耳蜗血管纹缘细胞培养方法的研究进展

血管纹(Stria Vascularis,SV)缘细胞(Mar-ginal cells,MCs)培养的方法包括原代分离细胞培养和组织块培养。由于MCs培养的环境受人工控制,便于以物理、化学、生物等因素为实验条件,因而在内耳生理、生化、胚胎发生等研究领域得到广泛应用,对研究内耳的生长、分化、生理功能及其影响因素有特殊的意义。本文对耳蜗SVMCs培养方法的研究进展作一综述。     

成年大鼠耳蜗血管纹缘细胞的体外培养

目的:建立成年大鼠耳蜗血管纹缘细胞(MC)的体外培养体系。方法:采用活体组织分离及培养技术,获取培养的大鼠耳蜗MC,透射电镜观察细胞超微结构,免疫组织化学法检测上皮细胞标志性中间丝角蛋白的表达及纯度,RT-PCR检测MC特征性的中间丝角蛋白mRNA的表达。结果:体外培养的MC呈不规则多角形,细胞增殖相互连接,紧密排列呈现培养上皮细胞典型的"铺路石"样外观,并可见由数百个MC密集生长组成的折光性强的球形结构(dome);透射电镜观察可见细胞周围有微绒毛样结构;免疫细胞化学检查,纯化后95%以上的MC细胞质呈桔黄色,而对照组无此着色。RT-PCR扩增出标志中间丝角蛋白基因表达的PCR产物,条带位于300~400 bp,与目的条带(368 bp)相符。结论:采用组织块培养技术成功建立成年大鼠耳蜗血管纹MC的体外培养体系,为进一步研究成熟期MC的功能提供合适的细胞模型。     

豚鼠血管纹缘细胞的原代培养

目的:建立豚鼠内耳血管纹缘细胞的体外培养系统,为研究缘细胞的功能提供良好的材料。方法:采用移植培养技术,将活体分离的血管纹组织块接种于无菌塑料培养皿中,于5％CO2 37℃恒温箱内培养,每周换液2次,观察细胞生长情况。利用免疫组织化学技术,将抗细胞角蛋白抗体和抗波形蛋白抗体用于检测培养细胞。制作透射电镜标本观察培养细胞的超微结构。结果:培养细胞成功生长4周,具有多角形细胞和梭形细胞两种不同形态,免疫组织化学反应显示角蛋白、波形蛋白染色于多角形细胞胞浆中,梭形细胞仅波形蛋白呈阳性反应。透射电镜示多角形细胞具有紧密接合和桥粒等上皮细胞的特征。结论:采用移植块培养技术,成功建立豚鼠内耳血管纹缘细胞的原代培养方法,为研究缘细胞的功能提供了合适的细胞模型。     

耳蜗血管纹边缘细胞培养方法的研究现状

耳蜗内淋巴是一种特殊的细胞外液，具有高K^＋、高渗、高正电位的特点，它在听觉传导过程中发挥着重要的作用。研究证实，血管纹（stria vascularis，SV）边缘细胞（marginal cells，MCs）是内淋巴形成的必要条件，对内淋巴正电位的维持也是不可缺少的。但是，由于SV组织取材的困难性以及其结构的复杂性，边缘细胞的生理和病理生理功能仍未完全阐明。细胞培养是克服上述限制的有效方法，因而在耳蜗组织的生理、生化等研究领域得到一些学者的尝试应用。本文对SV边缘细胞培养的研究现状作一综述。     

新生大鼠耳蜗血管纹缘细胞中ATP存在的证据

目的:研究体外培养的新生大鼠耳蜗血管纹缘细胞中存在ATP的证据,即细胞中是否存在ATP囊泡,体外培养液中能否检测到所释放的ATP。方法:采用出生1～3 d的Sprague-Dawley大鼠,进行体外血管纹缘细胞培养、纯化、鉴定。特异性标记ATP囊泡的喹丫因染色后在荧光显微镜下观察缘细胞中的ATP囊泡。采用生物发光法检测缘细胞细胞外液中所释放的ATP的浓度。结果:体外培养的新生大鼠耳蜗血管纹缘细胞,经流式细胞法检测上皮细胞标志性的角蛋白和波形蛋白的纯度,证实培养所获得的细胞为缘细胞。经喹丫因染色后在荧光显微镜下可见缘细胞细胞质中存在大量的绿色星点状染色。采用生物发光法检测缘细胞细胞外液中ATP的浓度,通过细胞荧光值可计算出ATP的浓度。结论:新生大鼠耳蜗血管纹缘细胞中存在ATP囊泡,并能分泌ATP。     

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.     

