蝾螈椭圆囊毛细胞换能、编码和突触传递的形态学基础

本实验研究椭圆囊毛细胞换能、编码和突触传递的形态和显微力学基础。以幼年蝾螈为对象，光镜和电镜观察椭圆囊囊斑的微细和超微结构。实验结果：（１）只有毛细胞的高静纤毛和动纤毛的头部和耳石膜接触，耳石膜的剪切力由直接和间接两种途径传递至静纤毛；（２）耳石膜和表皮板组成纤毛束上下两端的致密板状结构，皮板下微管起固定和支撑下板作用，在两板之间的无定形物质有缓冲和利于上板滑动的功能，这种安排是纤毛受力后偏曲的基础；（３）囊斑上皮存在着动纤毛排列方向不同的四种毛细胞，感受器依靠这些分布不同的毛细胞群进行信号编码；（４）多根传入神经末梢和１～２ 根传出神经末梢与毛细胞构成传入和传出突触，存在于毛细胞底部胞液内和传出神经末梢内的囊泡是突触传递的物质基础。     

数字图像分析技术在鸡胚卵黄囊膜血管形成模型中的应用

目的应用数字图像处理技术客观、准确和快速地对鸡胚卵黄囊膜(chick embryo yolk sac membrane,YSM)血管图像进行定量分析,为促血管和抗血管生成药物的评价和筛选提供有效可靠的技术支持。方法应用OPTPRO2007图像采集系统和Image-proplus6.0图像分析软件对鸡胚卵黄囊膜血管图像背景处理和血管面积及血管密度等参数进行客观、准确地测量。结果建立了有效的鸡胚卵黄囊膜数字图像血管自动分析方法和步骤并通过药物实验对鸡胚卵黄囊膜血管图像进行了参数检测和统计分析。结论应用OPTPRO2007图像采集系统和Image-proplus6.0图像处理分析软件对鸡胚卵黄囊膜图像血管进行自动分析的方法不仅操作简便而且可较准确、快速、客观地反映鸡胚血管新生情况,优于鸡胚尿囊膜血管发生模型的常规人工目测血管计数方法。     

Quality assessment of report gene green fluorescence protein in chicken embryo in vivo electroporation

目的 在成功建立鸡胚活体原位电转基因技术的基础上,探讨报告基因绿色荧光蛋白(GFP)的表达及其在鸡胚发育过程对胚胎形态结构影响,分析α-平滑肌肌动蛋白(α-SMA)和神经丝蛋白(NF)的表达情况.方法 活体原位电转基因技术将pCAGGS-GFP质粒转入带壳培养第3天和第3天去壳培养至第5天的鸡胚,在电转基因24h后荧光体视显微镜观察,选择对照组和阳性表达胚胎,每组各5个胚胎,冷冻切片后进行荧光免疫组织化学检测α-SMA和NF分别在脊髓和视顶部位的表达状况.结果 在脊髓和视顶不同发育阶段和转染的不同时间,实验组、对照组及GFP阳性表达区域和正常区域内α-SMA、NF两种蛋白表达不存在差异,胚胎形态结构也不存在差异.结论 原位电转GFP在鸡胚发育早期过程中不影响正常基因的表达及鸡胚胎发育形态结构的变化,可以作为报告基因.     

体外培养脑神经细胞的计算机测量

利用IBM PC/AT兼容机及PIP 1024 B图像板,开发了测量体外培养脑神经细胞的系统软件,该系统可以在6～7分钟内完成一个高倍视野的细胞测量,可自动连续测量24～40个视野。每个细胞的胞体面积、直径、周长,突起平均数及突起总长度,以平均值和标准差表示。可对组间结果进行比较及t值测定。应用该系统我们测定了鸡胚大脑半球神经细胞在低密度无血清培养条件下,1、2、3、5天各项指标的变化。     

神经网络对卵巢粘液性囊腺肿瘤细胞识别的研究

目的：探讨人工神经网络在识别卵巢粘液性囊腺肿瘤细胞的价值。方法：使用MATALB软件中的神经网络工具箱（Neural Network Toolbox）,分别设计两种神经网络并训练。网络以五个形态参数（细胞核面积、周长、最大直径、等效圆直径、似圆度）为输入,输出为正常、良性、交界和恶性四种形态类型。结果：训练好的神经网络可以准确的对卵巢肿瘤细胞形态分类。结论：人工神经网络对卵巢粘液性囊腺肿瘤的病理学鉴别有很好的应用前景;选择适合的网络,不仅看网络自身,还要看训练样本。     

