果蝇变态过程中的细胞凋亡和细胞自噬

程序化细胞死亡(programmed cell death,PCD)分为I型PCD细胞凋亡(apoptosis)和II型PCD细胞自噬(autophagy)。果蝇等完全变态昆虫有2种类型的器官:即细胞内分裂器官(如脂肪体、表皮、唾液腺、中肠、马氏管等)和有丝分裂器官(复眼、翅膀、足、神经系统等)。在昆虫变态过程中,细胞内分裂器官进行器官重建,幼虫器官大量发生细胞凋亡和细胞自噬到最后完全消亡,同时成虫器官由干细胞从新生成;而有丝分裂器官则由幼虫器官直接发育为成虫器官。在果蝇等昆虫的变态过程中,细胞凋亡和细胞自噬在幼虫器官的死亡和成虫器官的生成中发挥了非常重要的作用。文章简要介绍细胞凋亡和细胞自噬在果蝇变态过程中的生理功能和分子调控机制。     

家蚕幼虫-蛹变态期前胸腺和脂肪体细胞解离和程序性细胞死亡的比较分析

【目的】检测家蚕Bombyx mori变态期前胸腺细胞的解离、自噬与凋亡,并与脂肪体的进行对比,从而解析昆虫幼虫-蛹变态期过程中不同组织重塑的异同。【方法】以家蚕5龄期、游走期、预蛹期和蛹期前胸腺和脂肪体组织为材料,在光学显微镜下观察前胸腺和脂肪体细胞解离情况;分别利用Lyso-Tracker和TUNEL染色,在荧光共聚焦显微镜下观察细胞自噬和细胞凋亡的发生情况;利用qRT-PCR检测家蚕前胸腺中自噬发生标志基因Atg8的表达水平;利用透射电镜观察前胸腺和脂肪体细胞自噬小体和前胸腺线粒体;利用Caspase3酶活性检测试剂盒测定Caspase3酶活性;利用qRT-PCR检测前胸腺中蜕皮酮(ecdysone)合成相关基因Spo,Phm,Dib和Sad的表达水平;利用酶免疫试验(enzyme-immunoassay, EIA)测定前胸腺中蜕皮酮的含量,进而检测合成蜕皮酮的活力。【结果】在家蚕幼虫到蛹的变态发育过程中,在化蛹第1天家蚕前胸腺和脂肪体细胞中同时开始出现细胞解离;脂肪体细胞自噬和凋亡分别在游走期和预蛹第1天开始出现并逐渐增强;而前胸腺一直到化蛹第2天都没有发生明显的细胞自噬和凋亡;此外,前胸腺中线粒体的形态变化和蜕皮酮合成相关基因的转录水平均与对应时期前胸腺合成蜕皮酮的活力一致。【结论】在变态发育时家蚕不同组织消亡发生的时间不同,虽然前胸腺和脂肪体在化蛹第1天同时出现细胞解离,但是前胸腺直到化蛹第2天都不发生细胞自噬和凋亡,可能与其持续合成蜕皮酮的功能有关。本研究为昆虫幼虫-蛹变态发育时期组织消亡的深入研究提供了理论依据与工作基础。     

TGF-β1诱导马鹿茸MSCs软骨分化及c-myc基因的表达

鹿茸间充质干细胞(MSCs)是维持茸再生与骨化的重要组织,旨在研究鹿茸MSCs的软骨分化及原癌基因c-myc对该过程的调控作用。利用成年塔里木马鹿生长60 d的鹿茸第2代间充质干细胞(MSCs,P2),通过TGF-β1(10 ng/m L浓度)刺激,诱导塔里木马鹿茸间充质干细胞向软骨分化,采用免疫组化和阿利新蓝染色鉴定诱导结果,并通过q PCR方法检测软骨分化过程中c-myc基因的表达变化。结果显示,MSCs在诱导后的第9天开始出现细胞形态变化,由梭形向多角形转变,原来菊花状的分布逐渐向铺路石状变化,至14 d可观察到软骨陷窝,21 d软骨细胞基质明显,并开始出现细胞凋亡。非诱导组28 d细胞出现凋亡,细胞内发现空泡。35 d两组细胞凋亡明显,细胞折光性变差,间隙变大。阿利新蓝染色鉴定,诱导至第14天细胞基质中开始出现大量阳性染色。免疫组化实验检测,诱导至21 d的细胞基质中出现棕色Col II阳性反应物,随培养时间增加颜色加深,并集中分布在细胞及其周围基质中。在软骨分化进程中,第7、14、21和28天,诱导组c-myc基因表达与非诱导组相比显著下调(P0.05),但诱导至35 d,诱导组c-myc表达与非诱导组相比无显著差异(P0.05)。在TGF-β1刺激下,塔里木马鹿茸MSCs可以分化成软骨,原癌基因c-myc下调表达诱导鹿茸MSCs进入凋亡状态并分化为软骨细胞。     

