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。     

棉铃虫蛹期血淋巴的蜕皮甾类

目前为止仅在少数几种昆虫中研究过蛹期的蜕皮激素。关于蜕皮甾类的性质分析,结果也颇不一致。本文采用放射免疫分析、薄层层析、高压液相色谱及质谱对棉铃虫Heliothis armigera蛹血淋巴内的蜕皮激素进行了研究。结果如下:1.物理-化学方法证明蛹血淋巴内存在二种蜕皮甾类:蜕皮酮和20-羟基蜕皮酮。2.蛹期蜕皮甾类滴度呈一宽峰,高峰出现在化蛹后的第5天(3435ng/ml)。3.在高峰时,蜕皮酮与20-羟基蜕皮酮的比例为1:3.57,说明20-羟基蜕皮酮是主要的蜕皮甾类。4.比较雌雄两性蛹的蜕皮甾类滴度,未见明显差异。研究表明在棉铃虫中影响成虫发育的主要激素是20-羟基蜕皮酮而不是蜕皮酮。     

昆虫变态发育过程中的细胞自噬和凋亡

在昆虫变态期,幼虫组织发生退化或消亡,原因在于蜕皮甾醇激素（ecdysteroid）,即通常所说的蜕皮激素,诱导这些组织的细胞发生了自噬(autophagy)和凋亡(apoptosis)的程序性细胞死亡(programmed cell death,PCD)。一般情况下,自噬途径构成一种饥饿应激适应性以避免细胞的死亡,表现为低水平Cvt泡(Cvt vesicle)和自噬体(autophagosome)对部分胞质溶胶、蛋白聚集体和细胞器的吞噬和降解。昆虫进入变态发育时,由于蜕皮激素的激活,由遗传级联系统调控的PCD机制被启动,低水平的常态自噬转入高水平的自噬并同时诱发凋亡,细胞进入不可逆的死亡,导致幼虫组织在变态期退化或消亡。对果蝇Drosophila变态期PCD机制中最重要的发现是:（1）在自噬发生的PI3KⅠ- Tor 和 PI3KⅢ的分子通路中,由自噬相关蛋白Atg1引发的高水平自噬能够诱导凋亡;（2）蜕皮激素诱导表达的βFTZ-F1,E93,BR-C,E74A等转录因子不但激活凋亡的Caspases通路,还能诱导自噬的发生。     

小地老虎蜕皮腺的形态和结构

用电镜和光镜以及组织化学方法研究了小地老虎(Agrotis ypsilon)幼虫蜕皮腺的形态与结构,及其在5龄—6龄幼虫的蜕皮期和预蛹—蛹的变态期的变化,并对蜕皮腺分泌的蜕皮液进行了组织化学分析.结果表明:(1)幼虫期的蜕皮腺共有15对,其中12对位于胸部1—3节和腹部1—9节的背侧面,其余3对位于前、中、后胸基节窝的外侧,至化蛹30小时后逐渐解体;(2)蜕皮腺的分泌细胞均为“液泡型”,在幼虫每次蜕皮期间出现周期性变化,随着蜕皮过程的结束,细胞由分泌状态进入静止状态,体积逐渐变小,色泽由乳白色趋向透明,液泡变小或消失,内质网数量溅少,液泡内含物对PAS反应减弱;(3)蜕皮前蜕皮腺分泌细胞的内含物PAS强阳性,表明蜕皮液是粘多糖或糖蛋白性质的,它通过导管分泌到新旧表皮层之间.     

