大鼠耳蜗中三磷酸腺苷表达及来源的初步研究

目的 研究大鼠耳蜗中三磷酸腺苷(adenosine triphosphate,ATP)的表达部位、来源及其可能的存在形式.方法 运用喹丫因染色技术,观察培养的大鼠耳蜗血管纹缘细胞、全膜迷路铺片和新鲜分离的单离外毛细胞;用免疫组化荧光染色法,观察突触素(synaptophysin,SYN)和小泡相关膜蛋白-2(vesicle-associated membrane protein-2/synaptobrevin,VAMP-2)在大鼠耳蜗中的表达.结果 培养的缘细胞胞浆中存在星点状喹丫因染色,全膜迷路铺片血管纹以外的区域和单离毛细胞中均未发现喹丫因的特异性染色.SYN和VAMP-2在大鼠耳蜗的内螺旋束、外螺旋束、Deiters细胞内侧缘和螺旋神经元(spiral ganglion neurons,SGNs)有共同表达.结论 大鼠耳蜗缘细胞胞浆中存在大量囊泡状的ATP,而毛细胞、支持细胞中的ATP可能以非囊泡的形式储存.内螺旋束、外螺旋束和SGNs中有可能存在SYN染色的ATP囊泡.     

新生大鼠耳蜗K(o)lliker器支持细胞ATP释放的机制

目的 观察体外培养的新生大鼠耳蜗K(o)lliker器支持细胞是否存在并释放ATP,初步探讨其释放机制.方法 选取出生后ld的Sprague-Dawley大鼠,分离耳蜗膜迷路,采用机械分离与酶消化相结合的方法获得单离的K(o)lliker器支持细胞.观察膜迷路和K(o)lliker器支持细胞的喹丫因染色情况.采用生物发光法,通过影响K(o)lliker器支持细胞ATP代谢、改变细胞内外Ca2浓度、抑制细胞内磷脂酶信号通路及添加缝隙连接半通道阻断剂,观察K(o)lliker器支持细胞释放ATP浓度的变化.结果 用喹丫因染色体外培养的K(o)lliker器支持细胞,发现胞质中存在大量绿色星点状染色.采用生物发光法检测的ATP标准曲线呈明显的对数线性关系.随着巴佛洛霉素A1浓度增加,K(o)lliker 器支持细胞培养液中ATP浓度逐渐降低,而随着己二酸二癸酯浓度的增加,培养液中ATP浓度逐渐升高;在一定浓度范围内,随着细胞外Ca2浓度增加,K(o)lliker器支持细胞ATP的释放减少,而随着细胞内游离Ca2浓度增加,K(o)lliker器支持细胞释放ATP量增加;培养液中加入甘珀酸钠或乌热酸抑制半通道后可以显著的降低ATP释放.此外,抑制细胞内磷脂酶信号通路也可以减少ATP的释放.结论 体外培养的新生大鼠耳蜗K(o)lliker器支持细胞存在并释放ATP,细胞内、外液中Ca2+浓度的变化可能通过调节半通道的开放而影响其ATP的释放.     

新生大鼠耳蜗血管纹缘细胞释放ATP的机制

目的 证实体外培养的新生大鼠耳蜗血管纹缘细胞能够释放ATP,并进一步探讨缘细胞释放ATP的机制.方法 分离、培养新生大鼠耳蜗血管纹缘细胞,采用生物发光法分别检测巴佛洛霉素A1、己二酸二癸酯( didecyl adipate,DDA)、细胞外K+、毒胡萝卜素、细胞外Ca2、U73122及马兜铃酸钠对细胞外液中缘细胞ATP释放的影响.结果 随着巴佛洛霉素A1浓度的增加,细胞外液中ATP的浓度明显下降;当DDA浓度增加时,细胞外液中ATP的浓度几乎呈线性增加.随着细胞外液中的K+浓度的增高,缘细胞释放的ATP浓度呈现上升趋势,当细胞外液中的K+浓度为9.15 mmol/L时,ATP的释放量达到峰值,之后随着K+浓度的继续升高ATP的释放量呈下降趋势.随着毒胡萝卜素浓度的增加,缘细胞释放的ATP浓度呈现明显下降的趋势.当细胞外Ca2+浓度为0 mmol/L时,缘细胞仍然释放ATP;Ca2+浓度增加与ATP的释放呈负相关,但当细胞外的Ca2+浓度达到1.25 mmol/L以上时,ATP的释放量维持在一个较稳定的水平.U73122的浓度在0.25～1.25 μmol/L时,其与缘细胞释放的ATP浓度呈负相关.当马兜铃酸钠的浓度为12.5 ～ 100.0 μmol/L,缘细胞ATP释放呈明显下降的趋势;当其浓度＞100.0 μmol/L时,ATP释放浓度趋于平稳.结论 体外培养的新生大鼠耳蜗血管纹缘细胞能够释放ATP,其释放量与钙泵、K+通道状态以及细胞内信号传导通路相关酶的活性有关.     

