小肠黏膜下层无细胞基质作为阴道平滑肌细胞载体的可行性

摘要 背景：目前国内外用于阴道组织工程研究的主要支架材料聚乙醇酸存在降解过快等缺陷。天然脱细胞支架材料尤其是小肠黏膜下层逐渐成为组织工程研究的重点。 目的：探索用猪小肠黏膜下层基质作为组织工程学阴道细胞载体的可行性。 方法：取新西兰雌兔,分离出阴道平滑肌组织块,组织块+酶消化法原代培养阴道平滑肌细胞。体外培养传代后作为种子细胞接种于自制猪小肠黏膜下层基质体外联合培养,倒置显微镜动态观察细胞形态及生长增殖情况,分别于1,2,3,4周时取标本,行组织学检查。 结果与结论：①体外成功培养出阴道平滑肌细胞,倒置显微镜下,见培养的阴道平滑肌细胞呈现长梭状,细胞集结于培养皿上形成典型的“峰和谷”样构型。②黏膜下层无细胞基质外观呈白色,半透明,有一定韧性。苏木精-伊红染色未见细胞成分存在。③阴道平滑肌细胞-肠黏膜下层标本切片苏木精-伊红染色后,光镜下可见细胞成分逐渐增多,由表浅向深层部位生长。④阴道平滑肌细胞-肠黏膜下层标本切片采用抗兔平滑肌α-肌动蛋白单克隆抗体免疫组化染色后,均可见抗兔α-Actin的阳性细胞。结果初步证明了猪小肠黏膜下层基质可作为一种平滑肌细胞载体。 关键词：阴道平滑肌细胞;组织工程;猪小肠黏膜下层;细胞载体;支架材料 doi:10.3969/j.issn.1673-8225.2010.47.001     

脱细胞猪小肠黏膜下层支架复合软骨细胞构建组织工程软骨*☆

背景：关节软骨损伤后无论是否施加干预,都难以达到满意的修复效果。 目的：观察以猪自体软骨细胞为种子细胞复合脱细胞猪小肠黏膜下层构建组织工程软骨的可行性。 方法：将培养至第3代的猪膝关节软骨细胞接种于小肠黏膜下层膜上,复合培养48 h,构建细胞-载体复合物,光学显微镜、扫描电镜观察软骨细胞在小肠黏膜下层膜上的生长情况。 结果与结论：苏木精-伊红染色见细胞在小肠黏膜下层基质层表面呈单层或复层生长;免疫组织化学染色结果显示软骨细胞与小肠黏膜下层表面之间形成一条连续阳性表达条带;扫描电镜见软骨细胞在支架孔隙内贴壁良好生长。     

幼年豚鼠视网膜谷氨酸毒性损伤与碱性成纤维细胞生长因子的干预

兴奋性氨基酸引起的兴奋毒性是造成缺血性眼科疾患视力下降的主要因素，其主要病理改变是视网膜神经细胞尤其是神经节细胞（RGCs）的丢失。我们选用45~50d幼年豚鼠为实验对象，采用腹腔内注射谷氨酸钠的方式造模，应用HE染色方法观察结果显示，按3g/kg的剂量，连续给予谷氨酸钠7d，再存活10d时，神经节细胞数量明显减少，视网膜损伤适中，可作为损伤模型组。另外，实验采用幼年豚鼠谷氨酸中等毒性损伤模型（3g/kg/d，i.p., 连续7d），给予预防性腹腔注射bFGF（800U/kg/d）。免疫组化检测证实，谷氨酸损伤幼年豚鼠视网膜节细胞层抗凋亡基因bcl-2较少表达，视网膜节细胞层和内网状层促凋亡基因Caspase-3表达增强，生长抑素（SOM）的阳性产物主要分布于视网膜节细胞层(GCL)和内核层(INL)，但该部位前突触标志物突触小泡蛋白则表达较弱。腹腔注射bFGF后，上述改变被逆转。说明bFGF可通过调节幼年豚鼠视网膜SYP 、SOM和凋亡相关基因的表达，发挥对过量谷氨酸钠引起的幼年豚鼠视网膜损伤的保护作用。     

