P物质、脑啡肽在豚鼠耳蜗的分布——免疫组织化学观察

本研究应用免疫组织化学方法系统地观察了P物质(SP)、亮氨酸脑啡肽(L-ENK)在豚鼠耳蜗的分布以及SP、L-ENK免疫反应阳性神经纤维与Corti's器毛细胞之间的关系,结果表明:SP的免疫反应活性(SP-IR)存在于耳蜗螺旋神经节的部分神经细胞及传入神经纤维中,在Corti's器的毛细胞下方亦可见SP免疫反应阳性纤维;L-ENK的免疫反应活性(ENK-IR)存在于耳蜗的传出神经纤维中。节内螺旋束、内螺旋束、隧道螺旋束、横贯纤维均含有大量的L-ENK免疫反应阳性纤维,Cort's器中的L-ENK免疫反应阳性终末与毛细胞之间具有密切接触,由此提示,SP可能为听觉初级传入神经递质之一;L-ENK作为传出神经递质或调质对听觉传入起调控作用。     

传出神经递质ACh及耳蜗活性物质ATP对耳蜗外毛细胞的作用

本文综述了耳蜗传出神经对耳蜗外毛细胞的调控作用。从解决和组织化学等形态学角度分析了传出神经纤维在耳蜗的分布特点，并从形态和生理两方面进一步证实了ＡＣＨ是耳蜗传出神经递质之一。近年来，应用离体耳蜗毛细胞的膜片钳和荧光测钙技术，人们对于ＡＣＨ，ＡＴＰ对外毛细胞的调控作用，Ｃａ＾２＋在其中的介导作用及其ＡＣＨＲ和Ｐ２Ｒ的药理分型有了更多的认识，但尚未有统一明确的结论。     

过氧化氢对老年豚鼠耳蜗外毛细胞大电导钙激活钾通道电流的影响

本文旨在研究氧自由基(oxygen free radical)供体——过氧化氢(H2O2)对老年豚鼠耳蜗外毛细胞大电导钙激活钾通道(large-conductance Ca2+-activated potassium channels,BKCa channels)电流的影响,探讨氧自由基对老年豚鼠耳蜗外毛细胞BKCa通道电流的作用机制。采用急性酶分离方法分离耳蜗外毛细胞,用全细胞膜片钳记录通道电流,鉴别并分析通道特性,观察不同浓度H2O2对BKCa通道电流的影响。结果显示,在膜片钳全细胞模式下,可记录到一串幅值较大、快速激活、几乎不失活的电流,激活电压大于-40~-30 mV,电流随膜电位的增加而增强,电流幅值不断增大,并表现出外向整流的特性,无"rundown"现象;IbTX(100 nmol/L)可完全阻断通道活动,证实该电流为BKCa通道电流。BKCa通道电流表现出明显的H2O2浓度依赖性激活,电流幅值和峰值电流密度随H2O2浓度(1、2、4μmol/L)增加而增大。以上结果提示,外毛细胞可能存在能够调节胞内钙平衡的氧自由基/BKCa途径。     

川芎嗪抗链霉素耳毒性作用及其对豚鼠耳蜗外毛细胞钾通道的影响

本文旨在研究川芎嗪（tetramethylpyrazine,TMP）拮抗链霉素耳毒性作用及其对豚鼠耳蜗外毛细胞K^＋通道的影响,探讨TMP拈抗链霉素耳毒性的离子通道机制。60只豚鼠随机分为6组,应用听觉脑干反应（auditory brainstem response,ABR）技术检测豚鼠ABR听阈,观测TMP的抗链霉素耳毒作用;并采用全细胞膜片钳技术观察TMP对耳蜗外毛细胞Ca^2＋敏感艮电流的影响。结果显示,TMP明显降低链霉素导致的豚鼠ABR听阈升高,提示TMP具有抗链霉素耳毒性作用;TMP能明显增大豚鼠耳蜗外毛细胞Ca^2＋敏感艮电流,并呈浓度依赖关系。结果提示,TMP通过增大艮通道电导而拮抗链霉素耳毒性作用。     