三磷酸腺苷在新生大鼠耳蜗血管纹缘细胞中的释放机制

目的探究新生大鼠耳蜗血管纹缘细胞中ATP释放的机制。方法原代培养新生SD大鼠血管纹缘细胞,利用钙离子荧光探针、生物发光法测定ATP浓度和β-氨基己糖苷酶释放率测定法来研究ATP和GPN与P2YR-PLC-IP3通路诱导的内质网钙库反应之间的关系,以及内质网钙库与细胞外ATP浓度和β-氨基己糖苷酶释放率的关系。结果 ATP和GPN可通过P2YR-PLC-IP3通路诱发内质网钙库释放,可被P2YR-PLC-IP3通路拮抗剂抑制。缘细胞外ATP浓度和β-氨基己糖苷酶释放率与细胞内钙离子浓度呈正相关。结论缘细胞外ATP作用于P2Y嘌呤能信号系统触发内质网钙库释放,胞内钙离子诱导溶酶体胞吐作用释放ATP至胞外,从而发挥其生物学作用。     

耳蜗血管纹缘细胞三磷酸腺苷释放机制

三磷酸腺苷（adenosine triphosphate,ATP）被认为是耳蜗中一种重要的信号分子,可以作为共同递质和（或）神经调节物质发挥耳蜗的各种生理功能。目前已证实新生大鼠耳蜗血管纹缘细胞内的ATP囊泡为溶酶体,可以释放ATP。然而,缘细胞释放ATP的机制还未完全阐明,本文结合国内外文献就耳蜗缘细胞中ATP释放机制的研究现状做一综述。     

耳蜗缘细胞中三磷酸腺苷囊泡的研究现状

耳蜗外侧壁包括螺旋韧带和血管纹。国内外研究发现耳蜗血管纹缘细胞中存在大量的各种形状的囊泡结构，这些囊泡由单层或双层膜包被，并常含有绒毛状电子致密物，此囊泡可能与离子的转运、三磷酸腺苷（adenosine triphosphate，ATP）的释放密切相关。有人证实囊泡中的ATP不是来源干线粒体。有学者观察到了囊泡明显的胞吐活动，但是不能确定ATP是否通过胞吐作用分泌至内淋巴。缘细胞中的ATP囊泡是否就是溶酶体，还有待更深入的研究证实。现结合国内外文献，就耳蜗缘细胞中ATP囊泡的研究现状做一综述。     

大鼠耳蜗血管纹缘细胞氧化性损伤体外模型的建立

目的 建立大鼠耳蜗血管纹缘细胞氧化性损伤的体外模型.方法 在体外培养的大鼠耳蜗血管纹缘细胞中加入过氧化氢(H2O2),观察细胞形态结构变化;采用CCK-8(cell counting kit-8)法检测200、300、400、600、800μmoL/LH2O2作用0.5、1、2、4、16、24 h对血管纹缘细胞活性的影响;检测不同浓度H2O2作用2 h后血管纹缘细胞脂质过氧化产物丙二醛含量的变化;利用碘化丙锭染色流式细胞仪测定细胞的凋亡率;通过免疫印迹法(Western blot)检测凋亡因子半胱氨酸天冬氨酸蛋白酶3(cmpase-3)活化片段cleaved-caspase-3的表达.结果 H2O2作用后血管纹缘细胞出现核固缩、边缘化,胞质浓缩,被膜包裹、隆起,产生凋亡;随着H2O2浓度的增加、作用时间的延长,缘细胞活性降低;200 μmol/L的H2O2作用2 h,即可诱导缘细胞凋亡率升高,差异具有统计学意义(P<0.05);cleaved-caspase-3在正常缘细胞呈微弱表达,H2O2作用后cleaved-caspase-3表达增强,并随H2O2浓度的增高而增强,但当H2O2达到600 μmol/L时,表达开始减弱,800 μmol/L时仅见微弱表达.结论 利用H2O2可成功建立耳蜗血管纹缘细胞氧化性损伤的体外模型,caspase-3的激活参与了缘细胞的氧化损伤过程.     