兔骨髓基质细胞的体外培养及鉴定

背景：骨髓基质细胞的分离纯化、细胞培养、细胞标记、诱导因子、基因转染对细胞生物学影响、细胞载体构建及细胞复合物回植时间的选择等尚处于实验研究阶段。 目的：摸索一种较为简便且可有效获得骨髓基质细胞的体外培养方法。 设计、时间及地点：细胞学体外实验，于2006-06/2007-01在中国科学院北京基因组研究所完成。 材料：SPF级6周龄新西兰大白兔8只，由中国科学院遗传发育研究所实验动物中心提供。 方法：无菌条件下抽取兔骨髓1 mL，D-Hanks液稀释后，离心弃上清，DMEM培养基重悬，制备单细胞悬液，叠加在密度为1.077的等体积淋巴细胞分离液的液面上，离心后取界面云雾状的单个核细胞层，用含有20%胎牛血清的DMEM培养基重悬。以1×10/cm2接种后贴壁法纯化细胞，待70%~80%长满单层后消化传代。 主要观察指标：倒置显微镜下观察细胞生长情况，锥虫蓝染色计数活细胞，苏木精-伊红染色对细胞进行鉴定，MTT法检测细胞活性，观察传代培养的细胞冻存后复苏情况。 结果：接种24 h后细胞开始贴壁，呈短梭形或三角形，且有长短不等的细胞突起；3 d后贴壁细胞开始分裂增殖，部分区域形成细胞团簇；1周后大部分细胞呈成纤维状散落在培养瓶底生长；传代后细胞均匀分布，形态呈细长梭形，并沿一定方向排列，1 mL骨髓基质细胞悬液传3代后的贴壁细胞数可达5×106~1×107数量级。骨髓基质细胞成活率约为90%，经鉴定为单核细胞，接种后1~6 d细胞持续旺盛生长，至第8天细胞活性最高，随后生长活性下降。复苏冻存56 d的细胞，其生长良好，增长迅速。 结论：以淋巴细胞分离液梯度离心后可获得纯度较高的骨髓基质单核细胞，经体外扩增培养能够得到足够数量的种子细胞，冻存复苏后细胞活性无变化。     

碱性成纤维细胞生长因子对许旺细胞在小肠黏膜下层支架上黏附及增殖的影响*☆◆

摘要 背景：许旺细胞复合小肠黏膜下层是构建人工神经的可行方法,碱性成纤维细胞生长因子有促进许旺细胞增殖的作用。 目的：验证碱性成纤维细胞生长因子对许旺细胞在小肠黏膜下层支架材料表面的细胞增殖及黏附状态的影响。 方法：将体外分离培养的2代SD乳鼠许旺细胞接种于小肠黏膜下层支架材料上并加入50 μg/L的碱性成纤维细胞生长因子复合培养为实验组,以单纯的许旺细胞复合小肠黏膜下层作为对照组,分别用MTT法测定细胞增殖能力,细胞黏附率检测细胞在支架上的黏附情况,用流式细胞仪测定细胞分裂周期,并用苏木精-伊红染色及描电镜观察细胞形态及与材料的贴附情况。 结果与结论：MTT显示实验组的细胞增殖吸光度值明显高于对照组(P < 0.05),实验组的细胞黏附率为(69.47±3.17)%,对照组为(44.58±1.76)%(P < 0. 05),细胞周期显示实验组比对照组的G2/M+S期细胞百分含量高(P < 0.05),培养7 d后的苏木精-伊红染色及扫描电镜显示实验组比对照组材料上聚集的细胞数量多,细胞形态伸展更明显,细胞贴附力更好。因此碱性成纤维细胞生长因子可明显地改善许旺细胞在小肠黏膜下层上的增殖及黏附能力,能促进许旺细胞与小肠黏膜下层支架的复合而构建人工神经导管。 关键词：碱性成纤维细胞生长因子;许旺细胞;小肠黏膜下层;增殖;黏附 doi:10.3969/j.issn.1673-8225.2010.21.007     