乳腺癌细胞核形态定量分析与临床病理特征的关系

目的：观察乳腺癌细胞核形态参数,探讨其与ER、PR、HER-2表达和临床病理特征的关系。方法收集388例乳腺癌标本,根据ER、PR和HER-2三种抗体的免疫组化标记结果,将乳腺癌分为管腔A( Luminal A)型、管腔B( Luminal B)型、HER-2过表达型和基底细胞样( Basal-like)型,各组行HE染色后通过图像分析软件测量细胞核参数,应用统计学分析各组间的差异,并通过电话或住院病例随访。结果各组乳腺癌细胞核等圆直径、面积及边缘周长差异有统计学意义( P均<0.05);ER+/PR+病例细胞核形态定量与ER-/PR-病例比较,差异有统计学意义( P<0.05);ER-/PR-病例组织学分级多为Ⅲ级,生存率低于ER+/PR+病例(P<0.05)。 Luminal A型乳腺癌的无病生存期高于Basal-like型(P<0.05),总生存期高于HER-2过表达型(P<0.05)和Basal-like型(P<0.05)。结论乳腺癌细胞核形态定量差异有显著性,对其分子分型有一定的参考价值。 ER、PR和HER-2免疫组化标记结合细胞核形态学测量结果对乳腺癌的治疗和预后的评估有重要意义。     

大鼠心肌细胞形态学增龄变化的定量研究

目的　探讨大鼠心肌细胞的增龄变化规律。　方法　取雄性大鼠 30只 ,分为幼年组 (出生后 2 0～ 2 5d)、青年组 (3～ 5月 )和老年组 (13～ 15月 )。常规石蜡切片 ,磷钨酸苏木精染色 ,图像分析仪测量心肌细胞的胞面积、核面积、核浆比和核椭圆度。　结果　随着年龄增长 :1 心肌细胞面积、核面积增大 ,核浆比降低 ,核椭圆度增高。 2 心室的细胞面积大于心房 ,左心房、左心室的细胞面积大于右心房、右心室 ,室间隔的细胞面积介于左右心室之间。 3 心肌细胞增粗增长 ,闰盘逐渐清晰、复杂。　结论　细胞核的增长速度相对慢于细胞的增长速度 ;细胞核的形态由圆形逐渐变为椭圆形 ;圆形细胞核是细胞幼稚的重要特征之一 ;心房较心室更具幼稚性。     

鸡胚心室小梁发育与肌性室间隔的形成

用显微解剖术和扫描电镜方法观察第２０－２９期鸡胚心室小梁的发育及肌性室间隔的形成过程。心脏外部观，原始心室左右部无明显的囊袋样扩张。在原始心室内部，小梁完全形成后即呈出出左右部分的形态差异。小梁在原始心室中线偏右处开始聚集，随后渐融合在一起，形成肌性室间隔。本研究表明，心室小染不仅是一种有规律排列的结构，而且与肌性室间隔的形成有关。     

背侧抑制性轴突导向蛋白对鸡胚脊髓背侧中间神经元发育的影响

目的:探讨在鸡胚脊髓发育早期背侧抑制性轴突导向蛋白(draxin)对脊髓背侧中间神经元(dorsal in-terneurons,dI)迁移特性的影响。方法:应用免疫组织化学的方法观察鸡胚脊髓dI的发育特性;应用电穿孔的方法在鸡胚一侧脊髓内过表达draxin,观察draxin过表达后对鸡胚脊髓内dI神经元发育特性的影响。结果:随着胚胎的发育,鸡胚脊髓内dI3中间神经元首先在脊髓背侧区形成并逐渐向腹侧迁移,而dI2、dI4和dI6中间神经元没有形成明显的腹侧迁移特性;鸡胚脊髓内分别过表达分泌型和跨膜型draxin时,dI3中间神经元的腹侧迁移延迟,且在跨膜型draxin过表达时其受影响程度较高;而dI2、dI4和dI6中间神经元的迁移未受到明显影响。结论:Draxin参与鸡胚脊髓内dI3中间神经元腹侧迁移的调节。     