β-蜕皮素对家蚕丝腺成长及合成甘氨酸、丙氨酸有关酶系的调节控制

本文研究了植源性β-蜕皮素(简称MH)对家蚕丝腺成长及合成甘氨酸、丙氨酸有关酶系的影响,探讨了5龄不同时期添食β-蜕皮素后丝腺体的成长及后部丝腺和脂肪体中丙氨酸-酮丙二酸、丙氨酸-乙醛酸和鸟氨酸转氨酶活力的变化。通过实验观察到:(1)5龄早期(饷食)添食β-蜕皮素,龄期延长8—9小时,茧层量增加,后部丝腺和脂肪体内丙氨酸-酮丙二酸转氨酶、丙氨酸-乙醛酸转氨酶在处理后6-12小时内,比对照组增高40—50%。随之酶活力略下降,至5龄后期复又升高,明显超过对照组。脂肪体中鸟氨酸转氨酶在β-蜕皮素处理后6小时和48小时均低于对照组。12—24小时以及48小时后直至老熟则明显高于对照组。(2)5龄后期(V—136小时)口腔注射β-蜕皮素,龄期缩短12—18小时。脂肪体和丝腺体中丙氨酸-酮丙二酸转氨酶、丙氨酸-乙醛酸转氨酶活力明显高于对照组(一般增高20—40%),老熟时迅速下降。本文对有关保幼激素和β-蜕皮素对5龄家蚕两种靶细胞(丝腺细胞和脂肪细胞)的相互配合和制约以完成对蚕整体的调节控制,以及这两种昆虫激素应用于蚕业生产以控制5龄期的长短和增加蚕丝产量等方面进行了讨论。     

外源蜕皮激素对蓖麻蚕蛹发育的效应

本文报道蓖麻蚕蛹在室温28℃下的卵巢发育过程,以及外源20-羟基蜕皮酮对蚕蛹发育的影响。正常蛹在任何发育期内注射20-羟基蜕皮酮后,全部仍羽化成蛾,但蛹期延长约1至4天。无脑蛹经注射后出现蛹——蛾的变态,发育情况因剂量而不同:注射0.1微克后约有半数蛹发育成蛾;注射2微克羽化率较高,卵巢管的发育也最好;4微克或更高的注射量能使全部蛹发育成蛾,但卵巢管多少有些不正常。注射量超过5微克时,蛾体较小,颜色浅黄,没有或只有很少的鳞片。蛹的发育天数随剂量的增大而减少。经外源20-羟基蜕皮酮处理后,无论是有脑蛾或是无脑蛾的卵粒都明显地比正常蛾的卵粒大。当超过一定的注射量时,注射量越大,蚕蛾的自动蜕壳能力越差。     

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。     

带有丝素重链信号肽序列的家蚕丝胶蛋白启动子驱动DsRed的瞬时分泌表达

为了探讨家蚕Bombyx mori丝素蛋白重链信号肽序列在中部丝腺组织中是否具有功能活性,根据家蚕丝蛋白基因的启动子活性高、丝蛋白具有高效分泌的特性,构建了带有丝素重链基因fib-H信号肽的家蚕丝胶-1(ser-1)启动子(ser-HS),用ser-HS驱动DsRed基因构建了分泌型瞬时表达载体pSK-SerHS-DsRed-polyA。转染细胞实验显示,该载体能在家蚕BmN细胞中瞬时表达DsRed。家蚕注射载体后,可在中部丝腺腔中检测到红色荧光,表明瞬时表达的DsRed已分泌到丝腺腔内。据此提出克隆的fib-H信号肽序列在家蚕中部丝腺组织中具有信号肽的功能。     

银盾革蜱若虫发育期20-羟基蜕皮酮含量变化规律(英语)

本文利用高效液相色谱(HPLC)对银盾革蜱饥饿、吸血和饱血后若虫整体水平20-羟基蜕皮酮含量变化规律进行了研究.结果表示20-羟基蜕皮酮存在于若虫整个发育期,其含量变化具有两个峰.第一个峰小,出现在吸血期(吸血后5天),含量为2.34ng/只,该峰出现后,若虫快速饱血并从宿主体落下;第二个峰大,出现在蜕化期(饱血后8天),含量为16.17ng/只,此峰可能与上表皮沉积有关.从饱血后第6天至饱血后第8天,20-羟基蜕皮酮含量迅速增加,皮层溶离发生在饱血后7天左右.     

