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.     

腺病毒介导bcl-2基因转染对顺铂所致大鼠螺旋神经节细胞损伤的拮抗作用

目的 通过腺病毒(adenovirus,Ad)载体介导bcl-2基因转染体外培养的新生大鼠耳蜗螺旋神经节细胞(spiral ganglion cells,SGC),探讨bel-2蛋白过度表达对顺铂所致SGC损伤的拮抗作用.方法 体外培养新生大鼠SGC,携带绿色荧光蛋白(green fluorescent protein,GFP)基因的腺病毒载体Ad-GFP转染SGC,并行神经丝蛋白(NF200)免疫细胞化学染色鉴定,激光共聚焦扫描荧光显微镜下观察.携带bel-2基因的腺病毒载体Ad-bel-2转染SGC,蛋白质印迹法(Western Blot)检测bcl-2蛋白表达.设立Ad-bcl-2转染加顺铂组(A组),Ad-GFP转染加顺铂组(B组),顺铂组(C组)和正常对照组(D组),顺铂作用浓度为2μg,/ml;顺铂作用48 h后,行各组SGC计数,并通过lmageJ软件测量各组SGC轴突长度.结果 成功分离并培养新生大鼠SGC.激光共聚焦扫描荧光显微镜下观察到腺病毒载体可安全高效转染体外培养的SGC.Ad-bcl-2转染3 d后,Western Blot检测有外源性人bcl-2基因的高效表达,而Ad-GFP转染组和顺铂组未检测到表达.顺铂作用后,A、B、C组部分SGC细胞突起变短、萎缩,胞体缩小、变圆,甚至浮起.A组SGC数目明显多于B组和C组(P值均＜0.01),但少于D组(P＜0.05);A组SGC轴突长度明显长于B组和C组,但短于D组(P值均＜0.01).结论 腺病毒能够安全高效地转染体外培养的新生大鼠SGC.bel-2蛋白过表达对顺铂所致SGC损伤有一定的拮抗作用.     

神经营养因子3基因转染对庆大霉素性耳聋保护作用的实验研究

目的 :探讨阳离子脂质体介导的神经营养因子 3(NT3)基因转染对豚鼠庆大霉素性耳聋的保护作用。方法 :将携带外源性基因NT3的重组真核表达载体 pIRES2 EGFP NT3与阳离子脂质体复合物注入豚鼠耳蜗 ,术后给予庆大霉素肌肉注射 (12 0mg/kg) 14d ,分别于停药后 1d、2周进行听性脑干反应 (ABR)及畸变产物耳声发射 (DPOAE)测听并观察转染基因在耳蜗的表达和分布及耳蜗毛细胞受损情况。结果 :NT3组DP gram幅值降低较对照组明显减轻 ,ABR阈值升高亦无对照组显著 (P <0 .0 5 )。耳蜗基底膜FITC phalloidin染色见NT3组耳蜗毛细胞的缺失较对照组明显减轻。结论 :阳离子脂质体介导的NT3基因转染对豚鼠庆大霉素性耳聋具有一定程度的保护作用。     

阳离子脂质体介导NT-3基因转染豚鼠耳蜗的实验研究

目的 探讨阳离子脂质体介导的NT-3基因转染对豚鼠耳蜗形态及功能的影响。方法 克隆人NT-3 cDNA基因，构建携带绿色荧光蛋白(EGFP)报告基因的重组质粒pIPES2-EGFP-NT3。采用脂质体介导的方法，将重组质粒转染豚鼠耳蜗，分别于术后1天、2周、4周观察转染基因在耳蜗及脑干等部位的表达和分布情况。同时进行耳声发射及ABR测试，了解基因转染对豚鼠耳蜗听功能的影响。结果 成功克隆人NT-3 cDNA基因并构建重组质粒pIRES2-EGFP-NT3。重组质粒转染豚鼠耳蜗后，荧光显微镜下观察发现耳蜗螺旋神经节、神经纤维、Corti器、血管纹等均可见绿色荧光，但4周时荧光蛋白的表达强度较1天时明显减弱。而CY3标记的NT-3免疫荧光染色结果基本与EGFP的表达情况一致。此外，对侧耳蜗及第四脑室脉络膜处亦可见较强的绿色荧光。耳蜗基底膜铺片见内外毛细胞结构完整。转染前后豚鼠耳声发射及ABR阈值均无明显改变。结论 阳离子脂质体介导的NT-3基因转染对豚鼠耳蜗形态及功能无明显影响；外源性NT-3能够在较长时间内持续表达。     

