纳米银对果蝇化蛹、羽化、寿命及细胞凋亡和蛋白表达的影响

纳米银因具有强有效的抗菌作用而被广泛应用于医药领域,然而它存在潜在的生物毒性.本研究选取经典的模式生物黑腹果蝇,探究纳米银对生物体的毒性作用机理.测定不同浓度纳米银处理下的Oregon R、w1118、MTF突变体等品系的化蛹率、羽化时间、羽化率及寿命;用rpr-lacZ品系的lacZ活性测定不同纳米银浓度处理下翅成虫盘的细胞凋亡情况; 通过SDS-PAGE研究不同浓度纳米银浓度处理下MTF突变体中肠蛋白的表达差异,并利用质谱分析差异蛋白的氨基酸序列.结果显示: 纳米银处理浓度升高至200 μg·mL^-1及以上时,MTF突变体化蛹率和羽化率显著降低; 纳米银处理浓度升高至800 μg·mL^-1时,Oregon R和w1118化蛹率和羽化率显著降低,而化蛹时间和羽化时间与纳米银处理浓度无显著关系.不同浓度纳米银处理对Oregon R和w1118的寿命无影响,而200 μg·mL^-1纳米银显著降低MTF突变体平均寿命.随纳米银处理浓度升高,细胞凋亡也逐渐增多.SDS-PAGE及质谱分析表明,随纳米银浓度上升,质子泵ATP激酶、热激蛋白、糖代谢相关酶系等蛋白的表达量上升.本研究表明,高浓度的纳米银会促进细胞凋亡致使部分蛋白表达量发生改变,从而降低果蝇存活率,为后续进一步了解其毒性作用机理提供理论依据.     

黄鳝生殖干细胞在性逆转生殖发育过程中的变化

应用组织学方法及免疫组织化学技术显示,黄鳝性逆转生殖发育过程中,生殖干细胞(GSCs)定位分布于生殖褶中,黄鳝雌性发育阶段的GSCs分散或成团存在,间性及雄性发育阶段GSCs均区分为A、B两种不同类型,雌性发育阶段GSCs与A、B两类GSCs在超微结构上存在差异。结果表明,生殖褶中GSCs是黄鳝分化生殖腺中唯一具有有丝分裂能力的生殖细胞群,雌性发育阶段GSCs表现出卵原干细胞特征,间性及雄性发育阶段GSCs为精原干细胞。CD49整合素是黄鳝雌性发育阶段GSCs和A类GSCs的表征分子。     

L-精氨酸在实验室果蝇扩繁中的应用

目的实验旨在研究在玉米琼脂培养基中添加L-精氨酸,探究L-精氨酸对果蝇化蛹时间、羽化时间、羽化率及雌雄比例的影响。方法实验分为2组,分别为对照组和实验组,对照组为基础培养基,实验组在基础培养基加1%的L-精氨酸,每管中雌、雄1∶1放入7对果蝇,每组12个重复,接种72 h后移除亲本,统计各组果蝇化蛹时间、羽化时间、羽化数及各组雌雄个数。结果培养基中添加1%的L-精氨酸,白眼(w~(1118))、残翅(CyO)和野生型黑腹果蝇的化蛹数、羽化数分别比对照组提高313.64%、303.53%(P0.01),157.09%、182.76%(P0.01)和233.33%、239.03%(P0.01),添加1%的L-精氨酸后白眼和野生型果蝇的化蛹时间比对照组提前2 d。结论在本实验条件下,1%的L-精氨酸可提高实验室果蝇繁殖力,可应用于实验室果蝇的扩繁。     

Osa在果蝇卵巢生殖干细胞分化中的功能研究

果蝇卵巢生殖干细胞(Germline stem cell,GSC)是在活体(in vivo)研究干细胞命运调控的理想平台。表观遗传机制在果蝇卵巢GSC命运调控中发挥重要作用,其机理的探明需要研究并发现更多参与此过程的表观调控因子。为探究果蝇染色质重塑复合物BAP中特有的亚基Osa在果蝇卵巢GSC分化调控中的功能及其分子机理,利用GAL4/UAS二元表达系统结合RNAi技术在干细胞微环境组分护卫细胞(Escort cells,ECs)中特异性下调osa的表达,并通过免疫荧光染色法对相关指标进行检测。结果显示,敲减ECs中osa可致卵巢组织卵原区中未分化生殖细胞数目(Undifferentiated germ cells,UGCs)显著增多,同时BMP信号通路激活标志p Mad、Dad-lacZ阳性细胞数目显著增多,并观察到EC细胞形态异常,不能有效包裹生殖系细胞。推论Osa以BMP信号通路依赖方式参与果蝇卵巢GSC的分化调控,其作用机理还可能涉及EC细胞特定的形态学过程。     