Caspase细胞凋亡通路及其在水生无脊椎动物的研究进展

半胱天冬氨酸酶(caspase)普遍存在于真核生物,在细胞凋亡中具有重要作用,广泛参与胚胎发育、炎症反应、器官发生、变态过程及自稳态维持等多种生理过程。caspase级联反应将细胞外信号传递到细胞内,水解底物蛋白或激活转录因子发挥作用。目前,细胞凋亡通路在哺乳动物的作用机制已有大量的报道,但对水生无脊椎的研究相对较少。综述caspase细胞凋亡通路及其在水生无脊椎动物的研究进展。     

独叶草的根和节部及叶的解剖学研究

报道了独叶草根、节部和叶的剖解学特征。这些器官在解剖学上表现出的突出点是：根具２个以上的根毛区（与星叶草机相同）,中有少量的次生生长,皮层细胞中具内生真菌;变态叶的叶迹或为单迹单维管组织束,或为单迹２维管组织束,或２迹在向皮层外部延伸过程中合并为具２条维管组织束的单迹;叶柄维管束不存在厚壁的维管束鞘,且在由基部向顶部延伸的过程中常发生复杂的分枝及汇合;叶片具有同形的叶肉植物,叶脉维管束鞘具２层细胞     

东亚飞蝗(Locusta migratoria manilensis Meyen)腹听器在胚胎期内的发生

1.本文以东亚飞蝗(Locusta migratoria manilensis Meyen)为材料,仔细地研究了它的腹听器在胚胎期内的发生过程。 2.东亚飞蝗的腹听器中的剑鞘器是由胚胎的表皮细胞内陷,经过细胞分裂、分化而形成;因此,我们可以肯定剑鞘器中的感觉细胞是在胚胎某一时期从表皮起源的。 3.东亚飞蝗腹听神经节在胚胎期中发生时经过以下的步骤:(1)第6天初期的胚胎表皮细胞开始内陷;(2)第6天到第7天内陷口加深;(3)第7天和第8天,内陷的原腹听神经节呈梨状,近表皮的一端较大,细胞尚无分化;(4)第9天,原腹听神经节的细胞已开始分化为5种;横列的明区开始出现;(5)第10天,内陷口逐渐愈合;本天后期,轴丝已出现,剑鞘体隐约可见,鞘顶结尚未形成;(6)第11天,轴丝、剑鞘体和鞘顶结已全部出现,剑鞘体数目约60～70个;(7)第12～13天,各种细胞进一步分化,与第14天孵出的蝗蝻的听 神经节大致相同,剑鞘体的数目约90～100个。 4.轴丝具有强烈的嗜银性,剑鞘体则缺乏。它们虽然大致同时出现,但我们就发生形态方面的事据推断,剑鞘体是由中间细胞分泌而成,轴丝是由感觉细胞所产生。     

自组装短肽在脊髓急性创伤早期减少细胞凋亡的研究

目的 为研究在脊髓急性损伤中,自组装短肽溶液对创伤后神经组织的保护作用及创伤后早期神经组织细胞凋亡的时间分布规律.方法 实验使用了手术切断大鼠脊髓胸段T8至T10作为脊髓损伤动物模型,使用末端脱氧核苷酸转移酶介导生物素标记(TUNEL)技术,检测实验后不同时间点的损伤脊髓组织细胞凋亡情况.结果 伤后2 h,损伤区及邻近段开始出现末端标记阳性细胞;伤后8 h,凋亡阳性细胞数达高峰;同时,自组装短肽实验组细胞凋亡较空白对照组少.结论 自组装短肽溶液对急性脊髓创伤组织具有保护作用.     