脑源性神经营养因子基因转染骨髓间充质干细胞体外诱导分化为神经元样细胞初步观察

目的探讨骨髓间充质干细胞（bone-marrow mesenchymal stem cells,BMscs）转染人脑源性神经营养因子（brain-derived neurotrophic factor,BDNF）基因后在体外分化为神经元样细胞的功能。方法克隆人BDNF基因并构建其真核表达载体;分离培养5只豚鼠BMscs,观测其形态并用流式细胞仪鉴定;将人BDNF基因电穿孔法转染BMscs,G418筛选后用维甲酸诱导分化,免疫细胞化学法对分化细胞进行鉴定。结果分离培养的细胞具有典型的BMSCs形态和标志,转染诱导后的细胞表达神经元特异性烯醇化酶、巢蛋白（Nestin）和胶质纤维酸性蛋白,并能分泌BDNF。结论BDNF基因转染的BMscs在体外能分化为神经元样细胞,电穿孔法能提高转染率,RA有促诱导作用。     

脑源性神经营养因子延迟应用对感音神经性聋影响的实验研究

目的探讨药物致聋后,延迟给予脑源性神经营养因子(brain-drived neurotrophic factor,BDNF)对耳蜗病理及听觉生理的影响。方法药物致聋大鼠20只,随机平均分为人工外淋巴液(artifical perilymph,AP)组和BDNF组。左侧耳蜗为实验侧,右侧耳蜗为对照侧。致聋30天后通过微渗透压泵向鼓阶内持续灌注AP或BDNF,期间进行电诱发听性脑干反应(electrically auditory brainstem response,EABR)阈值检测。持续给药28天后取耳蜗进行病理检查,观察Rosenthal管内螺旋神经节细胞(spiral ganglion cell,SGC)胞体密度及骨螺旋板缰孔内神经纤维密度。结果EABR阈值AP组持续升高,BDNF组阈值初期上升缓慢,后期开始下降,用药治疗14～28夭阈值低于AP组(P<0.01);耳蜗病理变化,SGC胞体密度及神经纤维密度左右侧对比,AP组双侧无差异(P>0.05),BDNF组左侧明显高于右侧(P<0.01);两组左侧比较,BDNF组高于AP组(P<0.01)。结论大鼠药物致聋30天后,鼓阶内BDNF持续给药对SGC胞体及树突均有保护作用,并可以相应改善电听觉敏感性。     

脑源性神经营养因子对耳蜗螺旋神经节的保护作用

目的　观察腺病毒携带的脑源性神经营养因子 (brainderivedneurotrophicfactor,BDNF)在豚鼠耳蜗中的表达 ,及噪声损伤后对螺旋神经节的保护作用。方法　 2 7只白色纯种豚鼠 ,暴露于135dBSPL ,4kHz的窄带噪声 4h。 7d后 ,12只经圆窗注入腺病毒携带BDNF(adenoviral mediatedBDNF ,ad BDNF) ,12只经圆窗注入ad LacZ ,3只注入人工外淋巴液。分别于 1、4、8周后取材 ,石蜡包埋中轴切片后 ,用免疫组化方法 (ABC法 )检测BDNF的表达。于光镜下计数螺旋神经节细胞。结果　在耳蜗各回中BDNF均有表达 ,4、8周组动物较 1周组动物表达弱。在 8周 ,注入ad BDNF组较ad LacZ组和人工外淋巴组螺旋神经节发生退行性病变的数目少 ,螺旋神经节细胞计数结果经统计学t检验 ,P <0 0 1,差异有显著性。结论　腺病毒携带的神经营养因子在耳蜗中能高效表达 ,在噪声损伤情况下腺病毒携带的脑源性神经营养因子对螺旋神经节有保护作用。该研究为基因治疗感音神经性聋提供了坚实的实验基础     