利诺吡啶对豚鼠耳蜗外毛细胞和Deiters细胞钾电流的抑制

为探讨KCNQ家族钾通道在耳蜗外毛细胞和Deiters细胞的功能性表达,我们观察并记录了KCNQ家族钾通道阻滞剂利诺吡啶对豚鼠耳蜗单离外毛细胞(outer hair cells,OHCs)和Deiters细胞总钾电流的影响。采用酶孵育加机械分离法分离豚鼠耳蜗单个OHCs和Deiters细胞：运用膜片钳技术,在全细胞模式下记录正常细胞外液中8个外毛细胞和5个Deiters细胞的总钾电流,并观察100μmol／L和200μmol／L利诺吡啶对外毛细胞和Deiters细胞总钾电流的影响。结果观察到,在正常细胞外液中的单离外毛细胞,可记录到四乙基二乙胺敏感的外向性钾电流和静息膜电位附近激活的内向性钾电流(the K^ current activated at negative potential,IKa)两种钾电流,而在单离Deiters细胞中只记录到外向整流性钾电流。在细胞外液中,加入100μmol／L利诺吡啶后,OHCs中的四乙基二乙胺敏感的钾电流峰电流成分被抑制,稳态电流幅值减小,且电流的失活时问常数明显延长;在细胞外液中加入100μmol／L和200μmol／L利诺吡啶后,OHCs的内向性钾电流IKa被完全抑制;而细胞外液中利诺吡啶终浓度为200μmol／L时,Deiters细胞的外向整流性钾电流幅值无明显变化。由此我们推测,KCNQ家族钾通道存在于豚鼠耳蜗外毛细胞,其介导的钾电流是四乙基二乙胺敏感的钾电流的组成部分,并构成全部的IKn,其功能是介导细胞内K^ 外流和防止细胞过度去极化;KCNQ家族钾通道不存在于豚鼠耳蜗Dciters细胞。     

豚鼠Ⅰ型前庭毛细胞胆碱能受体通道特性

本文旨在探讨豚鼠Ⅰ型前庭毛细胞上有无胆碱能受体存在,并对其相应的离子通道特性进行研究.应用全细胞膜片钳技术检测急性分离的豚鼠Ⅰ型前庭毛细胞对乙酰胆碱(acetylcholine, ACh)的反应.结果显示,7.5%(21/279)的Ⅰ型前庭毛细胞对10～1000μmol/L ACh敏感,引发明显的外向电流.该电流对ACh的反应呈浓度依赖性,半数激活浓度(EC50)为(63.78±2.31)μmol/L,但该电流为非电压依赖性.在-50mV钳制电压和正常细胞外液中,100μmol/L ACh激活一持久缓慢的外向电流,电流幅值为(170±15)pA,该电流幅值依赖于胞外钙离子浓度,可被胞外给予的钙依赖性钾通道拮抗剂TEA阻断.Ⅰ型前庭毛细胞的再次激活时间不小于1min.长时间暴露在ACh的情况下,受体离子通道不会发生自发性关闭.以上结果提示,部分豚鼠Ⅰ型前庭毛细胞上存在胆碱能受体,胞外给予ACh可激活一持久缓慢的外向电流,其胆碱能受体通道对于ACh的作用呈浓度依赖性和外钙依赖性、非电压依赖性或失敏性.本研究结果对于阐明前庭传出神经的功能及其作用机制,证实并揭示Ⅰ型前庭毛细胞上存在传出神经递质受体以及日后临床指导眩晕疾病的康复治疗具有重要的意义.     

用共聚焦显微镜观察ACh及ATP对豚鼠耳蜗外毛细胞Ca^2＋的调控

用激光扫描共聚焦显微镜研究了一般公认的耳蜗传出神经递质乙酰胆碱（ＡＣｈ）和三磷酸腺苷（ＡＴＰ）对豚鼠耳蜗外毛细胞（ＯＨＣｓ）胞内游离Ｃａ＾２＋浓度（Ｃａ＾２＋）的作用,ＯＨＣｓ用Ｃａ＾２＋敏感荧光染料Ｆｌｕｏ－３着色,胞内Ｃａ＾２＋的分布以细胞底部稍强。ＡＣｈ在ＯＨＣ底部引起Ｃａ＾２＋的缓慢上长并维持在一个较高水平。ＡＴＰ在整个ＯＨＣ引起一个急剧的Ｃａ＾２＋升高,升高幅度在ＯＨＣ顶部最大。随着ＡＴ     

