分阶段添加细胞诱导因子诱导豚鼠脂肪间充质干细胞定向分化内耳毛细胞

背景：感音神经性耳聋主要是由内耳毛细胞的缺失或受损造成,用脂肪间充质干细胞来再生修复内耳毛细胞是治疗听力损失的有效方法。目的：探讨体外定向诱导豚鼠脂肪间充质干细胞向内耳毛细胞样细胞分化的可行性。方法：体外分离培养豚鼠脂肪间充质干细胞至第3代,流式细胞仪检测细胞免疫表型,分阶段加入表皮生长因子、碱性成纤维细胞生长因子、胰岛素样生长因子1、全反式维甲酸、脑源性神经营养因子、神经营养因子3,观察诱导分化的效果。结果与结论：豚鼠脂肪间充质干细胞体外培养呈梭形,漩涡状贴壁生长,第3代细胞表面标记CD29与CD44表达呈现阳性,CD34与CD45表达呈现阴性。应用细胞因子诱导后细胞早期nestin和GFAP表达阳性,继续诱导10 d后表达毛细胞特异性标记物MyosinⅦa和Math1,说明细胞因子可定向诱导豚鼠脂肪间充质干细胞向内耳毛细胞分化。     

干细胞移植豚鼠耳蜗形态结构及听力的变化

背景：既往研究证明耳蜗微造孔注射病毒或非病毒载体行基因治疗对耳蜗结构及听功能的影响轻微.目的：观察骨髓间充质干细胞通过耳蜗微造孔术移植到正常豚鼠内耳后对耳蜗结构及听功能的影响.方法：正常豚鼠分3组,空白对照组未予任何干预;单纯耳蜗微造孔术组单纯行耳蜗微造孔术,不注射任何成分;骨髓间充质干细胞组鼓阶微造孔后注射经DAPI荧光标记的骨髓间充质干细胞.结果与结论：单纯耳蜗微造孔术组、骨髓间充质干细胞组经耳蜗微造孔术后7 d的听性脑干诱发电位阈值均较空白对照组提高,术后28 d恢复至正常水平;耳蜗石蜡切片苏木精-伊红染色显示实验动物耳蜗内结构无明显异常改变.骨髓间充质干细胞组耳蜗切片可见荧光信号多分布于耳蜗底周的外淋巴腔（鼓阶和前庭阶）,无堆积堵塞管腔情况.结果表明经鼓阶开窗行骨髓间充质干细胞移植对耳蜗形态结构和听力的影响是轻微的,干细胞移植后能在耳蜗内迁移并存活.     

干细胞移植豚鼠耳蜗形态结构及听力的变化

背景:既往研究证明耳蜗微造孔注射病毒或非病毒载体行基因治疗对耳蜗结构及听功能的影响轻微.目的:观察骨髓间充质干细胞通过耳蜗微造孔术移植到正常豚鼠内耳后对耳蜗结构及听功能的影响.方法:正常豚鼠分3组,空白对照组未予任何干预;单纯耳蜗微造孔术组单纯行耳蜗微造孔术,不注射任何成分;骨髓间充质干细胞组鼓阶微造孔后注射经DAPI荧光标记的骨髓间充质干细胞.结果与结论:单纯耳蜗微造孔术组、骨髓间充质干细胞组经耳蜗微造孔术后7 d的听性脑干诱发电位阈值均较空白对照组提高,术后28 d恢复至正常水平;耳蜗石蜡切片苏木精-伊红染色显示实验动物耳蜗内结构无明显异常改变.骨髓间充质干细胞组耳蜗切片可见荧光信号多分布于耳蜗底周的外淋巴腔(鼓阶和前庭阶),无堆积堵塞管腔情况.结果表明经鼓阶开窗行骨髓间充质干细胞移植对耳蜗形态结构和听力的影响是轻微的,干细胞移植后能在耳蜗内迁移并存活.     

干细胞治疗感音神经性聋的研究理论及其临床应用进展

背景:内耳毛细胞或螺旋神经元的丢失可引起感音神经性聋.内耳细胞死亡后,利用干细胞研究来恢复听力已成为近年来研究热点.目的:总结近年来干细胞向内耳细胞方向分化的体内外研究进展,对干细胞进行感音神经性聋后内耳细胞替代治疗的研究成果进行综述.方法:应用计算机检索Pubmed数据库(2000-01/2009-08),检索词为"inner ear,Stem cells";应用计算机检索CNKI数据库(2000-01/2009-08),检索词为"内耳、干细胞".结果与结论:计算机初检得到170篇文献,纳入与干细胞在感音神经性聋的细胞转化有密切关系的实验研究和综述,排除重复研究类文献,对符合条件的32篇文献进行汇总分析.不同类型干细胞具有向内耳细胞分化的能力,能够分化为神经源性细胞类型.干细胞移植到内耳结构中可以发生迁移、分化,并部分分化为损伤区域的细胞类型,为感音神经性聋听力恢复提供了一条治疗途径.如何使分化细胞发挥正常生理功能,移植到内耳后能否建立神经通路,是干细胞治疗感音神经性聋下一步研究方向.     