中华蜜蜂工蜂嗅叶的胚后发育

通过形态解剖、免疫组织化学、原位细胞凋亡检测技术,对中华蜜蜂工蜂嗅叶的胚后发育过程进行了系统的比较研究.结果表明:(1)中华蜜蜂工蜂的嗅叶由神经纤维网区和外围的中脑神经细胞体层组成,在幼虫早期,神经纤维网的腹外侧和背外侧区内有几个大型的成神经细胞,呈现出典型的不对称分裂模式;在随后的发育过程中,神经纤维网的体积逐渐增加,神经细胞逐渐分散,细胞层变薄;分裂细胞主要集中在背外侧细胞区;在3龄幼虫期 ,触角嗅觉神经元的传入纤维开始进入嗅叶;在蛹发育的第3天,神经纤维网内的神经纤维球开始出现;(2)嗅叶发育过程中的细胞凋亡发生较少,增殖细胞和凋亡细胞数量的相对比率基本恒定;细胞凋亡的高峰期出现在幼虫末期;蛹发育的第4天左右,凋亡细胞完全消失 ;(3)中华蜜蜂的嗅叶存在雌雄异形现象[动物学报 54(3):546-554,2008].     

水飞蓟宾诱导肺腺癌Anip973 细胞凋亡的分子机制研究

目的:探讨水飞蓟宾诱导肺腺癌Anip973细胞系细胞凋亡的分子机制。方法:采用MTT法、倒置显微镜和电子显微镜等形态学检测以及流式细胞仪(FCM)技术检测、DNALadder分析、凋亡分子PARP的表达检测细胞凋亡,同时进行凋亡相关蛋白Bax、Bcl-2、caspase-3和caspase-9表达活性分析。结果:(1)水飞蓟宾对人肺腺癌Anip973细胞系细胞的增殖有显著抑制作用;(2)水飞蓟宾作用Anip973细胞48h后,随着浓度的增加,倒置显微镜下可见细胞数目减少,胞体变小、变圆,到高浓度时出现较多的死亡细胞;(3)扫描电镜观察发现,随着水飞蓟宾作用浓度的增加,Anip973细胞中出现增多的凋亡细胞,凋亡细胞表现出典型的超微结构特征;(4)流式细胞仪检测的结果发现,随着药物作用时间的延长,Anip973细胞的G1期细胞比例增多,S期细胞明显减少,G2期细胞略有减少,并出现明显的凋亡峰。(5)水飞蓟宾作用后的Anip973细胞出现明显的DNALadder和PARP降解增加等凋亡特征;(6)水飞蓟宾作用后,Anip973细胞中的凋亡相关蛋白Bax表达增加、caspase-3和caspase-9酶活性增加,而Bcl-2表达降低。结论:水飞蓟宾在体外有抑制人肺腺癌细胞Anip973的增殖作用,并通过激活线粒体依赖的caspase凋亡通路,诱导其凋亡。     

蜕皮激素对家蚕脂肪体中BmATG8蛋白亚细胞定位与细胞核皱缩的调控

【目的】昆虫脂肪体是物质合成代谢、先天免疫的重要器官。ATG8蛋白的亚细胞定位是细胞自噬的主要指标之一,细胞核皱缩是细胞凋亡的形态标记之一,目前家蚕 Bombyx mori 中尚未在蜕皮和变态发育进程中对BmATG8蛋白的细胞生物学变化进行观察。本研究旨在同时检测家蚕脂肪体细胞中BmATG8蛋白亚细胞定位和细胞核皱缩的时空变化,研究蜕皮激素(20E)信号对两者的调控作用。【方法】利用免疫荧光和Hoechst染色方法,分别在家蚕幼虫4龄第2天至预蛹第2天、5龄第2天幼虫注射20E (10 μg/头)后以及对游走期幼虫脂肪体中20E受体基因 usp 进行RNAi后,检测家蚕脂肪体中BmATG8蛋白定位和细胞核形态变化。【结果】在家蚕幼虫蜕皮和幼虫-蛹变态发育时期,BmATG8蛋白高水平存在于脂肪体细胞中,同时细胞核发生皱缩。在正常摄食时期,20E处理(10 μg/头)能够诱导细胞中大量出现BmATG8蛋白且存在于细胞质中并诱导细胞核皱缩。对 usp 基因进行RNAi后,脂肪体细胞内的BmATG8蛋白显著减少,同时细胞核皱缩减弱。【结论】家蚕BmATG8蛋白不仅在幼虫-蛹变态时期细胞质中大量存在,而且在幼虫蜕皮时期也大量表达,与细胞核的皱缩同时出现,BmATG8蛋白在细胞质中的定位与细胞核皱缩两者均受到 20E信号通路的调控。本研究为BmATG8蛋白功能及其调控机制的深入研究提供了重要的科学依据。     