pEGFP-N1/MnSOD重组质粒的构建及体外表达

目的:构建大鼠源性锰超氧化物歧化酶(MnSOD)基因真核表达载体pEGFP-N1/MnSOD,并检测其在原代培养的大鼠耳蜗血管纹边缘细胞中的表达。方法:从大鼠心肌组织中扩增出SOD基因,克隆入融合表达载体pEGFP-N1中,用脂质体法转染大鼠耳蜗血管纹边缘细胞,激光共聚焦显微镜观察绿色荧光蛋白及目的基因的表达,流式细胞仪检测转染效率。结果:构建了大鼠源性MnSOD基因真核表达载体pEGFP-N1/MnSOD,并观察到转染/感染48h后,绿色荧光蛋白及目的基因在大鼠耳蜗血管纹边缘细胞中表达,目的基因的转染效率为23.47%。结论:成功构建了大鼠源性MnSOD基因真核表达载体pEGFP-N1/MnSOD,并使目的基因成功在大鼠耳蜗血管纹边缘细胞中表达,为内耳抗氧化基因治疗奠定了基础。     

新生小鼠膜迷路耳蜗外侧壁的组织培养

目的培养小鼠耳蜗螺旋韧带/血管纹来源的细胞,为体外研究提供细胞模型。方法显微解剖新生小鼠耳蜗螺旋韧带/血管纹组织,组织块外植培养,胰蛋白酶消化分离细胞,免疫组织化学染色,原位透射电镜观察鉴别细胞的来源。结果自外植耳蜗螺旋韧带/血管纹组织生长出上皮样细胞及成纤维样细胞。前者呈典型的上皮细胞形态,表达细胞角蛋白,并具血管纹边缘细胞的超微结构特征。后者呈成纤维细胞形态,表达波形蛋白,具有耳蜗螺旋韧带成纤维细胞的超微结构特征。结论培养出小鼠耳蜗血管纹边缘细胞及螺旋韧带成纤维细胞来源的原代上皮细胞及传代成纤维细胞,为体外研究提供了细胞模型及方法。     

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。     

Quinacrine staining of marginal cells in the stria vascularis of the guinea-pig cochlea: a possible source of extracellular ATP?

There is accumulating evidence for a purinergic humoral system involved in the control of cochlear function. Evidence of specific P₂ purinoceptors on cochlear tissues implies a role for extracellular adenosine triphosphate (ATP) in the cochlea. To further this hypothesis a study was undertaken to determine if there was any specific source of purine compounds in cochlear tissues. Cochlear tissues (the sensory epithelium and lateral wall) from the guinea pig were incubated with the acridine derivative quinacrine dihydrochloride (5×10⁻⁶ M in phosphate-buffered saline for 30 min at room temperature) which fluoresces on binding to high concentrations of ATP. Most cochlear tissues showed a diffuse green fluorescence slightly above the background level. However, a region of the marginal cells of the stria vascularis showed a specific punctate fluorescence. Optical sectioning of these cells by confocal microscopy revealed that the fluorescent structures in these marginal cells was confined to a region up to 10 μm from their endolymphatic surface. Similar cells studied by transmission electron microscopy showed membrane-bound vesicles located in the same region of the cell. These data imply that purine compounds are localized in discrete structures, perhaps vesicles, within the marginal cells which could serve as a source of extracellular ATP in the cochlea.     

Endocytosis of microperoxidase in the marginal cells of stria vascularis

OBJECTIVE: Endocytosis has been thought to control entry into the cell and play a crucial role in the development, immune response, neurotransmission, intercellular communication, signal transduction, and cellular and organismal homeostasis. We investigated the basic properties of endocytosis in the marginal cells of stria vascularis (SV) to discuss whether marginal cells have a potential to maintain the endolymph homeostasis. METHODS: We perfused microperoxidase (MPO), an endocytosis tracer, into the cochlear duct. After 5-60 min of endolymphatic perfusion, the tissues were fixed and the distribution of MPO within the marginal cell was observed by transmission electron microscopy. RESULTS: Endocytosis started already at 5 min after MPO perfusion. Small MPO-loaded endosomes were observed up to 30 min after MPO perfusion. The small tubulovesicular endosomes and the plasma membrane invagination were not decorated by an electron dense bristle structure. After endocytosis, MPO labeled preendosomes were quickly transported to the large vacuolar endosomes that connected with tubular endosomes. At 60 min after MPO perfusion, MPO-loaded large vesicles that have small vesicles in the lumen were observed. CONCLUSION: The time-course of MPO-loaded endosomes was similar to that of CF-loaded endosomes in the marginal cells of SV. The strial marginal cells have vigorous endocytotic activity both in clathrin-independent and clathrin-dependent endocytosis. This high activity of endocytosis in SV seems to be needed to maintain the homeostasis of endolymph via membranous channels and/or receptors regulations.     