感音神经性耳聋的影像学及病因学研究

听力障碍的病人很多,但有关听力障碍,尤其是感音神经性耳聋的研究,还处于初期阶段,听觉传导通路耳蜗以及蜗后的病变均可引起感音神经性耳聋,包括耳蜗、蜗神经、蜗神经核以及听觉中枢.本文主要探讨感音神经性耳聋的病因学及影像学表现.     

重度感音神经性耳聋行人工耳蜗植入术13例围术期护理

对13例重度感音神经性耳聋患儿行人工耳蜗植入术,并给予精心围术期护理,本组无一例患儿出现面瘫、感染、大出血及植入体移位等并发症.认为人工耳蜗可帮助患有重度感音神经性耳聋患儿恢复或获得听力,提高其语言交流能力.     

改良鼻饲法在脑卒中后意识障碍患者中的应用

对13例重度感音神经性耳聋患儿行人工耳蜗植入术,并给予精心围术期护理,本组无一例患儿出现面瘫、感染、大出血及植入体移位等并发症。认为人工耳蜗可帮助患有重度感音神经性耳聋患儿恢复或获得听力,提高其语言交流能力。     

1例双耳电子耳蜗植入术的护理

电子耳蜗植入术是目前治疗重度感音神经性耳聋的最有效方法。电子耳蜗系一种模拟耳蜗功能的声电换能助听装置，其一部分经手术植入耳蜗内，其余部分则如助听器戴在体外，电子耳蜗将编码的电信号越过受损或丧失的耳蜗毛细胞，通过植人的电极直接刺激残存的听神经元而恢复听觉。双侧电子耳蜗植入后．将给病人带来全新的立体声语言世界，病人可真切地感     

儿童分泌性中耳炎与语言发育迟缓的相关性研究

目的：探讨儿童期分泌性中耳炎所致感音神经性耳聋对患儿语言、认知和交流能力的影响。方法：对26例语言发育迟缓的分泌性中耳炎患儿，分析其高频听力损失和语言频率的关系。结果：患儿只有当高频听力损失达到一定程度，并波及到语频时，才会引起语言发育迟缓，如能早发现，早确诊，早进行听力语言康复，可以大大地减少因感音神经性耳聋所致的语言障碍。结论：儿童期分泌性中耳炎所致的感音神经性耳聋可以引起患儿语言发育迟缓。将分泌性中耳炎病程超过3个月作为感音神经性耳聋的高危儿童，开展听力筛查，可早发现感音神经性耳聋儿童，并及时配戴助听器和开展听觉语言康复训练。     

MRI对儿童感音神经性耳聋人工耳蜗植入术前的诊断价值

目的探讨磁共振成像在儿童感音神经性耳聋(SNHL)人工耳蜗植入术前的诊断价值及临床应用。方法回顾性分析80例临床诊断为SNHL拟行人工耳蜗植入的患儿MRI图像,结合内耳畸形的最新分类标准进行影像学分类诊断。结果 80例(160耳)发现中内耳畸形152耳,其中耳蜗畸形38耳,前庭畸形33耳,半规管畸形41耳,内耳道畸形40耳,前庭导水管扩大37耳,蜗神经畸形46耳。结论 MRI能对儿童感音神经性耳聋人工耳蜗植入术前提供丰富详细的解剖学信息,并进行分类诊断,对指导手术、评估预后等都具有重要的临床意义。     

Effects of Localized Neurotrophin Gene Expression on Spiral Ganglion Neuron Resprouting in the Deafened Cochlea

A cochlear implant may be used to electrically stimulate spiral ganglion neurons (SGNs) in people with severe sensorineural hearing loss (SNHL). However, these neurons progressively degenerate after SNHL due to loss of neurotrophins normally supplied by sensory hair cells (HCs). Experimentally, exogenous neurotrophin administration prevents SGN degeneration but can also result in abnormal resprouting of their peripheral fibers. This study aimed to create a target-derived neurotrophin source to increase neuron survival and redirect fiber resprouting following SNHL. Adenoviral (Ad) vectors expressing green fluorescent protein (GFP) alone or in combination with brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT3) were injected into the cochlear scala tympani or scala media of guinea-pigs (GPs) deafened via aminoglycosides for 1 week. After 3 weeks, cochleae were examined for gene expression, neuron survival, and the projection of peripheral fibers in response to gene expression. Injection of vectors into the scala media resulted in more localized gene expression than scala tympani injection with gene expression consistently observed within the partially degenerated organ of Corti. There was also greater neuron survival and evidence of localized fiber responses to neurotrophin-expressing cells within the organ of Corti from scala media injections (P < 0.05), a first step in promoting organized resprouting of auditory peripheral fibers via gene therapy.     