家蚕凋亡相关基因研究进展

细胞凋亡指细胞受基因调控的自主的细胞死亡过程,是细胞为维持内环境稳态的一种手段。家蚕Bombyx mori是重要的鳞翅目模式昆虫,对其细胞凋亡机制的研究具有代表性。家蚕细胞凋亡不仅参与了整个变态发育过程,而且在家蚕天然免疫反应中扮演着重要角色。在家蚕卵-幼虫-蛹-成虫各阶段通过凋亡基因的调控促进组织退化及冗余细胞的清除,并且在家蚕抗家蚕核型多角体病毒(Bm NPV)过程中,细胞凋亡的发生对Bm NPV增殖的抑制也有着重要作用。本文就近年来家蚕细胞凋亡诱导因素、细胞凋亡相关基因的研究现状及通路和细胞凋亡对家蚕发育影响的研究进展进行综述,为解决目前家蚕细胞凋亡机制研究不足、内质网通路涉猎少、各通路间联系不清晰等问题,以及深入研究家蚕细胞凋亡并解析家蚕变态发育机制和天然免疫反应提供参考。     

Some autophagic and apoptotic features of programmed cell death in the anterior silk glands of the silkworm, Bombyx mori

Programmed cell death has been subdivided into two major groups: apoptosis and autophagic cell death. The anterior silk gland of Bombyx mori degenerates during larval-pupal metamorphosis. Our findings indicate that two types of programmed cell death features are observed during this physiological process. During the prepupal period, pyknosis of the nucleus, cell detachment,and membrane blebbing occur and they are the first signs of programmed cell death in the anterior silk glands. According to previous studies, all of these morphological appearances are common for both cell-death types. Autophagy features are also exhibited during the prepupal period. Levels of one of the lysosomal marker enzymes-acid phosphatase-are high during this period then decrease gradually. Vacuole formation begins to appear first at the basal surface of the cell, then expands to the apical surface just before the larval pupal ecdysis. After larval-pupal ecdysis, DNA fragmentation, which is the obvious biochemical marker of apoptosis, is detected in agarose gel electrophoresis, which also shows that caspase-like enzyme activities occur during the programmed cell death process of the anterior silk glands. Apoptosis and autophagic cell death interact with each other during the degeneration process of the anterior silk gland in Bombyx mori and this interaction occurs at a late phase of cell death. We suggest that apoptotic cell death only is not enough for whole gland degeneration and that more effective degeneration occurs with this cooperation.     

Some autophagic and apoptotic features of programmed cell death in the anterior silk glands of the silkworm,Bombyx mori

Programmed cell death has been subdivided into two major groups: apoptosis and autophagic cell death. The anterior silk gland of Bombyx mori degenerates during larval-pupal metamorphosis. Our findings indicate that two types of programmed cell death features are observed during this physiological process. During the prepupal period, pyknosis of the nucleus, cell detachment and membrane blebbing occur and they are the first signs of programmed cell death in the anterior silk glands. According to previous studies, all of these morphological appearences are common for both cell death types. Autophagy features are also exhibited during the prepupal period. One of the lysosomal marker enzymes, acid phosphatase, levels are high during this period then decrease gradually. Vacuole formation begins to appear first at the basal surface of the cell, then expands to the apical surface just before the larval pupal ecdysis. After larval-pupal ecdysis, DNA fragmentation, which is the obvious biochemical marker of apoptosis, is detected in agarose gel electrophoresis which also shows that caspase-like enzyme activities occur during the programmed cell death process of the anterior silk glands. Apoptosis and autophagic cell death interact with each other during the degeneration process of the anterior silk gland in Bombyx mori and this interaction occurs at a late phase of cell death. We suggest that only apoptotic cell death not enough for whole gland degeneration and that more effective degeneration occurs with this cooperation.

Addendum to: Goncu E, Parlak O. Morphological changes and patterns of ecdysone receptor B1 immunolocalization in the anterior silk gland undergoing programmed cell death in the silkworm, Bombyx mori. Acta Histochem 2008; In press.     