胰蛋白酶及Ⅱ型胶原酶消化获取关节软骨细胞*

背景：近年来，众多研究欲采用体外分离培养的关节软骨细胞作为修复缺损的关节软骨的种子细胞，然而，获得纯化的具有生物活性的关节软骨细胞较为困难。 目的：拟运用胰蛋白酶与Ⅱ型胶原酶联合消化获取关节软骨细胞。 方法：从SD大鼠的正常股骨及胫骨关节表面获取关节软骨，先后运用0.25%的胰蛋白酶和0.2%Ⅱ型胶原酶消化，显微镜下见大量细胞游离后，弃去大块的未消化的关节软骨碎片，离心，去上清，PBS洗涤2次后，加入软骨细胞原代培养液进行培养、增殖。应用甲苯胺蓝及苏木精-伊红染色方法检验所得细胞是否为关节软骨细胞。 结果与结论：在严格掌握酶的浓度及消化时间的前提下，通过0.25%胰蛋白酶和0.2%Ⅱ型胶原酶联合酶解关节软骨的方法，成功从大鼠股骨及胫骨关节软骨内分离培养出细胞，并经过甲苯胺蓝染色及苏木精-伊红染色证实，所得的细胞为具有生物活性的关节软骨细胞。 关键词：关节软骨细胞；Ⅱ型胶原酶；胰蛋白酶；联合消化；甲苯胺蓝；苏木精-伊红染色     

体外诱导的人脂肪间充质干细胞源性软骨细胞体内成骨活性☆

目的：目前对间充质干细胞向软骨细胞诱导分化主要包括体内诱导和体外诱导两种方式，前者依靠体内微环境，随机性大且难以进行监控和调整。本实验探讨体外诱导人脂肪间充质干细胞向软骨细胞分化，以及诱导后细胞在裸鼠体内的成软骨能力。 方法：实验于2006-08/2007-05在中南大学湘雅医院中心实验室完成。①对象与材料：皮下脂肪组织来源于股骨颈骨折手术患者6例，年龄25~64岁，对实验及治疗均签署知情同意书，实验经医院医学伦理委员会批准。清洁级裸鼠5只，体内成软骨实验过程中对动物的处置符合动物伦理学标准。离心管容量20 mL，由GeneRay公司生产。②实验方法：将皮下脂肪组织剪碎，I型胶原酶消化法分离培养脂肪间充质干细胞，待细胞铺满瓶底80%时胰酶消化传代。传至第6代时收集 5×106个细胞，用含1%新生牛血清、高糖DMEM、10 μg/L转化生长因子β1、37.5 mg/L维生素C、6.25 mg/L胰岛素、6.25 mg/L转铁蛋白、10-7 mol/L地塞米松的软骨细胞诱导剂重悬于无支架离心管内。③实验评估：诱导过程中观察细胞聚集状态。诱导2周后采用苏木精-伊红染色、RT-PCR、Western blot对细胞形态及功能变化进行检测。将诱导后细胞与纤维蛋白原凝胶混合，植入裸鼠皮下，3周后切取植入组织块，苏木精-伊红染色及II型胶原免疫组化染色观察体内成软骨情况。 结果：①诱导后细胞聚集状态：诱导2 d后脂肪间充质干细胞自行聚集为一条状细胞团，1周后细胞团聚集呈球状。②苏木精-伊红染色结果：未见有软骨陷窝形成。③RT-PCR检测结果：细胞团内Ⅱ型胶原蛋白和Sox9 mRNA均呈阳性表达。④Western blot检测结果：细胞团内有较强的Ⅱ型胶原蛋白和Aggrecan表达。⑤体内成软骨情况：植入裸鼠皮下的组织块内有大量软骨陷窝及II型胶原蛋白形成。 结论：①在无支架离心管内，脂肪间充质干细胞经软骨诱导剂作用后具有典型的软骨细胞表型，但未形成成熟软骨组织所具备的软骨陷窝。②诱导后细胞与凝胶复合种植于裸鼠体内，可形成具有典型软骨特征的组织。     

