腺病毒介导的BDNF及NT3基因对体外培养听神经元的营养作用研究

在听力损伤过程中有一大类的损伤是由于各种外界因素对于听神经的损伤而引起的。为了观察脑源性神经营养因子（ＢＤＮＦ）和神经营养素－３（ＮＴ３）对听神经元的营养作用,并研究基因治疗在听神经损伤治疗中的可行性。在体外建立了听神经元的培养系统,利用ＬａｃＺ重组腺病毒感染培养的听神经元来研究重组腺病毒介导的外源基因的转染效率,通过Ｘ－Ｇａｌ染色来显示被感染的阳性细胞,在加入１００病毒感染复数（ＭＯＩ）的Ａｄ－     

顺铂对小鼠耳蜗螺旋神经节细胞凋亡及caspase-3表达的影响

目的:建立小鼠顺铂(CDDP)耳毒性模型,研究不同剂量顺铂对小鼠耳蜗螺旋神经节细胞凋亡及caspase-3表达的影响。方法:采用末端脱氧核苷酸转移酶介导的缺口末端标记(TUNEL)技术检测螺旋神经节细胞的凋亡;应用免疫组织化学Envision法检测caspase-3在螺旋神经节中的表达;同时结合听脑干反应(ABR)测试,观察用药前后小鼠听力的变化。结果:不同剂量顺铂组小鼠体重和听力明显下降,与对照组比较均有显著性差异(P0.05,P0.01);并且随着顺铂给药剂量的增加,小鼠耳蜗螺旋神经节中TUNEL阳性细胞数增多,以及caspase-3表达明显增强。结论:应用小鼠能建立可靠的顺铂耳毒性模型;顺铂可导致耳蜗螺旋神经节细胞凋亡,而且此凋亡过程中有caspase-3的参与,进一步证实了凋亡可能是顺铂耳毒性机制之一。     

腺病毒介导BDNF基因对无血清培养SH-SY5Y细胞的生物学作用研究

目的 :为了有效的将腺病毒介导的脑源性神经营养因子 (BDNF)用于神经损伤的保护治疗。方法 :在体外用BDNF重组腺病毒 (Ad BDNF)对SH SY5Y细胞进行感染 ,在无血清培养条件下对细胞的生长分化进行了形态学的观察 ,利用MTT法检测不同浓度的Ad BDNF对SH SY5Y细胞的促存活作用 ,并对细胞凋亡作用进行了检测。结果和结论 :腺病毒介导的BDNF可有效的促进感染后的SH SY5Y细胞的存活 ,生长和分化 ,并可有效的抑制无血清状态下细胞凋亡的发生     

逆转录病毒介导的人NT—3在嗅神经鞘细胞中的表达及活性检测

本文构建了pRev-TRE-NT-3的逆转录病毒表达载体,通过Ecopack293细胞将其与pRevTet-On分别进行包装,制备了重组缺陷型的hNT-3和Tet-on逆转录病毒,用这两种病毒感染原代培养大鼠神经嗅神经鞘细胞（olfctory ensheathing cells,OECs),并经强力霉素（Dox）诱导,获得Dox浓度依赖性hNT-3表达的OECs,最后经过Western-Blot检测hNT-3的表达以及通过DRG与NT－3转染后OECs联合培养来对表达的hNT-3活性进行鉴定,结果：（1）hNT-3－pcDNA的多克隆位点,用EcoRI和HindⅢ酶切,PCR扩增,再用Sall和Hind Ⅲ切下全长编码的hNT-3cDNA,定向连接到pRev-TRE上,pRev-TRE全长为6．5kb,NT-3全长为0.78kb,pRev-TRE-NT-3重组体经过鉴定,确认插入子的方向和完整性,结果与预期相符。（2）hNT-3修饰的OECs培养上清经Western-blot检测,与hNT-3抗体结合的蛋白条带分子量约为28kd,hNT-3修饰过的OECs上清表达量明显高于未转染组OECs的表达量,且hNT-3表达量与Dox浓度有依赖性。（3）与hNT-3修饰的OECs联合培养背根神经节（dorsal root ganglion,DRG),有许多DRG细胞向外迁移,这些细胞折光性强,突起较长而纤细,并形成复杂的网络。而未修饰的OECs组, 只有少量的细胞迁移生长,细胞迁移和突起生长都很局限,空白对照组的DRG没有向外迁移的细胞和向外延伸的突起;且对照组的突起生长长度明显比NT－3修饰的实验组短（P＜0．01）。这些结果表明,Tet-On调控NT－3表达在OECs转染成功,为进一步体内移植修复损伤提供了理想的材料。     