重组2型腺相关病毒介导锰超氧化物歧化酶转染大鼠耳蜗血管纹缘细胞的研究

目的探讨以重组腺相关病毒（Recombinant adeno—associated virus of the serotypes2，rAAV2）为载体对体外培养的大鼠耳蜗血管纹缘细胞（Marginal cells，MCs）转染锰超氧化物歧化酶（Manganese Superoxide Dismutase，MnSOD），获得高表达MnSOD转基因缘细胞的可行性。方法实验组按感染复数（MOI）为10^4v.g.／cell对体外培养的血管纹缘细胞转染rAAV2-MnSOD—EGFP ,同时设转染rAAV2－EGFP缘细胞作为空载对照组，未转染细胞为空白对照组。荧光显微镜下每2d观察1次MCs的绿色荧光蛋白（Enhenced green fluorescent protein，EGFP）表达情况。比色法测定各组MCs的MnSOD活性。激光共聚焦显微镜下观察转染rAAV2－MnSOD—EGFP后MnSOD的表达，免疫印迹法（Western blot）定量分析MnSOD的表达。结果（1）各组MCs转染后，生长正常，空载对照组MCs转染rAAV2－EGFP后2天开始出现微弱EGFP的表达，1周后至高峰：实验组转染rAAV2－MnSOD—EGFP后4d出现EGFP的表达，10d至高峰，且持续表达于细胞培养的整个期间。EGFP的表达在荧光显微镜490nm波长激发光下呈黄绿色．弥漫于整个胞质。（2）对激光共聚焦显微镜及Western blot的检测结果进行分析，实验组较空载对照组和空白对照组的MCs中MnSOD的表达明显升高，差异有统计学意义。结论rAAV2－MnSOD—EGFP能有效地转染体外培养的MCs并持续高表达MnSOD。     

速尿对离体培养豚鼠血管纹毒性作用的观察

目的：观察速尿对离体培养的豚鼠血管纹组织的影响，探讨速尿耳毒性的作用机制。方法：20只花色豚鼠随机分成二组：速尿组（n=16），正常对照组（n=4）。应用组织块培养的方法，将血管纹组织培养24小时，随即对不同的实验组分别应用不同终浓度的的速尿（60、300、600、1250、2500μg/ml），分别继续培养30分钟和90分钟，观察培养的血管纹组织学结构。结果：速尿组在组织学方面均未出现血管纹水肿、缘细胞胞浆肿胀、细胞间隙扩大和中间细胞皱缩等病理改变，与对照组相比较血管纹结构无显著性差异。结论：速尿对体外培养的豚鼠血管纹组织无明显诱导水肿的作用，提示速尿耳毒性的产生，可能是间接的作用机制。     

大鼠耳蜗血管纹边缘细胞原代培养及心钠素的表达

目的探讨心钠素（atrial natriuretic peptide,ANP）在原代培养大鼠耳蜗血管纹边缘细胞（marginal cell,MC）中是否表达。方法应用细胞培养技术原代培养MC,并进行鉴定、纯化,应用免疫细胞化学和逆转录-聚合酶链反应（RT-PCR）检测原代培养的MC中ANP及其mRNA的表达情况。结果培养细胞经免疫细胞化学检测其CK-18蛋白表达阳性,证明其为上皮来源的MC。免疫细胞化学检测在原代培养的边缘细胞胞体内发现ANP免疫反应阳性颗粒,RT-PCR方法扩增出编码ANP的目的条带。结论本实验表明原代培养的边缘细胞具有合成和分泌ANP的能力,提示边缘细胞在维持内耳微环境稳定方面具有调节作用。     