镁铝合金最大剂量的致敏试验*☆

摘要 背景：镁合金作为潜在的新型医用可降解生物金属材料受到越来越多的关注,作为植入物需与人体具有良好的生物相容性。 目的：评价镁铝合金(AZ31B)的致敏性。 方法：白化豚鼠35只,随机分为生理盐水阴性对照组10只和体积分数为5%甲醛阳性对照组10只,镁铝合金浸提液组15只。根据《GB-T 16886.10-2005 医疗器械生物学评价 第10部分刺激与迟发型超敏反应试验》最大剂量致敏试验步骤进行皮内诱导、局部诱导和激发。激发阶段去除贴附物后6,24,48,72 h的豚鼠皮肤反应按Magnusson和Kligman等级进行分级。激发阶段去除贴附物后72 h后对皮肤进行活检,行苏木精-伊红染色和光镜下观察。 结果与结论：生理盐水阴性对照组和镁铝合金浸提液组激发阶段去除贴附物后24,48,72 h皮肤无致敏反应,而甲醛阳性对照组在这任一时间点均有中度以上红斑。活检皮肤光镜下镁铝合金浸提液组未见皮肤水肿,皮肤棘细胞层水肿,血管周围、弥漫的真皮和表皮单核细胞浸润,见散在少量的嗜碱性细胞。结果提示镁铝合金浸提液在致敏方面具有生物安全性。 关键词：镁铝合金;致敏;豚鼠;生物相容性;可降解材料     

羊膜脱细胞基质的制备及其生物相容性

目的：羊膜是一种天然的细胞外基质，具有抗原性低、排异反应小特点。实验拟进行羊膜脱细胞基质的制备，观察其作为组织工程支架材料的生物相容性。 方法：实验于2007-03/08在河北医科大学解剖教研室实验中心完成。实验材料：4～6周龄健康清洁级SD大鼠,体质量100～ 150 g，由河北医科大学实验动物中心提供。实验过程中对动物处置符合动物伦理学标准。新鲜人羊膜取自石家庄市第四医院，采取前征得产妇知情同意，实验经医院伦理委员会批准。实验方法：①取新鲜人羊膜冲洗后以1%tritonX-100溶液振荡24 h，0.25%胰蛋白酶37 ℃振荡4 h，充分漂洗，冷冻干燥，环氧乙烷消毒，并进行苏木精-伊红染色和扫描电镜检测。②体外分离培养SD大鼠骨髓间充质干细胞，并复合培养于羊膜脱细胞基质上，以不含细胞的培养基作为空白对照，以普通培养基培养作为阴性对照，倒置显微镜下观察生长情况，并进行苏木精-伊红检测。四甲基偶氮唑盐法测定羊膜脱细胞基质浸提掖对骨髓间充质干细胞毒性；将羊膜脱细胞基质植入SD大鼠背部皮下，检测其组织相容性。 结果：①制备的羊膜脱细胞基质为白色菲薄、半透明的膜状物，柔韧性好，经苏木精-伊红和扫描电镜检测无细胞残留。苏木精-伊红染色可见羊膜表面细胞黏附生长良好，细胞伸展，形态与正常培养细胞无差异。②实验组第2、4、6、8天的吸光度值低于对照组，相对增殖率随培养时间延长而增加，平均相对增殖率为89.5%，细胞毒性评分均为1级。③皮下埋置术后动物全部成活，伤口无红肿、渗液等炎症反应，移植物均未见坏死、纤维化等。光镜下，术后1周移植物周围可见以淋巴细胞为主的炎症细胞浸润，羊膜卷尚完整；术后2周，淋巴细胞减少，偶见新生毛细血管；术后4周，部分移植物已经降解，基本未见淋巴细胞。 结论：经去污剂和酶消化法制备的羊膜脱细胞基质生物相容性良好，是一种理想的组织工程支架材料。     

新生C57小鼠耳蜗螺旋神经节细胞的原代培养*****☆

背景：目前在原代细胞培养领域内,对耳蜗螺旋神经节细胞培养条件的报道各有差异,个别方法重复性较差,不利于实际应用。 目的：原代培养并鉴定新生C57小鼠螺旋神经节细胞。 方法：显微解剖分离新生C57小鼠蜗轴组织,经胰酶消化+差速贴壁+化学药物相结合方法培养;倒置相差显微镜及苏木精-伊红染色观察细胞生长状态,免疫组织化学染色鉴别细胞来源。 结果与结论：蜗轴组织细胞纯化后,胞体呈椭圆形或三角形,有细长的突起,Nuen染色胞核呈棕黄色阳性反应,β3-Tubulin染色细胞胞浆与轴突均呈棕黄色阳性反应。提示实验成功培养出小鼠螺旋神经节细胞。     

Morphological and physiological development of olfactory receptor cells in rainbow trout (Salmo gairdneri) embryos

