗毛细胞损伤后保持听觉上皮完整以及毛细胞再生中的作用关注较少。本文就支持细胞在内耳发育、毛细胞损伤后的作用等研究做一综述。     

The expression of vascular endothelial growth factor (VEGF) and VEGF‐C in early laryngeal cancer: relationship with radioresistance

The expression of vascular endothelial growth factor (VEGF) and VEGF‐C in early laryngeal cancer: relationship with radioresistance Angiogenesis is essential for tumour growth and invasion. Vascular endothelial growth factor (VEGF) is a prime mediator of tumour angiogenesis. VEGF‐C is a closely related protein that effects lymphatic endothelial cells and may be important in the process of lymphatic metastasis. The purpose of this study was to evaluate the expression of these cytokines in patients with T1 and T2a glottic, squamous cell carcinoma, in comparison with normal epithelial control tissue, to ascertain any association with radioresistance. Twenty‐two tumours treated by radiotherapy (13 radiosensitive, nine radioresistant) and seven normal control tissues were studied. The minimum follow‐up was 2 years after radiotherapy. Expression of VEGF and VEGF‐C was evaluated by immunohistochemistry of formalin‐fixed, paraffin‐embedded biopsy specimens. Analysis was carried out using a quantitative computer image analyser. Both VEGF and VEGF‐C were detectable in tumour and normal control specimens. There was increased expression in tumour specimens of both VEGF (P = 0.03) and VEGF‐C (P < 0.001). In addition, the expression of VEGF‐C was associated with tumours of higher histological grade (P = 0.021). There was, however, no difference in VEGF and VEGF‐C expression between radioresistant and radiosensitive tumours. The expression of VEGF and VEGF‐C is increased in early laryngeal squamous cell carcinoma (SCC). However, measuring the expression of these proteins cannot predict radioresistance in this tumour group.     

Treatment of Maxillary Sinus Carcinoma: Clinical Results Using the Kitasato Modality

The conventional therapeutic regimen for maxillary sinus carcinoma consists of dissection of the maxilla, full-dose irradiation and extensive chemotherapy. However, the results obtained with this treatment are often poor. Even when patients recover, their quality of life is significantly reduced as a result of deformity of facial structures and swallowing and articulation dysfunctions. A retrospective analysis of 68 patients with maxillary sinus carcinoma treated with the Kitasato modality between 1975 and 1999 was conducted. All patients underwent pergingival maxillary sinus surgery combined with pre- and postoperative irradiation therapy with standardized total doses of 16 Gy; the postoperative irradiation was given in combination with regional intra-arterial infusion chemotherapy administered via the superficial temporal artery. All visible tumor lesions were removed where possible in order to preserve or facilitate cellular immunity after surgery. The cumulative 5-year survival rates were 85.7% for Stage II patients, 88.1% for Stage III, 76.6% for Stage IVA and 75.0% for Stage IVB.     

Tongue Base Reduction with Temperature-controlled Radiofrequency Volumetric Tissue Reduction for Treatment of Obstructive Sleep Apnea Syndrome

In recent years a considerable effort has been made to establish the use of different surgical techniques for the treatment of obstructive sleep apnea syndrome (OSAS). Nevertheless, treatment of hypopharyngeal obstruction due to tongue base hypertrophy remains in many ways an unsolved problem. The aim of this study was to evaluate the safety and efficacy of tongue base reduction with temperature-controlled radiofrequency volumetric tissue reduction in the treatment of OSAS. Twenty patients with OSAS and tongue base hypertrophy were treated with radiofrequency tissue ablation. An intensified treatment protocol was used, delivering 2,800 J per treatment session under local anesthesia. Two nights of polysomnography testing were performed before and after treatment. Daytime sleepiness, snoring and postoperative morbidity were assessed using questionnaires. Mean respiratory disturbance index (RDI) was reduced from 32.1 to 24.9/h after a mean of 3.4 treatment sessions. Six patients (33%) were cured after the procedure (reduction in RDI of &#83 50% and a postoperative RDI of <15/h) and ten (55%) showed an improvement of >20% in their RDI. Daytime sleepiness and snoring improved significantly. Peri- and postoperative morbidity was low; one severe complication occurred (tongue base abscess). We were able to achieve similar cure and responder rates to those reported in a recently published pilot study but with a reduced number of treatment sessions. We believe that this technique may improve patient acceptance and have beneficial cost implications.     