Primary culture of vital marginal cells from cochlear explants of the stria vascularis

Summary Explants of the stria vascularis and spiral ligament were dissected from guinea pig cochleae and were successfully cultivated for several weeks. After 2 days, fibroblast-like cells of the spiral ligament covered the bottom of the cell culture dish around the explant. Marginal cells of the stria vascularis proliferated and grew on the luminal surface towards the border of the explant at a rate of 15 m/day. At day 6 in culture the proliferating marginal cells reached the border of the explant and then advanced to the bottom of the cell-culture dish. There the marginal cells replaced fibroblast-like cells and built an epithelial hexagonal-shaped monolayer. Light microscopic and transmission electron microscopic investigations revealed that the cultured cells were viable and that typical morphological characteristics of marginal cells were preserved. Cultivation of these cells provides a unique model for studies of physiological properties of marginal cells of the stria vascularis.     

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.     

Changes in the stria vascularis of the guinea pig due to cis-platinum

Summary The microscopical and ultramicroscopical changes in the stria vascularis due to cis-diamminedichloroplatinum II (DDP) were studied. Sixteen healthy adult guinea pigs were used for the experiment. A standard dosage DDP (1.5 mg/kg/d) was administered over a period of 5–20 days. A clear degeneration pattern was found (ranging from no changes to cystic degeneration with protrusion of marginal cells followed by loss of marginal cells). DDP seems to be especially toxic for marginal cells in stria vascularis in the guinea pig.Supported by grants from the Heinsius Houbolt Foundation     

Respiratory quotient of stria vascularis of guinea pig in vitro

Summary The respiratory quotient of the stria vascularis was measured in vitro by means of Cartesian diver microgasometry. A value of about 1.2 was found when the incubation medium was phosphate-buffered serum substitute with glucose as the sole substrate. This value suggests that endogenous lipids and amino acids do not contribute significantly to strial respiratory metabolism and that carbohydrate is the primary fuel in vitro. A high activity of the hexose monophosphate pathway may be responsible for raising the respiratory quotient above unity.Supported by the grant NS 06575 from the National Institutes of Health and the grant 77-16842 from the National Science Foundation     

The resting potential of marginal cells in stria vascularis explants

Summary In order to examine the intracellular potential of stria vascularis marginal cells (MCs) under direct visual control, a short-term organ culture of guinea pig stria vascularis (SV) was developed. The experimental conditions allowed exposure of the luminal surface of the SV to artificial endolymph or perilymph. Using conventional microelectrodes and inverted bright-field microscopy, impalement of MCs from the endolymphatic side through the luminal cell membrane was achieved. With artificial perilymph at the luminal side a small positivity at the cell surface and low negative intracellular potentials were recorded at room temperature (23°C). The initial recording was –6.5 ± 5.0 mV while the stable recording was –4.0 ± 3.6 mV. Similar results were obtained at a bath temperature of 37°C. Furthermore, subtotal exposure of the luminal cell surface to artificial endolymph did not result in a significant potential change.     

Ultrastructure of guinea pig stria vascularis processed by rapid freezing and freeze substitution

The rapid-freezing and freeze-substitution method fixes a specimen as if it were prepared before excision. We used this method to study the stria vascularis of guinea pigs using electron microscopy. Findings were essentially the same as those obtained with conventional chemical fixation, although freeze substitution made it possible to observe the membrane structures in a smoother and more linear manner. This method seems to be the procedure of choice for studying the instantaneous movement and behavior of cells.     

Cell membrane alterations in the stria vascularis of the guinea pig after ethacrynic acid treatment studied by freeze-fracture

Summary A freeze-fracture examination of the stria vascularis during the first 2 h after injection of ethacrynic acid was performed. This showed a re-distribution of the particles on the membrane fracture faces of both marginal and intermediate cells. As oedematous spaces developed, particle-poor, vesicle-like structures were found associated with both cell types. The tight junctions at the apices of the marginal cells and around basal cells were unaffected.This work was supported by the Wellcome Trust in the form of a Research Fellowship