Mechanisms of programmed cell death in the midgut and salivary glands from Bradysia hygida (Diptera: Sciaridae) during pupal–adult metamorphosis

Programmed cell death is involved with the degeneration/remodeling of larval tissues and organs during holometabolous development. The midgut is a model to study the types of programmed cell death associated with metamorphosis because its structure while degenerating is a substrate for the formation of the adult organ. Another model is the salivary glands from dipteran because their elimination involves different cell death modes. This study aimed to investigate the models of programmed cell death operating during midgut replacement and salivary gland histolysis in Bradysia hygida. We carried out experiments of real‐time observations, morphological analysis, glycogen detection, filamentous‐actin localization, and nuclear acridine orange staining. Our findings allow us to establish that an intact actin cytoskeleton is required for midgut replacement in B. hygida and nuclear condensation and acridine orange staining precede the death of the larval cells. Salivary glands in histolysis present cytoplasmic blebbing, nuclear retraction, and acridine orange staining. This process can be partially reproduced in vitro. We propose that the larval midgut death involves autophagic and apoptotic features and apoptosis is a mechanism involved with salivary gland histolysis.     

Fenoxycarb modulates ecdysone receptor B1 and programmed cell death of the anterior silk gland of silkworm Bombyx mori

Fenoxycarb, O‐ethyl N‐(2‐(4‐phenoxyphenoxy)‐ethyl) carbamate has been shown to be one of the most potent juvenile hormone analogues against a variety of insect species. In the present study, topical application of fenoxycarb to fifth‐instar larvae of the silkworm Bombyx mori (Lepidoptera: Bombycidae) was performed immediately after the fourth ecdysis (on day 0), day 3 and day 6 of the instar and then its effects on the anterior silk glands (ASG) and ecdysone receptor B1 (EcR‐B1) protein were investigated during larval pupal development. Fenoxycarb application increased the instar length and prevented metamorphic events, depending on the application time. The ASGs of B. mori undergo programmed cell death during the larval–pupal metamorphosis and an insect steroid, 20‐hydroxyecdysone (20E), triggers this cell death. The exact mechanism by which 20E and juvenile hormone regulates programmed cell death in insect tissues is poorly understood. To gain insights into how juvenile hormone regulates metamorphic events like programmed cell death in the anterior silk glands, we analyzed the progression of programmed cell death with morphological observations and biochemical experiments like acid phosphatase activity and DNA electrophoresis. Then we examined the EcR‐B1 protein levels and their relationships with programmed cell death. Our results indicated that fenoxycarb modulates programmed cell death of the anterior silk glands and EcR‐B1 protein level, depending on the application time. Fenoxycarb may exhibit its effects in at least two different ways: (i) acting on prothoracic gland secretory activity; and/or (ii) regulation of EcR‐B1 expression in the anterior silk glands for programmed cell death process.     

An in vitro analysis of ecdysteroid-elicited cell death in the prothoracic gland of Manduca sexta

The prothoracic glands of the tobacco hornworm, Manduca sexta, secrete the precursor of the insect molting hormone and normally undergo programmed cell death (PCD) during pupal-adult metamorphosis, between days 5 and 6 after pupation. This phenomenon can be elicited prematurely in vitro by the addition of 20-hydroxyecdysone (20E) to the gland cultures. To induce nuclear condensation in vitro in the glands from day-1 pupae, the effective dose range of 20E is 0.7-7 micrograms/ml and the minimum exposure period is 24 h. Prothoracic glands from different stages of pupal-adult development express different responsiveness to exogenous ecdysteroids. By utilizing terminal deoxynucleotidyl-transferase-mediated dUTP nick-end-labeling (TUNEL) and the apoptotic DNA laddering method together with transmission electron microscopy, it has been demonstrated that the ecdysteroid-induced cell death of the prothoracic glands occurs via not only apoptosis but also autophagy, i.e., the induced dying cells show both severe nuclear fragmentation and autophagic vacuole formation, characteristics typical of apoptotic and autophagic cell death. The composite data indicate that ecdysteroids regulate directly both apoptotic and autophagic mechanisms of PCD of the prothoracic glands.     