The morphological and functional differentiation of the olfactory receptor cells were investigated in developing rainbow trout (Salmo gairdneri) embryos by means of light and electron (transmission and scanning) microscopy and electrophysiology. Ciliated receptor cells first appeared when the olfactory placode was folded to form a groovelike structure rostrad to the eye at stage 24 (day 18; 18 days postfertilization). Ciliated receptor cells predominated until immature microvillar receptor cells developed in stage 28 (day 26) embryos. At stage 29, the day of hatching, the anterior edge of the olfactory epithelium contained only ciliated receptor cells, and the midregion contained both ciliated and microvillar receptor cells. Spontaneous neural firing activity was recorded from the olfactory mucosa as early as stage 25. The neural responses to amino acids were initially recorded from stage 26 embryos, containing sparse ciliated receptor cells with a few short cilia. The D-enantiomers of amino acids were less effective. From these results we concluded that in rainbow trout the olfactory receptor cell has two separate morphological forms, ciliated and microvillar. These are ontogenetically distinct; the ciliated receptor cells preceded the microvillar. The ciliated receptor cells respond to amino acid stimulation.     

Projections of submucous neurons to the myenteric plexus in the guinea pig small intestine

The distribution of submucous neurons that project to the myenteric plexus of the guinea pig small intestine was established by retrograde transport of the carbocyanine dye 1,1′-didodecyl-3,3,3′,3′-tetramethyl indocarbocyanine perchlorate (DiI) from myenteric ganglia in organ culture in combination with immunohistochemistry. Following the application of DiI to the serosal surface of a single myenteric ganglion, from 2 to 15 DiI-labelled nerve cell bodies were labelled in the submucous plexus up to 7.9 mm circumferentially, 4.5 mm orally, and 3.4 mm aborally to the DiI application site. No cells were labelled in preparations in which connections between myenteric and submucous plexuses had been severed prior to DiI application. Cells that were immunoreactive for vasoactive intestinal polypeptide (VIP) or for substance P (SP) accounted for about 75% and 11% of DiI-labelled cells, respectively. Neither neuropeptide Y- nor calretinin-immunoreactive submucous neurons were labelled by DiI, indicating that these classes of neurons do not project to the myenteric plexus. Retrograde tracing from the myenteric plexus with Neurobiotin revealed that labelled VIP-immunoreactive neurons had several short, filamentous processes and a single long axon that could be followed through the circular muscle to myenteric ganglia without branches to the mucosa. The previously described projection of submucous, SP-immunoreactive putative sensory neurons to the myenteric plexus was confirmed. However, this study has identified a considerably larger population of presumed interneurons that are immunoreactive for VIP that likely transmit information from the submucous plexus to the myenteric plexus and presumably coordinate activity between the two ganglionated plexuses. J. Comp. Neurol. 399:255–265, 1998. © 1998 Wiley-Liss, Inc.     

Inflammatory demyelination induces ependymal modifications concomitant to activation of adult (SVZ) stem cell proliferation

Ependymal cells (E1/E2) and ciliated B1cells confer a unique pinwheel architecture to the ventricular surface of the subventricular zone (SVZ), and their cilia act as sensors to ventricular changes during development and aging. While several studies showed that forebrain demyelination reactivates the SVZ triggering proliferation, ectopic migration, and oligodendrogenesis for myelin repair, the potential role of ciliated cells in this process was not investigated. Using conventional and lateral wall whole mount preparation immunohistochemistry in addition to electron microscopy in a forebrain‐targeted model of experimental autoimmune encephalomyelitis (tEAE), we show an early decrease in numbers of pinwheels, B1 cells, and E2 cells. These changes were transient and simultaneous to tEAE‐induced SVZ stem cell proliferation. The early drop in B1/E2 cell numbers was followed by B1/E2 cell recovery. While E1 cell division and ependymal ribbon disruption were never observed, E1 cells showed important morphological modifications reflected by their enlargement, extended cytoskeleton, and reinforced cell–cell junction complexes overtime, possibly reflecting protective mechanisms against ventricular insults. Finally, tEAE disrupted motile cilia planar cell polarity and cilia orientation in ependymal cells. Therefore, significant ventricular modifications in ciliated cells occur early in response to tEAE suggesting a role for these cells in SVZ stem cell signalling not only during development/aging but also during inflammatory demyelination. These observations may have major implications for understanding pathophysiology of and designing therapeutic approaches for inflammatory demyelinating diseases such as MS.     