卡那霉素联合速尿致聋豚鼠的Math1基因治疗

目的 观察卡那霉素和速尿联合致聋豚鼠耳蜗鼓阶导入Math1基因后的形态学及功能改变,探讨Mathl基因治疗药物中毒性耳聋的可行性.方法 健康成年豚鼠经硫酸卡那霉素(500 mg/kg)和速尿(50 mg/kg)联合致聋,将听性脑干反应(ABR)反应阈＞95 dB SPL的豚鼠按随机数字表法分为空白对照组(不做任何处置,3只),手术对照组(右耳单纯鼓阶钻孔,3只),人工外淋巴液组(右耳鼓阶钻孔导入人工外淋巴液,3只),单纯病毒载体组[右耳鼓阶钻孔导入携带增强型绿色荧光蛋白基因(enhanced green fluorescent protein,EGFP)的重组腺病毒(Ad.EGFP),4只]、Math1基因治疗组[右耳鼓阶钻孔导入携带Math1及EGFP基因的重组腺病毒(Ad.Math1-EGFP),6只].各组动物分别于鼓阶注射前及注射后8周时行ABR测试,结束测试后处死动物,取出耳蜗组织行扫描电镜观察.结果 各组豚鼠不同频率(4、8、16、20 kHz)短纯音ABR阈值在不同检测时间段差异均无统计学意义,组间比较差异亦无统计学意义(P值均＞0.05).除Math1基因治疗组外,其余各组右耳耳蜗各回毛细胞形态和数目与左耳(自身对照)比较无明显差别.4只Math1基因治疗组豚鼠中,有2只右耳耳蜗第三回内、外毛细胞数量明显比左耳多,其中内毛细胞排列形态较外毛细胞整齐.结论 鼓阶显微注射导入Math1基因能使部分卡那霉素和速尿联合致聋豚鼠的耳蜗毛细胞修复或再生,但其听觉功能没有改善.     

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。     

A study of gentamicin injury mechanisms using cultured mouse cochlear spiral ganglion cells

Objective To study gentamicin injury mechanisms using postnatal mouse cochlear spiral gangcells (SGC).Methods SGCs were isolated using a combinatorial approach of enzymatic digestion and mechanical sep...     

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。     

庆大霉素对小鼠耳蜗螺旋神经节细胞损伤机制初步研究

目的探讨庆大霉素对小鼠耳蜗螺旋神经节细胞（spiral ganglion cell,SGC）的损伤机制。方法取出生后2～6天昆明种乳小鼠耳蜗,利用酶消化和机械分离相结合的方法分离出SGC,并进行体外培养。将培养4天的SGC用4%多聚甲醛室温固定30min,以小鼠源神经纤维细丝蛋白（Neurofilament-68/200kDa,NF-L＋H）单克隆抗体作为一抗,按照SP法（streptavidin-perosidase法,链霉菌抗生物素蛋白-过氧化物酶连结法）对所培养细胞进行免疫细胞化学染色,对SGC作出鉴定。将培养第4天的细胞分为4组：空白对照组及3种浓度的庆大霉素干预组（庆大霉素终浓度分别为50mg/L、100mg/L、150mg/L）,作用48h后收集各组细胞进行透射电镜分析。结果SGC原代培养获得成功,经NF-L＋H抗体免疫细胞化学染色,其胞质及突起均呈阳性反应,染成棕黄色,一般有相对生长的两个突起,为典型的双极神经元。在透射电镜下观察SGC超微结构,3种浓度的庆大霉素组SGC与空白对照组相比,均出现明显的形态学改变,这种改变与凋亡相关。结论庆大霉素对小鼠耳蜗SGC有直接毒性作用,可导致细胞凋亡,线粒体损伤在这一过程中可能具有重要意义。     

The effect of deafness duration on neurotrophin gene therapy for spiral ganglion neuron protection