stat3通过促进轴突生长间接抑制间神经丘早熟

鱼类侧线系统由感受水流的机械感受器和传导信息的侧线神经组成.stat3在斑马鱼侧线神经丘和侧线神经节中特异性表达,但stat3在斑马鱼后侧线系统发育过程中的功能仍然不清楚.本研究利用CRISPR/Cas9在斑马鱼中成功敲除stat3基因.然后,利用Tg(SqET20:GFP)转基因鱼追踪后侧线神经丘的发育.从4 dpf开始,stat3纯合突变体尾部的神经丘数量显著多于野生型.原位杂交结果显示,stat3纯合突变体后侧线神经上的mbp信号少于野生型.进一步用抗乙酰化-微管蛋白抗体以及TgBAC(Neurod1:EGFP)转基因鱼追踪后侧线轴突生长,发现stat3纯合突变体后侧线神经轴突末梢停在泄殖孔旁,不再支配尾部神经丘.综上结果表明:stat3通过调控轴突生长,间接地抑制间神经丘早熟.     

黑腹果蝇昼夜活动节律及行为时间分配

采用试验室单管观察记录的方法,对3,4,5日龄野生型黑腹果蝇Drosophila melanogaster Meigenw1118成虫每日活动节律进行研究。试验将果蝇活动划分为强活动(飞行和爬行)、弱活动(梳理、觅食等原地发生的运动)和静息(身体不发生移动的休息)3种类型。强活动和弱活动之和为总运动。研究结果显示,野生型黑腹果蝇w1118的昼夜活动表现为明显的双峰模态,晨峰和晚峰分别处于开、关灯前后;雌、雄果蝇总体活动无差异,关灯(18:30)前后雌蝇活动稍强于雄蝇,开灯(6:30)前后则相反;果蝇强活动的节律与总运动基本一致,而弱活动节律不明显;静息节律为单峰模式,其高峰期位于夜间1:00~5:00;雌蝇的静息活动显著多于雄蝇(P<0·05)。     

Henna基因突-变是导致北京紫眼果蝇眼部蝶呤含量减少的重要原因

苯丙氨酸羟化酶被认为参与黑腹果蝇眼部蝶呤代谢,但是始终都缺乏有力证据．Henna是苯丙氨酸羟化酶的编码基因,“北京紫眼”果蝇（Hnbp）是Henna基因的一个隐性突变体,Hn^bp突变形成的原因是Henna基因的第二外显子上具有插入片段．在Hn^bp中,其编码产物的Biopterin—Hydroxyl功能域中增加了15个氨基酸残基．蛋白预测结果显示,插入的残基改变了原有的蛋白结构,这种变化很可能降低了苯丙氨酸羟化酶与四氢生物蝶呤结合的能力．为了恢复突变表型,利用UAS—GAL4转基因体系使野生型Henna基因在Hn^bp的背景下表达．在双拷贝的转基因系GMR—GAL4 UAS—Henna／UAS—Henna;Hn^bp／Hn^bp中,突变表型得到完全恢复：果蝇的眼色由突变体的紫色恢复到野生型的红色,蝶呤含量也从突变体的30％升至98％,与野生型差异不显著（P〉0．05）．上述实验结果充分说明,Henna基因突变是导致Hn^bp突变体眼部喋呤含量降低的重要原因,为苯丙氨酸羟化酶参与果蝇眼部蝶呤代谢提供了体内实验的直接证据．     

Fus在斑马鱼生长发育及两性生长异形中的功能（英文）

为研究肉瘤融合基因(fus)在鱼类中的功能,在斑马鱼中利用CRISPR/Cas9基因突变技术使fus基因产生移码突变。fus~(-/-)纯合突变体斑马鱼发育正常并且完全可育,但体长和体重在幼鱼和成年阶段都小于野生型斑马鱼。此外,相较于野生型斑马鱼, fus~(-/-)纯合突变体斑马鱼不存在雌鱼身体比例大于雄鱼的性别异形现象。在fus~(-/-)纯合突变体斑马鱼早期幼鱼发育阶段,一些生长相关基因包括gh1、ghra、igf1、igf2a、stat5.1和socs6的表达水平显著低于野生型斑马鱼但其运动能力并未受到影响。因此,不同于fus基因在哺乳动物中的功能,它在斑马鱼中不参与运动神经元的发育,但在调节鱼类躯体生长发育与两性生长异形方面有着重要的功能。     