Cervical Reflex Induced by Click Stimuli in Cats

We studied click-evoked potentials in the anterior horn of the spinal cord in 17 cats. A concentric needle electrode was inserted into the anterior horn of the spinal cord at levels C3-C6. Potentials evoked with 105 dB SPL clicks were recorded with a peak latency of 4.89-5.10 ms only at the C3 level. These responses were observed 45-60 dB SPL above the auditory brainstem response (ABR) threshold, and no potentials were evoked by stimulation of the contralateral ear. Average was performed 100 times with changes in stimulation frequency of 1-20 Hz. The amplitude of the potentials decreased with increasing stimulus frequency, but there were no changes in ABRs. The responses disappeared after destruction of the medial vestibulospinal tract at the obex level, but ABRs were still recorded. The spinal nucleus of the accessory nerves was located in the anterior horn of the spinal cord at levels C1-C6, and the sternocleidomastoid muscle motoneurons were found at levels C1-C3. The click-evoked potentials recorded in this study reflect responses of the spinal nucleus of accessory nerves through the vestibulospinal tract to click stimulation. The responses have the same characteristics as vestibular-evoked myogenic potentials that can be recorded using surface electrodes over the sternocleidomastoid muscles of humans.     

Lack of significant estrogen and progesterone receptor expression in nasal telangiectasias in hereditary hemorrhagic telangiectasia: an immunohistochemical analysis

Obstructive sleep apnea syndrome (OSAS) is characterized by snoring and apnea during sleep leading to decreased oxygen saturation and disturbed sleep, excessive daytime sleepiness and neuropsychological disturbances. This study investigates cognitive neuropsychological abilities in a group of 53 OSAS patients before and after treatment with uvulopalatopharyngoplasty. General intellectual ability, verbal learning and memory as well as executive functioning were measured at baseline and 6 months postoperatively. After surgery there were significant improvements in verbal learning and memory (mean change - 39, SD 57.3, p <0.001), recall (mean change - 24.3, SD 39.3, p <0.001) and executive functioning (as assessed by percentage of errors on the Wisconsin Card Sorting Test; mean change-9.1, SD 15.7, p <0.001). These improvements were in accordance with improvements in the degree of sleep apnea, the oxygen desaturation index (mean change -9.7, SD 15.9, p <0.001) and arterial minimum oxygen saturation (mean change 4.5%, SD 10.2%, p <0.01). Surgical treatment seems to improve verbal learning, memory and recall and executive functions in parallel with better oxygenation in OSAS.     

Measurement of chemosensory function

Although hundreds of thousands of patients seek medical help annually for disorders of taste and smell, relatively few medical practitioners quantitatively test their patients' chemosensory function, taking their complaints at face value. This is clearly not the approach paid to patients complaining of visual, hearing, or balance problems. Accurate chemosensory testing is essential to establish the nature, degree, and veracity of a patient's complaint, as well as to aid in counseling and in monitoring the effectiveness of treatment strategies and decisions. In many cases, patients perseverate on chemosensory loss that objective assessment demonstrates has resolved. In other cases, patients are malingering. Olfactory testing is critical for not only establishing the validity and degree of the chemosensory dysfunction, but for helping patients place their dysfunction into perspective relative to the function of their peer group. It is well established, for example, that olfactory dysfunction is the rule, rather than the exception, in members of the older population. Moreover, it is now apparent that such dysfunction can be an early sign of neurodegenerative diseases such as Alzheimer's and Parkinson's. Importantly, older anosmics are three times more likely to die over the course of an ensuring five-year period than their normosmic peers, a situation that may be averted in some cases by appropriate nutritional and safety counseling. This review provides the clinician, as well as the academic and industrial researcher, with an overview of the available means for accurately assessing smell and taste function, including up-to-date information and normative data for advances in this field.