豚鼠I型前庭毛细胞胆碱能受体通道特性

本文旨在探讨豚鼠I型前庭毛细胞上有无胆碱能受体存在,并对其相应的离子通道特性进行研究。应用全细胞膜片钳技术检测急性分离的豚鼠I型前庭毛细胞对乙酰胆碱（acetylcholine,ACh）的反应。结果显示,7．5％（21／279）的I型前庭毛细胞对10-1000μmol／L ACh敏感,引发明显的外向电流。该电流对ACh的反应呈浓度依赖性,半数激活浓度（EC50）为（63.78±2．31）μmol／L,但该电流为非电压依赖性。在-50mV钳制电压和正常细胞外液中,100μmol／L ACh激活-持久缓慢的外向电流,电流幅值为（170±15）pA,该电流幅值依赖于胞外钙离子浓度,可被胞外给予的钙依赖性钾通道拮抗剂TEA阻断。I型前庭毛细胞的再次激活时间不小于1min。长时间暴露在ACh的情况下,受体离子通道不会发生自发性关闭。以上结果提示,部分豚鼠I型前庭毛细胞上存在胆碱能受体,胞外给予ACh可激活-持久缓慢的外向电流,其胆碱能受体通道对于ACh的作用呈浓度依赖性和外钙依赖性、非电压依赖性或失敏性。本研究结果对于阐明前庭传出神经的功能及其作用机制,证实并揭示I型前庭毛细胞上存在传出神经递质受体以及日后临床指导眩晕疾病的康复治疗具有重要的意义。     

大黄素对豚鼠结肠带细胞内腺嘌呤核苷酸的影响

大黄素 (Ｅｍｏｄｉｎ)是中药大黄 (Ｒｈｕｂａｒｂ)有效成分蒽醌类衍生物之一。我们已报道 ,大黄素通过促进豚鼠结肠带平滑肌细胞膜电位去极化 ,增强细胞的电兴奋性和收缩活动 ;大黄素作用受ＫＡＴＰ通道特异激动剂ｃｒｏｍａｋａｌｉｍ的抑制 ,提示其作用与抑制ＫＡＴＰ通道活性相关。本文进一步研究了大黄素对豚鼠结肠带细胞内ＡＴＰ、ＡＤＰ和ＡＭＰ的影响。1　材料与方法(1)标本制作和实验条件　健康豚鼠 (310± 2 5 )ｇ雌雄不限。制备分离的豚鼠结肠带标本 ,长 10ｍｍ ,固定于浴槽中 ,自由收缩的一端与肌张力传感器相连。用改良的Ｋｒ…     

酸性磷酸酶在豚鼠耳蜗中的显示

采用组织化学方法,与耳蜗膜迷路铺片技术和耳蜗冰冻切片技术相结合。对豚鼠耳蜗进行了观察。正常耳蜗中的酸性磷酸酶主要集中分布在内、外毛细胞的表皮板下,血管纹细胞和Boettcher氏细胞。并对酸性磷酸酶与溶酶体的关系以及毛细胞富含酸性磷酸酶的生理学意义进行了讨论。     

On the origin of the Hirudinea and the demise of the Oligochaeta

The phylogenetic relationships of the Clitellata were investigated with a data set of published and new complete 18S rRNA gene sequences of 51 species representing 41 families. Sequences were aligned on the basis of a secondary structure model and analysed with maximum parsimony and maximum likelihood. In contrast to the latter method, parsimony did not recover the monophyly of Clitellata. However, a close scrutiny of the data suggested a spurious attraction between some polychaetes and clitellates. As a rule, molecular trees are closely aligned with morphology-based phylogenies. Acanthobdellida and Euhirudinea were reconciled in their traditional Hirudinea clade and were included in the Oligochaeta with the Branchiobdellida via the Lumbriculidae as a possible link between the two assemblages. While the 18S gene yielded a meaningful historical signal for determining relationships within clitellates, the exact position of Hirudinea and Branchiobdellida within oligochaetes remained unresolved. The lack of phylogenetic signal is interpreted as evidence for a rapid radiation of these taxa. The placement of Clitellata within the Polychaeta remained unresolved. The biological reality of polytomies within annelids is suggested and supports the hypothesis of an extremely ancient radiation of polychaetes and emergence of clitellates.     