水杨酸钠对螺旋神经节Caspase 3mRNA及蛋白表达的影响

目的：水杨酸是阿司匹林的活性成分,是导致耳鸣的主要原因。而本实验主要探讨水杨酸钠对耳蜗螺旋神经节（SGN）调亡相关基因Caspase 3的mRNA及蛋白表达水平的影响,并初步探讨水杨酸钠对耳蜗毒性的机制。方法：分离取出大鼠蜗轴螺旋管,用酶消化后原代培养SGN,采用荧光定量PCR法检测5 mM水杨酸钠处理前后（1 h,3 h,6 h）Caspase 3 mRNA的变化,WesternBlot检测其蛋白的变化情况。结果：5 mM水杨酸钠处理细胞后1 h,Caspase 3 mRNA表达水平没有明显改变（P〉0.05）,但是当水杨酸钠处理3 h后,其表达水平明显上调（P〈0.05）,并呈时间依赖关系。而其蛋白表达水平也同样明显升高（P〈0.05）。结论：本实验取蜗轴螺旋管进行原代培养,获得较多的SGN,而水杨酸钠能上调这些原代培养SGN的Caspase 3 mRNA及蛋白的表达,导致SGN调亡,这对水杨酸钠耳蜗毒性的研究有一定应用价值。     

谷氨酸受体在噪声所致豚鼠螺旋神经节细胞损伤中的作用

本文旨在研究谷氨酸及其受体在噪声致豚鼠螺旋神经节细胞损伤中的作用。实验分为在体和离体两部分。（1）在体实验：豚鼠分为生理盐水（NS,10μL）组,NS（10μL）＋噪声组和犬尿喹啉酸（kynurenic acid,KYNA,5mmol／L,10μL）＋噪声组,每组15只。用微量注射器经完整圆窗膜表面给予NS或KYNA：暴露于白噪声110dBSPL,1h。在圆窗给药前及噪声暴露后测试听觉脑干诱发电位（auditory brainstem response,ABR）阈值及Ⅲ波幅值,听神经复合动作电位（compound action potential,CAP）阈值及N1波幅值和潜伏期,测试后取基底膜进行透射电镜观察。（2）离体实验：观察高浓度谷氨酸对急性分离的豚鼠螺旋神经节细胞的影响。结果显示,NS＋噪声组豚鼠ABR及CAP阈移显著高于KYNA＋噪声组,且Ⅲ波和NI波幅值明显降低,潜伏期明显延长。NS＋噪声组豚鼠毛细胞及传入神经末梢急性水肿和线粒体结构破坏：KYNA＋噪声组豚鼠的毛细胞和传入神经末梢无明显变化。离体胞外施加谷氨酸可引起螺旋神经节细胞逐渐出现水肿、变性,最后死亡。本实验提示,噪声暴露可引起豚鼠听功能损伤,毛细胞／传入神经突触的结构破坏和螺旋神经节细胞变性、死亡：这种损伤可能与噪声暴露引起谷氨酸的过度释放有关;谷氨酸通过其受体介导致使螺旋神经节细胞损伤,谷氨酸受体的广谱拮抗剂KYNA可减轻噪声对螺旋神经节细胞的损伤。     