烟草水提物对果蝇寿命及有关基因转录的影响

探究烟草的水浸泡液对果蝇寿命与衰老情况的影响以及其中的分子机制。取羽化12 h内的新生W^1118系雄果蝇,分别培养于添加不同浓度烟草浸出液的培养基中处理,每日统计果蝇死亡情况;每7日提取对照组与最高浓度处理组果蝇全RNA,通过Real-time PCR检测过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、去泛素化酶(Rpn11)和去乙酰化酶(Sirt6)等与寿命有关基因的表达水平变化。结果显示,烟草影响使雄果蝇寿命显著缩短,且影响随烟草提取液浓度增加影响逐渐增大;烟草影响下果蝇的抗氧化基因出现显著上调,泛素化调节基因与去乙酰化基因等表达水平的变化均出现显著改变。表明烟草对果蝇的寿命情况有不利影响,可能通过引起氧化胁迫导致果蝇早衰。     

Fus在斑马鱼生长发育及两性生长异形中的功能

为研究肉瘤融合基因(fus)在鱼类中的功能, 在斑马鱼中利用CRISPR/Cas9基因突变技术使fus基因产生移码突变。fus-/-纯合突变体斑马鱼发育正常并且完全可育, 但体长和体重在幼鱼和成年阶段都小于野生型斑马鱼。此外, 相较于野生型斑马鱼, fus-/-纯合突变体斑马鱼不存在雌鱼身体比例大于雄鱼的性别异形现象。在fus-/-纯合突变体斑马鱼早期幼鱼发育阶段, 一些生长相关基因包括gh1、ghra、igf1、igf2a、stat5.1和socs6的表达水平显著低于野生型斑马鱼但其运动能力并未受到影响。因此, 不同于fus基因在哺乳动物中的功能, 它在斑马鱼中不参与运动神经元的发育, 但在调节鱼类躯体生长发育与两性生长异形方面有着重要的功能。     

Dynamics of the male germline stem cell population during aging of Drosophila melanogaster

Drosophila melanogaster has emerged as an important model system for the study of both stem cell biology and aging. Much is known about how molecular signals from the somatic niche regulate adult stem cells in the germline, and a variety of environmental factors as well as single point mutations have been shown to affect lifespan. Relatively little is known, however, about how aging affects specific populations of cells, particularly adult stem cells that may be susceptible to aging-related damage. Here we show that male germline stem cells (GSCs) are lost from the stem cell niche during aging, but are efficiently replaced to maintain overall stem cell number. We also find that the division rate of GSCs slows significantly during aging, and that this slowing correlates with a reduction in the number of somatic hub cells that contribute to the stem cell niche. Interestingly, slowing of stem cell division rate was not observed in long-lived methuselah mutant flies. We finally investigated whether two mechanisms that are thought to be used in other adult stem cell types to minimize the effects of aging were operative in this system. First, in many adult tissues stem cells exhibit markedly fewer cell cycles relative to transit-amplifying cells, presumably protecting the stem cell pool from replication-associated damage. Second, at any given time not all stem cells actively cycle, leading to 'clonal succession' from the reserve pool of initially quiescent stem cells. We find that neither of these mechanisms is used in Drosophila male GSCs.     

Gbb/Bmp signaling is essential for maintaining germline stem cells and for repressing bam transcription in the Drosophila testis

Stem cells are responsible for replacing damaged or dying cells in various adult tissues throughout a lifetime. They possess great potential for future regenerative medicine and gene therapy. However, the mechanisms governing stem cell regulation are poorly understood. Germline stem cells (GSCs) in the Drosophila testis have been shown to reside in niches, and thus these represent an excellent system for studying relationships between niches and stem cells. Here we show that Bmp signals from somatic cells are essential for maintaining GSCs in the Drosophila testis. Somatic cyst cells and hub cells express two Bmp molecules, Gbb and Dpp. Our genetic analysis indicates that gbb functions cooperatively with dpp to maintain male GSCs, although gbb alone is essential for GSC maintenance. Furthermore, mutant clonal analysis shows that Bmp signals directly act on GSCs and control their maintenance. In GSCs defective in Bmp signaling, expression of bam is upregulated, whereas forced bam expression in GSCs causes the GSCs to be lost. This study demonstrates that Bmp signals from the somatic cells maintain GSCs, at least in part, by repressing bam expression in the Drosophila testis. dpp signaling is known to be essential for maintaining GSCs in the Drosophila ovary. This study further suggests that both Drosophila male and female GSCs use Bmp signals to maintain GSCs.     