兔角膜缘上皮干细胞体外扩增及生物学鉴定

背景：角膜缘干细胞体外培养的关键在于建立稳定的体外培养体系，包括角膜缘干细胞的定位、培养条件、载体选择和鉴别方法等。 目的：探索兔角膜缘上皮干细胞体外扩增方法，并对其生物学特性进行鉴定。 方法：采用兔角膜缘组织块培养法，以人羊膜为载体，在体外进行兔角膜缘上皮干细胞原代和传代培养。倒置显微镜观察其体外生长特征；苏木精-伊红染色以及扫描电镜观察其形态；AE5和P63单克隆抗体免疫组织化学染色鉴定其蛋白表达。 结果与结论：采用组织块培养法可在体外获得角膜缘上皮干细胞，能成功传代培养且保持较高增殖潜能。培养于去上皮羊膜上的干细胞可融合成片，呈“拉网”现象。原代角膜缘上皮干细胞AE5单克隆抗体染色阳性率低于5%，P63染色阳性达90%；随传代次数增加AE5染色阳性率增高，P63染色阳性率降低。结果显示兔角膜缘组织块培养法可以在体外成功获得角膜缘上皮干细胞，原代和传代细胞均具有干细胞特性，以羊膜为载体培养可形成角膜移植片。     

The development of ependyma in the human fetal brain: an immunohistological and electron microscopic study

The stratified inner layer of the embryonic fetal brain, the ventricular zone (VZ), contains glial fibrillary acidic protein (GFAP)-positive cell bodies of radial glia. The adult cerebral ventricle is lined by a single layer of cuboidal, ciliated common ependymal cells which are, immunohistologically, GFAP negative. In late gestation, the ventricular lining is formed by tanycytes, ependymal cells with short, intensely GFAP-positive basal fibres. The development of ependyma was examined, morphologically and immunohistologically, in human fetal brain from between 11 weeks gestation to 6 months post-term to determine the relationship between the radial glia cell, tanycyte and common ependymal cell. This study was not able to show whether tanycytes were formed from radial glia or were formed from a previously uncommitted population of VZ cells. The study did show, however, that tanycytes probably mature into common ependymal cells following acquisition of cilia and loss of basal fibres. Electron microscopic data indicate that tanycytes have features suggestive of a secretory and/or transport function.     

THE SUPRAHABENULAR RECESS IN THE RAT: A QUANTITATIVE STUDY OF CILIATED CELLS, SUPRAEPENDYMAL CELLS AND SOME SPECIFIC FEATURES OF SUPRAEPENDYMAL FIBRES

Tulsi R.S. & Dreosti I.E. (1981) Neuropathology and Applied Neurobiology 7, 21–36
The suprahabenular recess in the rat: a quantitative study of ciliated cells, supraependymal cells and some specific features ofsupraependy-mal fibres
Using light microscopy, scanning and transmission electron microscopy, an extensive quantitative analysis of ciliated ependymal cells, supraependymal cells and supraependymal fibres in the suprahabenular recess of twenty-two adult Wistar rats was carried out. In addition, six animals were used to determine the origin of supraependymal fibres and their relationship to ependyma. The findings were as follows: ciliated cells–mean 3–32 ± 0–26 per 1000 μm² (range 0–10); supraependymal cells–5–91 ± 1–37 per recess (range 2–16). Average number of cilia per cell in different animals ranged from 31 to 33. Sex did not influence the density of ciliated ependyma or supraependymal cells. Evidence was obtained that some supraependymal fibres are closely associated with aberrant commissural fibres. The supraependymal fibres enter the suprahabenular recess and form gap junctions, and other types of junctions, with subjacent ependyma.     