On the ontogeny of the haptor and the evolution of the Monogenea

Data on the ontogeny of the posterior haptor of monogeneans were obtained from more than 150 publications and summarised. These data were plotted into diagrams showing evolutionary capacity levels based on the theory of a progressive evolution of marginal hooks, anchors and other attachment components of the posterior haptor in the Monogenea (Malmberg, 1986). 5 + 5 unhinged marginal hooks are assumed to be the most primitive monogenean haptoral condition. Thus the diagrams were founded on a 5 + 5 unhinged marginal hook evolutionary capacity level, and the evolutionary capacity levels of anchors and other haptoral attachement components were arranged according to haptoral ontogenetical sequences. In the final plotting diagram data on hosts, type of spermatozoa, oncomiracidial ciliation, sensilla pattern and protonephridial systems were also included. In this way a number of correlations were revealed. Thus, for example, the number of 5 + 5 marginal hooks correlates with the most primitive monogenean type of spermatozoon and with few sensillae, many ciliated cells and a simple protonephridial system in the oncomiracidium. On the basis of the reviewed data it is concluded that the ancient monogeneans with 5 + 5 unhinged marginal hooks were divided into two main lines, one retaining unhinged marginal hooks and the other evolving hinged marginal hooks. Both main lines have recent representatives at different marginal hook evolutionary capacity levels, i.e. monogeneans retaining a haptor with only marginal hooks. For the main line with hinged marginal hooks the name Articulon-choinea n. subclass is proposed. Members with 8 + 8 hinged marginal hooks only are here called Proanchorea n. superord. Monogeneans with unhinged marginal hooks only are here called Ananchorea n. superord. and three new families are erected for its recent members: Anonchohapteridae n. fam., Acolpentronidae n. fam. and Anacanthoridae n. fam. (with 7 + 7, 8 + 8 and 9 + 9 unhinged marginal hooks, respectively). Except for the families of Articulonchoinea (e.g. Acanthocotylidae, Gyrodactylidae, Tetraonchoididae) Bychowsky's (1957) division of the Monogenea into the Oligonchoinea and Polyonchoinea fits the proposed scheme, i.e. monogeneans with unhinged marginal hooks form one old group, the Oligonchoinea, which have 5 + 5 unhinged marginal hooks, and the other group form the Polyonchoinea, which (with the exception of the Hexabothriidae) has a greater number (7 + 7, 8 + 8 or 9 + 9) of unhinged marginal hooks. It is proposed that both these names, Oligonchoinea (sensu mihi) and Polyonchoinea (sensu mihi), will be retained on one side and Articulonchoinea placed on the other side, which reflects the early monogenean evolution. Except for the members of Ananchorea [Polyonchoinea], all members of the Oligonchoinea and Polyonchoinea have anchors, which imply that they are further evolved, i.e. have passed the 5 + 5 marginal hook evolutionary capacity level (Malmberg, 1986). There are two main types of anchors in the Monogenea: haptoral anchors, with anlages appearing in the haptor, and peduncular anchors, with anlages in the peduncle. There are two types of haptoral anchors: peripheral haptoral anchors, ontogenetically the oldest, and central haptoral anchors. Peduncular anchors, in turn, are ontogenetically younger than peripheral haptoral anchors. There may be two pairs of peduncular anchors: medial peduncular anchors, ontogentically the oldest, and lateral peduncular anchors. Only peduncular (not haptoral) anchors have anchor bars. Monogeneans with haptoral anchors are here called Mediohaptanchorea n. superord. and Laterohaptanchorea n. superord. or haptanchoreans. All oligonchoineans and the oldest polyonchoineans are haptanchoreans. Certain members of Calceostomatidae [Polyonchoinea] are the only monogeneans with both (peripheral) haptoral and peduncular anchors (one pair). These monogeneans are here called Mixanchorea n. superord. Polyonchoineans with peduncular anchors and unhinged marginal hooks are here called the Pedunculanchorea n. superord. The most primitive pedunculanchoreans have only one pair of peduncular anchors with an anchor bar, while the most advanced have both medial and lateral peduncular anchors; each pair having an anchor bar. Certain families of the Articulonchoinea, the Anchorea n. superord., also have peduncular anchors (parallel evolution): only one family, the Sundanonchidae n. fam., has both medial and lateral peduncular anchors, each anchor pair with an anchor bar. Evolutionary lines from different monogenean evolutionary capacity levels are discussed and a new system of classification for the Monogenea is proposed.In agreeing to publish this article, I recognise that its contents are controversial and contrary to generally accepted views on monogenean systematics and evolution. I have anticipated a reaction to the article by inviting senior workers in the field to comment upon it: their views will be reported in a future issue of this journal. EditorIn agreeing to publish this article, I recognise that its contents are controversial and contrary to generally accepted views on monogenean systematics and evolution. I have anticipated a reaction to the article by inviting senior workers in the field to comment upon it: their views will be reported in a future issue of this journal. Editor