Muscle degeneration in the posteclosional development of a Drosophila mutant, abnormal proboscis extension reflex C (aperC)

The aperC (abnormal proboscis extension reflex C) mutation in Drosophila melanogaster causes a defect in the proboscis extension reflex (PER) in aged flies. Young flies of the mutant show an apparently normal PER. When aperCTF36 mutants were reared at 25 degrees C, the flies became unable to extend the proboscis at Day 5 of eclosion, but within a few days, many of them recovered the PER. When reared at 18 degrees C, the mutants showed a defective PER, but did not show the recovery. At 29 degrees C, only a limited number of the mutant flies showed the defective PER. Histological inspection of the mutant revealed that the degeneration occurs in a pair of muscles, the rostral protractors, which are involved in the extension of the rostrum. The degeneration of the muscle was observed in the mutant by polarizing light microscopy. The cross striation disappeared from the central portion of the muscle fibers at Day 3. Birefringence of the fibers also disappeared. At Day 10 the degenerated muscle fibers showed regeneration. The PER was closely correlated with the degree of muscle degeneration and regeneration. Temperature-shift experiments indicated that the temperature-sensitive-period of the aper CTF36 mutation occurs around Days 2-4 after eclosion. Results indicate that the aperC+ gene regulates the posteclosional maintenance of the muscle fibers.     

Post-eclosion decline in 'knock-down' thermal resistance and reduced effect of heat hardening in Drosophila melanogaster

The dependency of knock-down resistance on age, from 0 to 12 days post eclosion, was studied in two lines of Drosophila melanogaster, one selected for knock-down resistance and one unselected control. Additionally, the inducible Hsp70 expression level was assessed at maintenance temperature and after a heat hardening treatment (1 h at 35 degrees C) at the same ages. Knock-down resistance decreased roughly linearly with age in both control and knock-down selected lines and in both sexes regardless of maintenance temperature. Hsp70 expression, however, fell only from day 0 to day 3 and was thereafter changed little. Acclimation did not result in detectable difference between lines. The effect of hardening was only significant for control flies. This suggests that the increased knock-down resistance of the selected line was reached at the expense of hardening ability.     

Expression of bovine superoxide dismutase in Drosophila melanogaster augments resistance of oxidative stress.

Superoxide dismutases (SOD) play a major role in the intracellular defense against oxygen radical damage to aerobic cells. In eucaryotes, the cytoplasmic form of the enzyme is a 32-kDa dimer containing two copper and two zinc atoms (CuZn SOD) that catalyzes the dismutation of the superoxide anion (O2-) to H2O2 and O2. Superoxide-mediated damage has been implicated in a number of biological processes, including aging and cancer; however, it is not certain whether endogenously elevated levels of SOD will reduce the pathological events resulting from such damage. To understand the in vivo relationship between an efficient dismutation of O2- and oxidative injury to biological structures, we generated transgenic strains of Drosophila melanogaster overproducing CuZn SOD. This was achieved by microinjecting Drosophila embryos with P-elements containing bovine CuZn SOD cDNA under the control of the Drosophila actin 5c gene promoter. Adult flies of the resulting transformed lines which expressed both mammalian and Drosophila CuZn SOD were then used as a novel model for evaluating the role of oxygen radicals in aging. Our data show that expression of enzymatically active bovine SOD in Drosophila flies confers resistance to paraquat, an O2(-)-generating compound. This is consistent with data on adult mortality, because there was a slight but significant increase in the mean lifespan of several of the transgenic lines. The highest level of expression of the active enzyme in adults was 1.60 times the normal value. Higher levels may have led to the formation of toxic levels of H2O2 during development, since flies that died during the process of eclosion showed an unusual accumulation of lipofuscin (age pigment) in some of their cells. In conclusion, our data show that free-radical detoxification has a minor by positive effect on mean longevity for several strains.     