Intraspinal bronchogenic cyst: ultrastructural study of the lining epithelium

Summary This report describes the ultrastructural characters of the lining epithelium of a symptomatic intraspinal bronchogenic cyst at the C5-T2 level of a 21-year-old female. Six distinct cell types were recognized: ciliated cells, non-ciliated cells, and goblet cells that reached the lumen, and basal cells, Kulchitsky cells and undifferentiated cells that were basally located and did not reach the lumen. The microvilli of non-ciliated cells were coated with granulofibrillary material. Discharge of granular contents from goblet cells was noted. Abnormal cilia, particularly compound cilia, were frequent. Complex interdigitations of cytoplasmic membrane with prominent desmosomes were present in the pseudostratified region. Kulchitsky cells contained characteristic membrane-bound dense-core neurosecretory granules. Intraepithelial unmyelinated axons were observed but none were closely associated with Kulchitsky cells. The types of cells forming the lining epithelium of the present cyst and their topographic distribution within the epithelium are very similar to those of the normal tracheobronchial epithelium.     

Submucous rather than myenteric neurons are activated by mucosal biopsy supernatants from irritable bowel syndrome patients

Background We previously showed that colonic mucosal biopsy supernatants from patients with irritable bowel syndrome (IBS) activate neurons of the human submucous plexus, an area with densely packed immune cells. Based on the concept that mucosa‐nerve signaling is altered in IBS, we tested in this study whether the nerve sensitizing effect of IBS mucosal biopsy supernatants is more prominent in the submucous than myenteric plexus. Methods Fast neuroimaging with the voltage‐sensitive dye Di‐8‐ANEPPS was used to record activity of guinea‐pig submucous and myenteric neurons after application of constipation (C)‐ and diarrhea (D)‐IBS supernatants (three each) and four supernatants from healthy control subjects. Results are based on recordings from 4731 neurons. Key Results Control supernatants did not evoke significant responses in submucous or myenteric neurons. In contrast, all IBS supernatants evoked a significant spike discharge (median 3.6 Hz) in 46% of submucous neurons. This activation was significantly stronger than in the myenteric plexus where even twice the amount of supernatants evoked a lower spike frequency (median 2.1 Hz) in only 8.5% of neurons. Pharmacological studies revealed serotonin, histamine, and proteases as components mediating neuronal activation. Individual application of these components revealed that only serotonin evoked a significantly stronger activation of submucous compared with myenteric neurons. Conclusions & Inferences Direct neuronal activation by IBS mucosal biopsy supernatants is primarily a feature of submucous rather than myenteric neurons. This is associated with a stronger excitation of submucous neurons by serotonin. The plexus‐specific effects support the concept that altered mucosa‐nerve signaling underlies disturbances in IBS.     

Immunocytochemical study of somatostatin,neurotensin, GABA,and glycine in rat spinal dorsal horn

The development of the olfactory organ of the zebrafish, from the forming of the early placode to the adult organ, was investigated by electron microscopy and DiI labeling. The olfactory placode is formed by a subepidermal layer of cells. These cells differ from those of the epidermis as well as from brain cells, and they do not mingle either with epidermal or with brain cells. No migration of cells from the brain or the epidermis towards the subepidermal cell layer has been observed. The cells of the subepidermal layer seem to form all cell types of the olfactory mucosa, i.e., basal cells, ciliated and microvillous receptor cells, supporting cells, and ciliated nonsensory cells. Axons grow into the forebrain at a very early stage when the epidermis still covers the placode completely. Dendrites grow out when the epidermis separates, building the olfactory pit. This process implicates neither cell lysis nor cell degeneration. The olfactory pit forms a rosette with a midline raphe and olfactory lamellae. The incurrent nostril is separated from the excurrent nostril by a funnel-shaped structure. Differentiation of the olfactory placode in the embryo is accomplished very quickly, whereas the development into the adult organ during larval stages is a slow process. © 1993 Wiley-Liss, Inc.     

人胎幽门括约肌内NOS阳性神经元发育的研究

用ＮＡＤＰＨ－ｄ组织化学法对第３个月龄至足月（１０个月胎龄）人胎幽门括约肌内ＮＯＳ阳性神经元的发育进行了观察。结果显示：第５个月胎龄时,肌间神经节处的圆形细胞中部分细胞出现较弱的ＮＯＳ阳性反应。第６个月胎龄时,该处圆形细胞ＮＯＳ阳性反应增强,并分化形成梭形ＮＯＳ阳性神经细胞,部分细胞呈条索状排列向内环肌层方向延伸,有的到达粘膜下层。第７个月胎龄时,肌间神经节细胞胞体明显增大,细胞数增加,胞质增多,