Carbohydrates in the guinea pig stria vascularis demonstrated with lectin-gold techniques.

Soybean agglutinin (SBA), Helix pomatia agglutinin (HPA), Ricinus communis agglutinin-II (RCA-II), Limax flavus agglutinin (LFA) and wheat germ agglutinin (WGA) were employed to determine the localization of specific carbohydrates on thin sections of lowicryl K4M embedded guinea pig striae vasculares using the lectin-gold and glycoprotein-gold techniques. SBA, HPA and RCA-II gold labeling was observed in many of the cytoplasmic vesicles, endosomes and apical tubules located in the supranuclear region as well as on the microvilli and micropinocytotic invaginations of the luminal plasma membrane of the marginal cells. LFA labeling was found on the basal plasma membrane of the marginal cells as well as in the basement membrane of the perivascular spaces. WGA binding sites were detected along the plasma membrane of all types of cells constituting the stria vascularis. Our present results revealed that the membranes of internalization and many of the cytoplasmic vesicles, endosomes and apical tubules in the supranuclear region of the marginal cells are associated together and it is suggested that these structures may be related to the regulation of endolymph.     

Migration of cochlear lateral wall cells

The role of apoptosis and proliferation in maintenance of cochlear lateral wall cells was examined. The methods employed for detection of apoptosis were the Hoechst fluorescence stain and TUNEL (TdT-mediated dUTP-biotin nick-end-labeling) assay, and proliferations were 5-bromo-2'-deoxyuridine (BrdU) incorporation and presence of the proliferating cell nuclear antigen. The incidence of apoptosis in the strial marginal cell was 50% greater (32.9+/-3.7%) than strial intermediate and basal cells but similar to spiral ligament cells. Although division of marginal strial cells was rarely detected, a significant number of proliferating cells in the remaining stria vascularis and spiral ligament were observed. These data implied that replacement of marginal cells arose elsewhere and could be followed by a BrdU-deoxythymidine pulse-chase study. At 2 h post injection, nuclear BrdU in marginal cells was not detected; however, by 24 h post injection, 20-25% of marginal cell nuclei were BrdU-positive. These observations are consistent with the hypothesis that marginal cells were replaced by underlying cells. Cell migration appears to be an important mechanism for preserving the function and structure of the stria vascularis.     

内耳免疫反应中细胞凋亡的研究

目的探讨内耳免疫反应过程是否引起细胞凋亡以及Fas和FasL、Bcl-2和Bax的表达情况。方法选用雌性白色豚鼠16只，随机分为实验组和对照组各8只，以钥孔蛾血蓝蛋白全身免疫后，实验组以相同抗原进行内耳免疫，对照组内耳注射等量的磷酸盐缓冲生理盐水，在内耳免疫7d后处死动物，取内耳免疫侧耳蜗做石蜡切片。通过电镜和脱氧核糖核苷酸末端转移酶介导的缺口末端标记技术(terminal-deoxynucleotidyl transferase mediated nick end labeling，TUNEL)检测内耳凋亡细胞，免疫组化检测内耳Fas和FasL以及Bcl-2和Bax的表达。结果透射电镜观察发现实验组术后7d内耳外毛细胞、血管纹细胞及螺旋神经节细胞都出现了凋亡细胞的特征性改变，而对照组未发现具有上述特征的细胞。实验组内耳Corti器毛细胞，血管纹的缘细胞和螺旋神经节细胞存在TUNEL染色阳性细胞，TUNEL染色阳性细胞具有凋亡细胞的典型形态学特征，对照组内耳的任何结构中都没发现TUNEL染色阳性细胞。免疫组化染色实验组Corti器、螺旋神经节细胞、血管纹和螺旋韧带Fas和FasL蛋白表达阳性，而对照组只有螺旋神经节细胞和血管纹有较弱的Fas蛋白表达，FasL蛋白表达阴性。实验组Corti器、螺旋神经节细胞、侧壁Bcl-2蛋白表达阴性，对照组的Corti器、侧壁和螺旋神经节细胞Bcl-2蛋白表达阳性。实验组Corti器、侧壁和螺旋神经节细胞Bax蛋白表达阳性，对照组只有螺旋神经节细胞Bax蛋白表达弱阳性，Corti器、侧壁表达阴性。结论内耳免疫反应可诱导细胞凋亡发生，Fas-FasL是此过程的信号转导途径之一，Bcl-2和Bax蛋白在其中起了重要调节作用。     