Inner ear transgene expression after adenoviral vector inoculation in the endolymphatic sac

Gene transfer has been performed in a variety of organs. In the mammalian inner ear, viral vectors have been used to introduce exogenous reporter genes via the scala tympani into the cochlea. While scala tympani inoculation is clinically feasible, it is not without risks. Moreover, transgene expression has so far been restricted to the cochlear tissues in the perilymphatic spaces that are contiguous with the scala tympani. To achieve gene transfer of vestibular organs and cells surrounding the endolymphatic space, and to extend the clinical utility of inner ear gene therapy, we developed a new surgical approach for vector inoculation. A replication-deficient adenoviral vector, Ad.RSVntlacZ, was injected into the guinea pig endolymphatic sac. A large number of blue (LacZ-positive) cells was observed in the endolymphatic sac and duct, the vestibule, and the ampulla. Blue cells were also detected in the cochlea, mainly in cells bordering the endolymphatic space: marginal cells in the stria vascularis and supporting cells in the organ of Corti. These findings indicate that inoculation of viral vectors into the endolymphatic sac can provide efficient gene transfer into a variety of cell types that are not accessible via scala tympani inoculation.     

Noninvasive in vivo delivery of transgene via adeno-associated virus into supporting cells of the neonatal mouse cochlea

There are a number of genetic diseases that affect the cochlea early in life, which require normal gene transfer in the early developmental stage to prevent deafness. The delivery of adenovirus (AdV) and adeno-associated virus (AAV) was investigated to elucidate the efficiency and cellular specificity of transgene expression in the neonatal mouse cochlea. The extent of AdV transfection is comparable to that obtained with adult mice. AAV-directed gene transfer after injection into the scala media through a cochleostomy showed transgene expression in the supporting cells, inner hair cells (IHCs), and lateral wall with resulting hearing loss. On the other hand, gene expression was observed in Deiters cells, IHCs, and lateral wall without hearing loss after the application of AAV into the scala tympani through the round window. These findings indicate that injection of AAV into the scala tympani of the neonatal mouse cochlea therefore has the potential to efficiently and noninvasively introduce transgenes to the cochlear supporting cells, and this modality is thus considered to be a promising strategy to prevent hereditary prelingual deafness.     

骨髓间充质干细胞对衰老模型大鼠肺损伤的修复作用

背景：研究发现外源性骨髓间充质干细胞能够定居于肺脏组织,参与肺组织的再生,但其修复衰老肺损伤方面的报道较少。目的：观察骨髓间充质干细胞减轻D-半乳糖所致肺脏组织损伤的作用。方法：选择SD大鼠30只,随机分为3组,包括对照组、衰老模型组、细胞治疗组,每组10只。衰老模型组和细胞治疗组大鼠每日皮下注射D-半乳糖,连续4个月制备衰老模型。细胞治疗组通过尾静脉输注3×10^6个骨髓间充质干细胞,每周1次,连续4周;对照组和衰老模型组给予同等剂量的细胞培养液。采用表达绿色荧光蛋白的慢病毒载体标记骨髓间充质干细胞,以确定骨髓间充质干细胞在大鼠肺脏的植入情况。测定3组大鼠肺脏组织中超氧化物歧化酶活性和丙二醛水平;苏木精-伊红染色观察3组大鼠肺脏组织结构的差异。结果与结论：绿色荧光蛋白标记的骨髓间充质干细胞移植给大鼠后,能向肺脏组织迁移并存活。与衰老模型组相比,细胞治疗组肺脏的超氧化物歧化酶活性明显升高,丙二醛水平明显降低。各组大鼠的组织病理切片显示,模型组大鼠的正常肺泡结构破坏,出现气腔扩大、肺气肿改变;而细胞治疗组大鼠的肺脏损伤有明显修复。结果可见骨髓间充质干细胞对衰老大鼠的肺脏损伤有修复作用,从而发挥其抗衰老作用。     

尾静脉移植人脂肪干细胞在脑缺血大鼠脑内的存活

背景:间充质干细胞移植可显著改善缺血性脑血管病神经功能。目的:观察尾静脉途径移植人脂肪源间充质干细胞对局灶性脑缺血大鼠神经功能的影响。方法:制作局灶性脑缺血大鼠模型,随机分为实验组和对照组,实验组在造模后3d尾静脉注射5×106脂肪源间充质干细胞,对照组不进行细胞移植。结果与结论:两组大鼠神经功能缺损行为学评分随着细胞移植后时间延长均逐渐降低。移植后第14,21天,实验组大鼠行为学评分较对照组显著降低(P<0.05),且实验组改善程度明显优于对照组(P<0.01);移植的人脂肪源间充质干细胞在局灶性脑缺血大鼠血管周边和缺血周边区聚集并存活。说明尾静脉移植的脂肪源间充质干细胞可在宿主脑内存活,并改善神经功能。     