SOCS3基因重组腺病毒的构建及其在猪脂肪细胞中的表达

本研究旨在构建细胞因子信号转导抑制因子3(Suppressor of cytokine signaling 3,SOCS3)的重组腺病毒表达载体,获得有感染性的病毒颗粒。以pcDNA3-SOCS3质粒为模板扩增SOCS3基因,将其亚克隆至腺病毒穿梭载体pAdTrack-CMV,经测序验证后,重组的穿梭质粒用PmeI酶切线性化后转化到BJ5183感受态细菌中与其内的骨架载体pAdEasy-1进行同源重组,获得的重组质粒pAd-SOCS3,经PacI线性化后转染至HEK293细胞中进行包装和扩增,纯化后用TCID50法测定病毒滴度。以重组的病毒感染原代培养的猪脂肪细胞后,荧光显微镜下观察报告基因GFP的表达,RT-PCR和Western blotting检测细胞内SOCS3 mRNA和蛋白的表达。重组腺病毒载体pAd-SOCS3经酶切及PCR鉴定正确,病毒滴度为1.2×109PFU/mL;感染原代培养的猪脂肪细胞后,荧光显微镜观察可见报告基因GFP的表达;RT-PCR和Western blotting检测到细胞中SOCS3 mRNA和蛋白的表达显著提高。本研究成功构建了SOCS3基因的重组腺病毒,感染原代培养的猪脂肪细胞可稳定表达SOCS3蛋白,为深入研究SOCS3的功能奠定了基础。     

雌激素对老年C57BL/6J小鼠耳蜗螺旋神经节细胞凋亡的影响*

目的:观察雌激素对于老年C57BL/6J小鼠耳蜗螺旋神经节细胞凋亡的影响,并探讨雌激素对老年性耳聋保护作用的可能机制。方法:将40只C57BL/6J雌鼠分为以下4组(10只/组): 3 m组(3月龄组),12 m组(12月假手术组); 12 m OVX组(12月去卵巢组),在9月龄行双侧卵巢切除术,正常饲养至12月龄;12 m OVX+E2组(雌激素干预组)在9月龄行双侧卵巢切除术,经过1月洗脱期后,给予皮下注射雌激素100 μg/(kg·d),持续2月至12月龄,其余各组小鼠正常喂养。至12 m OVX+E2组小鼠给药结束后,尾静脉采血,酶联免疫吸附( ELISA) 法检测各组小鼠血清雌激素水平;听性脑干反应(ABR)检测各组小鼠听力阈值变化;用2%戊巴比妥钠麻醉小鼠,断颈后取双侧耳蜗,石蜡包埋切片,苏木精-伊红HE染色观察各组小鼠耳蜗螺旋神经节形态学变化;TUNEL 染色观察各组小鼠耳蜗螺旋神经节神经元凋亡情况;取耳蜗螺旋神经节,应用实时荧光定量PCR(QRT-PCR)检测各组小鼠耳蜗螺旋神经节凋亡蛋白Caspase-3、Bax、Bcl-2 mRNA 表达水平。结果:12 m组与3 m组相比,小鼠听力阈值升高(P＜0.01),耳蜗螺旋神经节细胞出现缺失严重及异常凋亡(P＜0.01);12 m OVX组小鼠听力阈值高于12 m组(P＜0.01),且螺旋神经节细胞缺失加重,细胞凋亡增多(P＜0.01),凋亡蛋白 Caspase-3、Bax mRNA水平升高(P＜ 0.01),抗凋亡蛋白Bcl-2 mRNA水平降低(P＜0.01);外源性给予雌激素12 m OVX+E2组,较12 m OVX组,听力阈值降低(P＜0.01),细胞凋亡减少,凋亡蛋白 Caspase-3、Bax mRNA水平降低(P＜0.01),抗凋亡蛋白Bcl-2 mRNA水平升高(P＜0.01)。结论:雌激素可抑制老年C57BL/6J小鼠耳蜗螺旋神经节细胞凋亡,从而实现对老年性耳聋的保护作用。     

Caspase-3在老年豚鼠耳蜗螺旋神经节细胞的表达

目的检测caspase-3在老年豚鼠耳蜗的表达。方法实验分两组:实验组和对照组,实验组豚鼠年龄为33至35个月之间,对照组豚鼠年龄为2至3个月。用免疫组织化学方法检测caspase-3在两组豚鼠耳蜗的表达。结果Caspase-3在实验组耳蜗的表达呈阳性,阳性区域主要存在于耳蜗螺旋神经节细胞。在对照组耳蜗的表达呈阴性。结论Caspase-3在老年豚鼠耳蜗螺旋神经节细胞中呈阳性表达,提示caspase-3在豚鼠耳蜗老化过程中起重要作用。     