鼠尾胶原在原代培养耳蜗血管纹边缘细胞中的应用

目的：建立利用自制的鼠尾胶原培养大鼠耳蜗边缘细胞的方法。方法：制作鼠尾胶原,并观察利用自制的鼠尾胶原所培养出大鼠耳蜗边缘细胞的效果。结果：自制的鼠尾胶原能正常地培养出大鼠耳蜗边缘细胞,细胞角蛋白18表达阳性,免疫组织化学结果和扫描电镜证实所培养的细胞具有典型的分泌上皮细胞特征。结论：成功建立了利用自制鼠尾胶原培养大鼠耳蜗边缘细胞的方法,并且制作简便,成本低廉。     

Ultrastructure of the stria vascularis and Reissner's membrane in experimental hydrops

A time-sequence study was made of the early ultrastructural changes of the stria vascularis and Reissner's membrane in the guinea pig after obliteration of the endolymphatic sac and duct. Pathological alterations of both the stria vascularis and Reissner's membrane were found to start in the apex of the cochlea. The morphological changes of the stria vascularis were characterized by an increase of vesicles in the marginal cells and by intercellular edema, followed by vacuolization and atrophy of marginal and intermediate cells. In Reissner's membrane extensive gaps in the mesothelial cell layer were observed together with intracellular pathology of the epithelial cells. The significance of these ultrastructural changes in the stria vascularis and Reissner's membrane with regard to the pathophysiology of the endolymphatic hydrops is discussed.     

Freeze-etch visualizations of some elements in the stria vascularis

A quick-freeze, deep-etch study was made to observe the three-dimensional aspect of both membranes and organelles in the stria vascularis of the guinea pig. Our replicas showed various membrane characteristics of the vesicles, e.g., a clathrin basket on the inner surface membrane, aggregation of membrane particles on the protoplasmic fracture face, and regularly arranged surface protuberances on the outer surface membrane. In the cell processes of marginal cells, there were not only bundles of microtubules but also another type of fibril. Many gap junctions were observed between marginal, intermediate, and basal cells. In some replicas, the inner surfaces of the gap junctions were covered with particle protrusions with some ordered arrangements. The functional roles of these elements in the stria vascularis are discussed.     

An ultrastructural study of the neuromuscular junctions of the cat intrinsic laryngeal muscles. II. Synapse formation by autonomic nerves

The purpose of this study is to identify the origin of the nerve terminals of unknown origin observed at the previously denervated neuromuscular junctions in the cat intrinsic laryngeal muscles. The results were as follows: 1. Until 3 weeks after the transection of the recurrent laryngeal nerve, no nerve terminals were found at the neuromuscular junctions of the intrinsic laryngeal muscles except for the cricothyroid muscle, and no nerve fibres were detected in the Schwann tubes formed by Schwann cells and perineural cells. In addition, autonomic nerves around the vessels in the muscles were markedly decreased. 2. At 6 weeks, accompanied by an increase of autonomic nerves around the vessels, nerve fibres and nerve varicosities containing a number of large granular vesicles were observed in the Schwann tubes. 3. From 9 to 30 weeks, nerve terminals containing large granular vesicles were found at the neuromuscular junctions in all cases, even though the superior laryngeal nerve or the vagal nerve was transected on the ipsilateral side. 4. A spontaneous discharge was recognized in 6/8 cases after 6 weeks, but an evoked electromyogram could not be recognized. 5. The synaptic vesicles of the nerve terminals were labelled by 5-hydroxydopamine (5-OHDA), which was used as a marker for the sympathetic nerve. From these results, it was indicated that if the transected recurrent laryngeal nerve was prevented from regenerating, the autonomic nerves around the vessels entered into the Schwann tubes and reached the denervated neuromuscular junctions, instead of the motor nerve. The effect of autonomic nerves on muscle fibres was discussed.