脂肪间充质干细胞移植对心肌梗死后炎症反应及心室重构的影响简

背景：脂肪间充质干细胞的免疫调节作用是否能够用于心肌梗死的治疗,以及发挥这一作用的最佳时机如何,目前研究较少。目的：观察移植的脂肪间充质干细胞对心肌梗死后炎症反应及心室重构的影响,探讨脂肪间充质干细胞治疗心肌梗死的可能机制。方法：酶消化法分离培养大鼠脂肪间充质干细胞,40只大鼠结扎左冠状动脉前降支建立心肌梗死模型后随机数字表法均分为假手术组、对照组（注射 HG-DMEM）、心梗后3 h,7 d 移植脂肪间充质干细胞组。移植后14 d采用ELISA法检测血浆肿瘤坏死因子α和白细胞介素10的水平,移植后28 d行超声心动图检测左室舒张末期内径、收缩末期内径、射血分数和短轴缩短率。结果与结论：细胞移植后第14天,与对照组相比,3 h移植组和7 d移植组血浆促炎细胞因子肿瘤坏死因子α的水平明显降低（P＜0.01）,抗炎细胞因子白细胞介素10的水平明显升高（P＜0.01）;细胞移植后第28天,与对照组比较,脂肪间充质干细胞移植组左室舒张末期内径和收缩末期内径明显减小,心脏缩小,射血分数和短轴缩短率明显提高,心功能改善,且3h移植组较7d移植组作用更趋明显。说明心肌梗死早期进行脂肪间充质干细胞移植,能够显著抑制梗死后炎症反应,调节炎症细胞因子网络平衡,延缓心室重构,改善心脏功能。     

脂肪干细胞与骨髓间充质干细胞成软骨能力的比较

背景：脂肪干细胞与骨髓间充质干细胞是软骨组织工程中应用较多细胞,二者在生物学特性上有诸多相似之处。目的：比较脂肪来源和骨髓来源的2种间充质干细胞的成软骨分化能力。方法：选取3月龄新西兰大白兔,取腹部脂肪,分离提取脂肪干细胞。取兔双侧股骨,采用贴壁筛选法分离提取骨髓间充质干细胞。绘制第3代脂肪干细胞和骨髓间充质干细胞的生长曲线,比较2种细胞的倍增时间。对第3代脂肪干细胞和骨髓间充质干细胞进行成软骨诱导,分别对诱导14d的2种细胞行甲苯胺蓝染色和Ⅱ型胶原免疫组化染色。结果与结论：骨髓间充质干细胞原代细胞呈聚集样生长,而脂肪干细胞原代细胞呈单个、散在生长。脂肪干细胞增殖速度要快于骨髓间充质干细胞,倍增时间短于骨髓间充质干细胞h。2种细胞成软骨诱导14d后,均表达糖胺聚糖和Ⅱ型胶原,且骨髓间充质干细胞成软骨诱导后表达Ⅱ型胶原水平高于脂肪干细胞。说明脂肪干细胞与骨髓间充质干细胞体外增殖皆迅速且稳定,但是脂肪干细胞的生长增殖速度更快。单层培养时,特定条件下,脂肪干细胞与骨髓间充质干细胞均能向软骨细胞转化,但是骨髓间充质干细胞比脂肪干细胞具有更高的潜能。     

Conditioning the Cochlea to Facilitate Survival and Integration of Exogenous Cells into the Auditory Epithelium

The mammalian auditory epithelium (AE) cannot replace supporting cells and hair cells once they are lost. Therefore, sensorineural hearing loss associated with missing cells is permanent. This inability to regenerate critical cell types makes the AE a potential target for cell replacement therapies such as stem cell transplantation. Inserting stem cells into the AE of deaf ears is a complicated task due to the hostile, high potassium environment of the scala media in the cochlea, and the robust junctional complexes between cells in the AE that resist stem cell integration. Here, we evaluate whether temporarily reducing potassium levels in the scala media and disrupting the junctions in the AE make the cochlear environment more receptive and facilitate survival and integration of transplanted cells. We used sodium caprate to transiently disrupt the AE junctions, replaced endolymph with perilymph, and blocked stria vascularis pumps with furosemide. We determined that these three steps facilitated survival of HeLa cells in the scala media for at least 7 days and that some of the implanted cells formed a junctional contact with native AE cells. The data suggest that manipulation of the cochlear environment facilitates survival and integration of exogenously transplanted HeLa cells in the scala media.