Paeoniflorin protects spiral ganglion neurons from cisplatin‐induced ototoxicity: Possible relation to PINK1/BAD pathway

The objective of this study was to elucidate whether paeoniflorin (PF) exerted an effect on cisplatin‐induced spiral ganglion neuron (SGN) damage, with special attention given to the role of PINK1/BAD pathway in this process. Middle cochlear turn culture and C57BL/6 mice were utilized to identify the character of PF in vitro and in vivo. We found that cisplatin treatment led to SGN damage, in which reactive oxygen species (ROS) generation increased, PINK1 expression decreased, BAD accumulation on mitochondria raised and mitochondrial apoptotic pathway activated. Conversely, we demonstrated that PF pre‐treatment obviously mitigated cisplatin‐induced SGN damage. Mechanistic studies showed that PF could reduce ROS levels, increase PINK1 expression, decrease the BAD accumulation on mitochondria and, thus, alleviate the activated mitochondrial apoptosis in SGNs caused by cisplatin. Overall, the findings from this work reveal the important role of PF and provide another strategy against cisplatin‐induced ototoxicity.     

Survival, synaptogenesis, and regeneration of adult mouse spiral ganglion neurons in vitro

The inner ear spiral ganglion is populated by bipolar neurons connecting the peripheral sensory receptors, the hair cells, with central neurons in auditory brain stem nuclei. Hearing impairment is often a consequence of hair cell death, e.g., from acoustic trauma. When deprived of their peripheral targets, the spiral ganglion neurons (SGNs) progressively degenerate. For effective clinical treatment using cochlear prostheses, it is essential to maintain the SGN population. To investigate their survival dependence, synaptogenesis, and regenerative capacity, adult mouse SGNs were separated from hair cells and studied in vitro in the presence of various neurotrophins and growth factors. Coadministration of fibroblast growth factor 2 (FGF-2) and glial cell line-derived neurotrophic factor (GDNF) provided support for long-term survival, while FGF-2 alone could strongly promote neurite regeneration. Fibroblast growth factor receptor FGFR-3-IIIc was found to upregulate and translocate to the nucleus in surviving SGNs. Surviving SGNs formed contacts with other SGNs after they were deprived of the signals from the hair cells. In coculture experiments, neurites extending from SGNs projected toward hair cells. Interestingly, adult mouse spiral ganglion cells could carry out both symmetric and asymmetric cell division and give rise to new neurons. The authors propose that a combination of FGF-2 and GDNF could be an efficient route for clinical intervention of secondary degeneration of SGNs. The authors also demonstrate that the adult mammalian inner ear retains progenitor cells, which could commit neurogenesis.     

Effects of noradrenaline on the GABA response in rat isolated spiral ganglion neurons in culture

In the present study, the modulatory effects of noradrenaline (NA) on the GABA response were investigated in the isolated cultured spiral ganglion neurons of rat by using nystatin perforated patch recording configuration under voltage-clamp conditions. NA reversibly depressed GABA response in a concentration-dependent manner and neither changed the reversal potential of the GABA response nor affected the apparent affinity of GABA to its receptor. alpha2-adrenoceptor agonist and antagonist, clonidine and yohimbine mimicked and blocked the NA action on the GABA response, respectively. N-[2(methylamino)ethyl]-5-isoquinoline sulfonamide dihydrochloride (H-89), a protein kinase A inhibitor, mimicked the effect of NA on the GABA response. NA failed to affect the GABA response in the presence of both cAMP and protein kinase A modulator. However, NA still depressed the GABA response even in the presence of both phorbol-12-myristate-13-acetate, a protein kinase C activator and chelerythrine, a protein kinase C inhibitor. These results suggest that the NA suppression of the GABA response is mediated by alpha2-adrenoceptor which reduces intracellular cAMP formation through the inhibition of adenylyl cyclase. Therefore, NA input to the spiral ganglion neurons may modulate the auditory transmission by affecting the GABA response.