Asymmetrical development of bones and soft tissues during eye migration of metamorphosing Japanese flounder,<Emphasis Type="Italic"> Paralichthys olivaceus</Emphasis>

The symmetrical body of flatfish larvae dramatically changes into an asymmetrical form after metamorphosis. Eye migration results in the most significant asymmetrical development seen in any vertebrate. To understand the mechanisms involved in eye migration, bone and cartilage formation was observed during metamorphosis in laboratory-reared Japanese flounder, Paralichthys olivaceus, by using whole-body samples and histological sections. Most of the hard tissues of the cranium (parasphenoid, trabecular cartilage, supraorbital canal, and supraorbital bar) exist symmetrically in the larval period before metamorphosis and develop by twisting in the same direction as that in which the eye migrates. An increase in skin thickness beneath the eye was observed only on the blind side at the beginning of eye migration; this was the first definitive difference between the right and left sides of the body. The pseudomesial bar, a peculiar bone present only in flatfishes, developed from this thick skin and grew dorsad. Novel sac-like structures were found and named retrorbital vesicles. The retrorbital vesicle of the blind side grew larger and faster than that of the ocular side when the right eye moved most dramatically, whereas no difference was observed between the volume of right and left connective tissue in the head. The asymmetrical presence and growth of the pseudomesial bar together with inflation of the retrorbital vesicle on the blind side may be responsible for right eye migration during metamorphosis in the Japanese flounder.     

Isolation of SFRS3 gene and its differential expression during metamorphosis involving eye migration of Japanese flounder Paralichthys olivaceus

Both eyes of flatfishes are located on one side of the body due to asymmetrical eye migration. The molecular mechanisms underlying such asymmetry is poorly understood. As an initial step, we have adopted suppression subtractive hybridization for the identification of upregulated genes during metamorphosis involving eye migration in Japanese flounder, Paralichthys olicaceus. One of the upregulated genes was identified as the splicing factor arginine/serine rich-3 (SFRS3). Sequence analysis of SFRS3 revealed that it encodes a protein of 168 amino acids containing the typical eukaryotic RNA recognition motif (RRM) and an arginine/serine-rich region. The overall amino acid sequences of the Japanese flounder SFRS3 was highly conserved with that of other organisms. The expression of flounder SFRS3 gene increased sharply from the beginning of metamorphosis and reached a high level of expression at stage H of metamorphosis 43 days after hatching. The SFRS3 gene upregulation was mainly limited to the head region, particularly in the rapidly proliferative tissues, the lateral ethmoid and "skin thickness" on blind side, which are thought as two proliferative tissues to push the eye movement. In spite of the upregulated expression of SFRS3 during metamorphosis, its role in metamorphosis involving eye migration requires further studies.     

Adult-type pigment cells,which color the ocular sides of flounders at metamorphosis,localize as precursor cells at the proximal parts of the dorsal and anal fins in early larvae

Flounders form left-right asymmetry in body coloration during metamorphosis through differentiation of adult-type melanophores and xanthophores on the ocular side. As the first step in investigating the formation of flounder body coloration asymmetry, in this study, we aimed to determine where the precursors of adult-type chromatophores distribute in larvae before metamorphosis. In Paralichthys olivaceus and Verasper variegatus, GTP cyclohydrolase 2 (gch2), a common marker of melanoblasts and xanthoblasts, was found to be transiently expressed in cells located along the bilateral skeletal muscles at the basal parts of the dorsal and anal fins of premetamorphic larvae. When V. variegatus larvae were fed with a strain of Artemia collected in Brazil, this gch2 expression was abolished and the differentiation of adult-type melanophores was completely inhibited, while the density of larval melanophores was not affected. In a cell trace test in which the cells at the basal part of the dorsal fin were labeled with DiI at the premetamorphic stage, adult-type melanophores labeled with DiI were found in the skin on the ocular side after metamorphosis. These data suggest that, in flounder larvae, adult-type melanophores are distributed at the basal parts of the dorsal and anal fins as unpigmented precursor cells.     