Cell death in the salivary glands of metamorphosing calliphora vomitoria

The salivary gland cells of Calliphora vomitoria larvae initiate and complete their own destruction in a programmed manner at the onset of metamorphosis. On entering the post-feeding period the larvae come to rest and the polytene salivary gland cells show a significant increase in DNA synthesis followed closely by a surge of mRNA synthesis accompanied by increasing protein production. During this prelude to cell death the new mRNA gives rise to at least 10 new proteins. The first new proteins having a MWt between 30 and 100kD appear by day 8 of the life-cycle and a number persist until the advent of cell death on day 9. Other new proteins appear in a cascade of production during day 8 and in vitro translation of mRNA produced at this time shows a new 55kD protein appearing before cell destruction. Significantly no evidence of DNA degeneration or laddering associated with classical apoptosis was observed, on the contrary considerable DNA synthesis in the form of chromosomal endoduplication or "genomic amplification" was seen; selective gene expression being apparently controlled at translational level. Overall the results strongly suggest a synthetically mediated programmed cell death in the metamorphosing salivary glands of the blow-fly which is distinct from apoptosis.     

Morphological evidence that salivary gland degeneration in the American dog tick,Dermacentor variabilis (Say), involves programmed cell death

During the preoviposition and oviposition periods of ixodid ticks, the salivary glands degenerate. It is unclear whether this is a necrotic or a programmed cell death event. We used an in situ TUNEL technique to determine if salivary gland degeneration involves apoptosis. Salivary glands were dissected from replete females at days 3, 5, 8, 11, 13, and 33 post-detachment. There were no differences in tick weight at detachment, suggesting that changes were not due to engorgement abnormalities. The onset of apoptosis began at day 5 and continued through oviposition at day 33. The greatest amount of nuclei containing fragmented DNA was observed on day 8 post-detachment, suggesting this was the peak occurrence of programmed cell death. Further, the temporal organization of programmed cell death suggests that the granule-secreting acini undergo apoptosis first, and that during the first week of oviposition the type I acini do not exhibit programmed cell death. These data suggest that the type I acini may still function in maintaining off-host hydration state of ovipositing females. These data provide morphological evidence that salivary gland degeneration involves a temporal programmed cell death event.     

Real-time observation of autophagic programmed cell death of<Emphasis Type="Italic"> Drosophila</Emphasis> salivary glands in vitro

Autophagy, a form of programmed cell death (PCD) that is morphologically distinguished from apoptosis, is thought to be as prevalent as apoptosis, at least during development. In insect metamorphosis, the steroid hormone 20-hydroxyecdysone (ecdysone) activates autophagic PCD to eliminate larval structures that are no longer needed. However, in comparison with apoptosis, there are not many studies on the regulation mechanisms of autophagy. To provide a useful model for studying autophagic PCD, I established an in vitro culture system that enables real-time observation of the autophagic cell destruction of Drosophila salivary glands. The new system revealed that de novo gene expression was still required for the destruction of salivary glands dissected from phanerocephalic pupae. This indicates the usefulness of the system for exploring genes that participate in the last processes of autophagic PCD.Edited by N. Satoh     

AP-1, but not NF-kappa B, is required for efficient steroid-triggered cell death in Drosophila

Extensive studies in vertebrate cells have assigned a central role to Rel/NF-kappa B and AP-1 family members in the control of apoptosis. We ask here whether parallel pathways might function in Drosophila by determining if Rel/NF-kappa B or AP-1 family members contribute to the steroid-triggered death of larval salivary glands during Drosophila metamorphosis. We show that two of the three Drosophila Rel/NF-kappa B genes are expressed in doomed salivary glands and that one family member, Dif, is induced in a stage-specific manner immediately before the onset of programmed cell death. Similarly, Djun is expressed for many hours before salivary gland cell death while Dfos is induced in a stage-specific manner, immediately before this tissue is destroyed. We show that null mutations in the three Drosophila Rel/NF-kappa B family members, either alone or in combination, have no apparent effect on this death response. In contrast, Dfos is required for the proper timing of larval salivary gland cell death as well as the proper induction of key death genes. This study demonstrates a role for AP-1 in the stage-specific steroid-triggered programmed cell death of larval tissues during Drosophila metamorphosis.