A cochlear implant can restore hearing function by electrically exciting spiral ganglion neurons (SGNs) in the deaf cochlea. However, following deafness SGNs undergo progressive degeneration ultimately leading to their death. One significant cause of SGN degeneration is the loss of neurotrophic support that is normally provided by cells within the organ of Corti (OC). The administration of exogenous neurotrophins (NTs) can protect SGNs from degeneration but the effects are short-lived once the source of NTs has been exhausted. NT gene therapy, whereby cells within the cochlea are transfected with genes enabling them to produce NTs, is one strategy for providing a cellular source of NTs that may provide long-term support for SGNs. As the SGNs normally innervate sensory cells within the OC, targeting residual OC cells for gene therapy in the deaf cochlea may provide a source of NTs for SGN protection and targeted regrowth of their peripheral fibers. However, the continual degeneration of the OC over extended periods of deafness may deplete the cellular targets for NT gene therapy and hence limit the effectiveness of this method in preventing SGN loss. This study examined the effects of deafness duration on the efficacy of NT gene therapy in preventing SGN loss in guinea pigs that were systemically deafened with aminoglycosides. Adenoviral vectors containing green fluorescent protein (GFP) with or without genes for Brain Derived Neurotrophic Factor (BDNF) and Neurotrophin-3 (NT3) were injected into the scala media (SM) compartment of cochleae that had been deafened for one, four or eight weeks prior to the viral injection. The results showed that viral transfection of cells within the SM was still possible even after severe degeneration of the OC. Supporting cells (pillar and Deiters' cells), cells within the stria vascularis, the spiral ligament, endosteal cells lining the scala compartments and interdental cells in the spiral limbus were transfected. However, the level of transfection was remarkably lower following longer durations of deafness. There was a significant increase in SGN survival in the entire basal turn for cochleae that received NT gene therapy compared to the untreated contralateral control cochleae for the one week deaf group. In the four week deaf group significant SGN survival was observed in the lower basal turn only. There was no increase in SGN survival for the eight week deaf group in any cochlear region. These findings indicated that the efficacy of NT gene therapy diminished with increasing durations of deafness leading to reduced benefits in terms of SGN protection. Clinically, there remains a window of opportunity in which NT gene therapy can provide ongoing trophic support for SGNs.     

Neurotrophins and electrical stimulation for protection and repair of spiral ganglion neurons following sensorineural hearing loss

Exogenous neurotrophins (NTs) have been shown to rescue spiral ganglion neurons (SGNs) from degeneration following a sensorineural hearing loss (SNHL). Furthermore, chronic electrical stimulation (ES) has been shown to retard SGN degeneration in some studies but not others. Since there is evidence of even greater SGN rescue when NT administration is combined with ES, we examined whether chronic ES can maintain SGN survival long after cessation of NT delivery. Young adult guinea pigs were profoundly deafened using ototoxic drugs; five days later they were unilaterally implanted with an electrode array and drug delivery system. Brain derived neurotrophic factor (BDNF) was continuously delivered to the scala tympani over a four week period while the animal simultaneously received ES via bipolar electrodes in the basal turn (i.e., turn 1) scala tympani. One cohort (n=5) received ES for six weeks (i.e., including a two week period after the cessation of BDNF delivery; ES(6)); a second cohort (n=5) received ES for 10 weeks (i.e., a six week period following cessation of BDNF delivery; ES(10)). The cochleae were harvested for histology and SGN density determined for each cochlear turn for comparison with normal hearing controls (n=4). The withdrawal of BDNF resulted in a rapid loss of SGNs in turns 2-4 of the deafened/BDNF-treated cochleae; this was significant as early as two weeks following removal of the NT when compared with normal controls (p<0.05). Importantly, there was not a significant reduction in SGNs in turn 1 (i.e., adjacent to the electrode array) two and six weeks after NT removal, as compared with normal controls. This result suggests that chronic ES can prevent the rapid loss of SGNs that occurs after the withdrawal of exogenous NTs. Implications for the clinical delivery of NTs are discussed.