爆震和白噪声损伤后雏鸡耳蜗毛细胞再生及增殖细胞核抗原表达的研究

本实验分别用爆震和白噪声损伤雏鸡耳蜗，在光镜、扫描和透射电镜下，结合听性脑干反应测试，观察了雏鸡耳蜗毛细胞损伤和恢复的超微结构及听阈改变；又用抗增殖细胞核抗原单克隆抗体免疫组织化学技术观察了雏鸡耳蜗基底乳头的细胞增殖。结果发现：爆震白噪声暴露后雏鸡耳蜗毛细胞听毛散乱、倒伏，胞体增大，胞浆外溢，线粒体肿胀，听性脑干反应阈值升高，爆震组较噪声组损伤严重．到白噪声暴露后第5d、爆震后第7d，支持细胞顶部增宽，长出新生的毛细胞，部分透明细胞长出听毛，分化为新生毛细胞。噪声组14d、爆震组21d，雏鸡听性脑干反应阈值恢复正常，耳蜗基底乳头的形态也接近正常；部分支持细胞和透明细胞可见抗增殖细胞核抗原免疫组织化学染色阳性．本实验结果表明：爆震和白噪声损伤后雏鸡耳蜗毛细胞可以再生，并伴有形态及听功能恢复，部分支持细胞和透明细胞分裂增殖，可能分化为新生的毛细胞。     

Atoh1 null mice show directed afferent fiber growth to undifferentiated ear sensory epithelia followed by incomplete fiber retention.

Inner ear hair cells have been suggested as attractors for growing afferent fibers, possibly through the release of the neurotrophin brain-derived neurotrophic factor (BDNF). Atoh1 null mice never fully differentiate hair cells and supporting cells and, therefore, may show aberrations in the growth and/or retention of their innervation. We investigated the distribution of cells positive for Atoh1- or Bdnf-mediated beta-galactosidase expression in Atoh1 null and Atoh1 heterozygotic mice and correlated the distribution of these cells with their innervation. Embryonic day (E) 18.5 Atoh1 null and heterozygotic littermates show Atoh1- and BDNF-beta-galactosidase-positive cells in comparable distributions in the canal cristae and the cochlea apex. Atoh1-beta-galactosidase-positive but only occasional Bdnf-beta-galactosidase-positive cells are found in the utricle, saccule, and cochlea base of Atoh1 null mutant mice. Absence of Bdnf-beta-galactosidase expression in the utricle and saccule of Atoh1 null mice is first noted at E12.5, a time when Atoh1-beta-galactosidase expression is also first detected in these epithelia. These data suggest that expression of Bdnf is dependent on ATOH1 protein in some but does not require ATOH1 protein in other inner ear cells. Overall, the undifferentiated Atoh1- and Bdnf-beta-galactosidase-positive cells show a distribution reminiscent of that in the six sensory epithelia in control mice, suggesting that ear patterning processes can form discrete patches of Atoh1 and Bdnf expression in the absence of ATOH1 protein. The almost normal growth of afferent and efferent fibers in younger embryos suggests that neither fully differentiated hair cells nor BDNF are necessary for the initial targeted growth of fibers. E18.5 Atoh1 null mice have many afferent fibers to the apex of the cochlea, the anterior and the posterior crista, all areas with numerous Bdnf-beta-galactosidase-positive cells. Few fibers remain to the saccule, utricle, and the base of the cochlea, all areas with few or no Bdnf-beta-galactosidase-positive cells. Thus, retention of fibers is possible with BDNF, even in the absence of differentiated hair cells.     

Vestibular defects in head-tilt mice result from mutations in Nox3, encoding an NADPH oxidase

The vestibular system of the inner ear is responsible for the perception of motion and gravity. Key elements of this organ are otoconia, tiny biomineral particles in the utricle and the saccule. In response to gravity or linear acceleration, otoconia deflect the stereocilia of the hair cells, thus transducing kinetic movements into sensorineural action potentials. Here, we present an allelic series of mutations at the otoconia-deficient head tilt (het) locus, affecting the gene for NADPH oxidase 3 (Nox3). This series of mutations identifies for the first time a protein with a clear enzymatic function as indispensable for otoconia morphogenesis.     

Vestibular function of saccule in cats as indicated by the response of Deiters' nucleus to static tilts

Summary In ketamine anesthetized cats, the contralateral labyrinth, the ipsilateral utricle, the ipsilateral horizontal and anterior semicircular canal nerves were completely destroyed, leaving the ipsilateral saccule intact. Neurons in the Deiters' nucleus, driven by electrical stimulation of the superior saccule, were responsive to static tilts in the antero-posterior direction. Results suggest that the saccule is capable of conveying information on head positional changes in the antero-posterior direction and that such information is retained at the level of the Deiters' nucleus.     