Vestibular Lesion-Induced Developmental Plasticity in Spinal Locomotor Networks during Xenopus laevis Metamorphosis

During frog metamorphosis, the vestibular sensory system remains unchanged, while spinal motor networks undergo a massive restructuring associated with the transition from the larval to adult biomechanical system. We investigated in Xenopus laevis the impact of a pre- (tadpole stage) or post-metamorphosis (juvenile stage) unilateral labyrinthectomy (UL) on young adult swimming performance and underlying spinal locomotor circuitry. The acute disruptive effects on locomotion were similar in both tadpoles and juvenile frogs. However, animals that had metamorphosed with a preceding UL expressed restored swimming behavior at the juvenile stage, whereas animals lesioned after metamorphosis never recovered. Whilst kinematic and electrophysiological analyses of the propulsive system showed no significant differences in either juvenile group, a 3D biomechanical simulation suggested that an asymmetry in the dynamic control of posture during swimming could account for the behavioral restoration observed in animals that had been labyrinthectomized before metamorphosis. This hypothesis was subsequently supported by in vivo electromyography during free swimming and in vitro recordings from isolated brainstem/spinal cord preparations. Specifically, animals lesioned prior to metamorphosis at the larval stage exhibited an asymmetrical propulsion/posture coupling as a post-metamorphic young adult. This developmental alteration was accompanied by an ipsilesional decrease in propriospinal coordination that is normally established in strict left-right symmetry during metamorphosis in order to synchronize dorsal trunk muscle contractions with bilateral hindlimb extensions in the swimming adult. Our data thus suggest that a disequilibrium in descending vestibulospinal information during Xenopus metamorphosis leads to an altered assembly of adult spinal locomotor circuitry. This in turn enables an adaptive compensation for the dynamic postural asymmetry induced by the vestibular imbalance and the restoration of functionally-effective behavior.     

Larval morphometry of the Japanese flounder,Paralichthys olivaceus

The larval development of the Japanese flounder,Paralichthys olivaceus, was surveyed using two types of morphometric analyses, modified allometry and polar coordinate analysis by principal component analysis (PCA). In the former, centroid size was used as a growth index instead of total length (TL), such enabling the determination of more detailed changes in each character than ordinary allometry based upon TL. Polar coordinate analysis disclosed two remarkable inflexions during the larval development ofP. olivaceus. Postlarvae ofP. olivaceus were found to undergo four developmental phases. From the point of view of metamorphosis, the phases were named drifting larva, premetamorphic larva, metamorphic larva and postmetamorphic larva, respectively. These phases were also tested by other characters related to flounder metamorphosis.     

Early life history stages of peacock flounder Bothus lunatus (Bothidae) from the western and central tropical Atlantic

Previously unknown larvae and poorly studied metamorphic and postmetamorphic stages of development of peacock flounder Bothus lunatus (Bothidae) with a length of 5.4 to 39.5 mm from the western and central tropical waters of the Atlantic Ocean are described, and their identification is substantiated. Small-and medium-sized larvae of this species (with a length of nearly 10 mm) at stages of pre-and postflexion, like all larvae of the genus Bothus at the corresponding stages of development, have a strongly flattened from the sides, moderately deep, and almost pigment-free transparent body; bear a long anterior ray in the dorsal fin; and have single melanophores in the end part of the body. They differ from larvae of other Atlantic species of the genus known at early stages of development in the shape of the anterior head profile, namely in the presence of a deep notch in front of the eyes. With growth, their body assumes a rounded shape. During metamorphosis of larval B. lunatus, the right eye shifts to the left side of the head through a slit formed during the separation of the origin of dorsal fin base from the cranium. This specific feature distinguishes them from larvae of the remaining species of the genus in which, during metamorphosis, the right eye passes to the left side through the hole in the head.     

Presumptive relationships between ventricular proliferaion and development of the lateral motor columns in the spinal cord of Rana pipiens larvae.

The extent of mitotic activity in the proliferative ventricular zone of the developing frog (Rana pipiens) spinal cord is a function of both the longitudinal cord level and the developmental stage. Counts of mitotic cells in the ventricular zone demonstrated higher levels of proliferation in the dorsal than ventral halves of the spinal cord with decreasing total proliferative activity from the early to late larval (tadpole) stages. Mitoses were virtually absent from the ventricular zone by the conclusion of metamorphosis. Changes in mitotic counts at different levels of the spinal cord can be correlated with the presence or absence of the brachial or lumbosacral pairs of lateral motor columns. A parallel also exists between the caudo-cephalic direction of motor column development and a similar progression of mitotic activity in the ventricular zone, a portion of which gives rise to the spinal motor neurons. It is suggested that proliferation in the ventricular zone during the larval frog stages contributes to the presumptive motor neuron population and migrates into the lateral motor columns during their later maturation.