冻干胡蜂蜂蛹对果蝇寿命和繁殖性能的影响

本实验通过研究胡蜂蜂蛹冻干品对果蝇寿命和繁殖力的影响,证明胡蜂蜂蛹具有延缓衰老和增强繁殖性能的作用。采用乙醚麻醉法,收集8h（小时）内羽化的成虫,选择个体大小相近的雌雄果蝇,在不同浓度的培养基中饲养,每两天观察统计一次果蝇死亡数,直到全部死亡。结果表明,雌雄果蝇在5．0％实验组与对照组的平均寿命比较均具有极显著差异性（P〈0．01）,雌雄果蝇在0．2％和1．0％实验组与对照组比较平均寿命差异性显著（P〈0．05）,由此可见胡蜂蜂蛹可延长果蝇平均寿命;0．2％,1．0％组的果蝇子代数显著高于对照组果蝇子代数（P〈0．05）,5．0％组果蝇子代数极显著高于对照组果蝇子代数（P〈0．01）,由此可见冻干胡蜂蜂蛹可增强果蝇的繁殖能力。     

A new strain of Drosophila that inhibits the development of symptoms in imagoes infected with sigma virus

A strain of Drosophila melanogaster bearing the mutant gene ebony has been found to slow the development of symptoms (carbon dioxide sensitivity) in adult flies inoculated with sigma virus, a member of the rhabdovirus group. This inhibition is made evident by comparing mean incubation times of the virus in ebony and wild-type (Oregon) flies. The increase in mean incubation time in ebony flies has ranged from about 3 to 8 days, depending on the virus strain, amount of virus injected, and the age of the flies at the time of inoculation. This delay in development of symptoms appears to be due to a dominant autosomal gene, although further work is needed to confirm this. When accumulation of infectious virus after inoculation is compared in ebony and Oregon flies, there seems to be no inhibition of multiplication in ebony at the level of the entire fly. The relationship of this work to current theories on the mechanism of symptom production by sigma virus is discussed.     

Hh signalling is essential for somatic stem cell maintenance in the Drosophila testis niche

In the Drosophila testis, germline stem cells (GSCs) and somatic cyst stem cells (CySCs) are arranged around a group of postmitotic somatic cells, termed the hub, which produce a variety of growth factors contributing to the niche microenvironment that regulates both stem cell pools. Here we show that CySC but not GSC maintenance requires Hedgehog (Hh) signalling in addition to Jak/Stat pathway activation. CySC clones unable to transduce the Hh signal are lost by differentiation, whereas pathway overactivation leads to an increase in proliferation. However, unlike cells ectopically overexpressing Jak/Stat targets, the additional cells generated by excessive Hh signalling remain confined to the testis tip and retain the ability to differentiate. Interestingly, Hh signalling also controls somatic cell populations in the fly ovary and the mammalian testis. Our observations might therefore point towards a higher degree of organisational homology between the somatic components of gonads across the sexes and phyla than previously appreciated.     

Action of genotypical surrounding of host Drosophila melanogaster on biological effects of endosymbiont Wolbachia (strain wMelPop)

In this work, a comparative study of the structure of symbiotic bacteria Wolbachia (strain wMelPop decreasing the fly lifespan) in genotypically different Drosophila melanogaster, as well as the effect of the bacteria on the host cell ultrastructure was investigated out. As a result of special crossings, the Drosophila melanogaster [w]Trl ³⁶² and [w]Trl ^en82 lines, which are carried of mutations for the gene Trithorax-like, are synthesized (lines infected with Wolbachia are designated as [w]). The Drosophila melanogaster line free of Wolbachia was obtained by treatment with antibiotics of the initially infected [w]w ¹¹¹⁸ line. The complex of the used methods and approaches has allowed us to perform a comparative study of the morphology of cell structures for the first time before and after the infestation of insects with bacteria and to evaluate effect of the bacteria on viability and fertility of flies of these lines. Electron microscopy analysis has shown that the embryos of the analyzed lines contain typical Wolbachia in contact with various host cell compartments; the ultrastructural organization of the bacteria indicates the preservation of their functional activity. In the cytoplasm of embryos that are mutant for the gene Trithorax-like, morphologically atypical mitochondria were revealed, as well as Wolbachia (wMelPop) of unusual morphology with a modified form of membtane envelopes. The presence of Wolbachia in ovarian cells of the female mutant fly lines has been found to produce no effect on the amount of the female-ovipositioned eggs. It has been established for the first time that lifespans of the infected and Wolbachia-free Drosophila melanogaster mutant lines TM3 containing chromosome 3 as a balancer are equal. However, it is significantly shorter in the imago of the [w]w ¹¹¹⁸ line than in flies of the mutant lines. This has allowed us to suggest that either the chromosome-balancer TM3 or mutation of the gene Trl play an important role in the host-symbiont interactions. On checking this suggestion, it was found that the lifespan of homozygotes [w]Trl ³⁶² and [w]Trl ^en82 after the infection of flies with bacteria decreased markedly and was close to the lifespan of [w]w ¹¹¹⁸ line. The obtained data indicate that the chromosome-balancer TM3 can have a significant effect on the symbiont-host interaction.