Spatial Expression of Otolith Matrix Protein‐1 and Otolin‐1 in Normally and Kinetotically Swimming Fish

Kinetosis (motion sickness) has been repeatedly shown to affect some fish of a given clutch following the transition from 1g to microgravity or from hypergravity to 1g. This susceptibility to kinetosis may be correlated with irregular inner ear otolith growth. Otoliths are mainly composed of calcium carbonate and matrix proteins, which play an important role in the process of otolith mineralization. Here, we examine the morphology of otoliths and the expression pattern of the major otolith proteins OMP‐1 and otolin‐1 in a series of hypergravity experiments. In the utricle, OMP‐1 is present in centripetal (medial) and centrifugal (lateral) regions of the meshwork area. In the saccule, OMP‐1 was expressed within a dorsal and a ventral narrow band of the meshwork area opposite to the periphery of the sulcus acusticus. In normal animals, the spatial expression pattern of OMP‐1 reaches more posteriorly in the centrifugal aspect and is considerably broader in the centripetal portion of the utricle compared to kinetotic animals. However, otolin‐1 was not expressed in the utricule. In the saccule, no differences were observed for either gene when comparing normal and kinetotically behaving fish. The difference in the utricular OMP‐1 expression pattern between normally and kinetotically swimming fish indicates a different otolith morphology and thus a different geometry of the otoliths resting on the corresponding sensory maculae. As the utricle is the endorgan responsible for sensing gravity, the aberrant morphology of the utricular otoliths, based on OMP‐1 expression, likely leads to the observed kinetotic behavior. Anat Rec, 298:1765–1773, 2015. © 2015 Wiley Periodicals, Inc.     

The delayed rectifier,Ikh, is the major conductance in type i vestibular hair cells across vestibular end organs

Hair cells were dissociated from the semicircular canal, utricle, lagena and saccule of white king pigeons. Type I hair cells were identified morphologically based on the ratios of neck width to cuticular plate width (NPR < 0.72) as well as neck width to cell body width (NBR < 0.64). The perforated patch variant of the whole-cell recording technique was used to measure electrical properties from type I hair cells. In voltage-clamp, the membrane properties of all identified type I cells were dominated by a predominantly outward potassium current, previously characterized in semicircular canal as I _KI. Zero-current potential, activation, deactivation, slope conductance, pharmacologic and steady-state properties of the complex currents were not statistically different between type I hair cells of different vestibular end organs. The voltage dependence causes a significant proportion of this conductance to be active about the cell’s zero-current potential. The first report of the whole-cell activation kinetics of the conductance is presented, showing a voltage dependence that could be best fit by an equation for a single exponential. Results presented here are the first data from pigeon dissociated type I hair cells from utricle, saccule and lagena suggesting that the basolateral conductances of a morphologically identified population of type I hair cells are conserved between functionally different vestibular end organs; the major conductance being a delayed rectifier characterized previously in semicircular canal hair cells as I _KI. Received: 19 July 1995/Received after revision: 19 October 1995 Accepted: 25 October 1995     

Central projections of portions of the vestibular ganglia innervating specific parts of the labyrinth in the rhesus monkey

In 40 monkeys attempts were made to determine the central projections of cell groups in the superior and inferior vestibular ganglia (SVG, IVG) which innervate distinctive parts of the labyrinth, by: (1) severing peripheral branches of the ganglia, (2) resecting portions of the ganglia, and (3) producing discrete partial lesions in the ganglia. Serial sections of the temporal bones were stained with Sudan black B, and Nauta-stained sections of the brain stem were cut transversely and horizontally. Section of peripheral branches of the vestibular ganglia produced no central degeneration and unreliable cell changes in the ganglia. Partial lesions of the SVG, innervating the cristae of the anterior and lateral canals, produced maximal degeneration in the superior and medial (rostral part) vestibular nuclei. Partial lesions of the SVG, innervating the macula of the utricle, produced maximal degeneration in the medial (caudal part) and inferior (mediodorsal part) vestibular nuclei. Some descending fibers related to the utricle entered the accessory cuneate nucleus. Discrete lesions in portions of the IVG indicate relationships between: (1) the crista of the posterior canal and the superior vestibular nucleus (caudomedial part), and (2) the saccule and dorsolateral parts of the inferior vestibular nucleus. Cells of the vestibular ganglia innervating distinctive parts of the labyrinth have unique, as well as common, central projections within the vestibular nuclear complex.