sox19b在斑马鱼胚胎眼睛发育中的作用

目的 通过抑制sox19b基因的表达,探讨sox19b基因在斑马鱼胚胎眼睛发育和形成中的作用。方法 通过显微注射sox19b 吗啉寡聚核苷酸（MO）抑制sox19b 基因的表达,观察胚胎发育过程中表型的变化;采用石蜡包埋组织切片及HE染色、RT-PCR和整封原位杂交等方法探讨敲除sox19b 后对斑马鱼胚胎眼睛发育的调控机制。 结果 敲除sox19b 基因后,斑马鱼胚胎眼睛发育受到影响,表现为眼睛变小或缺失,视网膜及晶状体结构发育异常(n =57/93);眼睛发育过程中重要调控基因 rx3、pax2a及 vsx2 等表达明显降低,进而影响眼睛的发育和形成。 结论 sox19b 基因作为转录调控因子,可以通过调节眼区转录因子的表达进而影响斑马鱼胚胎早期眼睛的发育和形成。     

斑马鱼胚胎发育过程中miR-9、miR-219及Dicer基因的时序性表达

目的探讨斑马鱼胚胎发育过程中miR-9、miR-219及Dicer基因时序性表达规律。方法取正常发育至受精后4、8、12、16、20、24、36、48和72 h(hpf)斑马鱼胚胎各约40枚,用Trizol法分别提取总RNA,茎环法反转录为c DNA,用Real-time PCR检测胚胎miR-9、miR-219及Dicer表达量。结果 miR-9、miR-219及Dicer表达水平在胚胎发育过程中呈现峰谷变化,不同发育阶段之间总的差异有统计学意义(P0.05)。与胚胎发育全程的基因平均表达水平相比,miR-9在8 hpf之前表达量较低(P0.05),36 hpf后表达量持续增高,48 hpf和72 hpf时表达量显著高于平均水平(P0.01)。miR-219在8 hpf之前表达量也处于较低水平(P0.05),20 hpf后迅速增高,24hpf时达到峰值,并持续到48 hpf(P0.01)。Dicer在胚胎早期(4 hpf)就有明显表达(P0.05),随后表达水平迅速下降,16 hpf时达到低点(P0.05),然后逐渐回升,直至胚胎末期。结论斑马鱼胚胎发育过程miR-9、miR-219及Dicer时序性表达有一定规律,miR-9主要在胚胎后期表达,miR-219主要在胚胎中期表达,Dicer主要在胚胎早期表达,其次在胚胎后期,提示miR-9、miR-219及Dicer参与胚胎发育调控过程。     

zdhhc17对斑马鱼集中延伸运动的调节及其机制 

目的 通过抑制zdhhc17基因的表达,探讨其在斑马鱼胚胎发育过程中的作用及机制。方法 通过显微注射zdhhc17吗啉反义寡聚核苷酸(MO)抑制zdhhc17翻译,观察斑马鱼胚胎发育过程中相关表型的变化。利用斑马鱼整封原位杂交技术,检测集中延伸运动相关标记基因（dlx3b、ntl、myod1、 tbx6等）的表达变化。利用RT-PCR技术,检测集中延伸运动相关调控因子mRNA表达水平的变化。 结果 敲除zdhhc17基因后,斑马鱼胚胎集中延伸运动出现严重缺陷,主要表现为尾部弯曲变短（82/118）,且集中延伸运动相关标记基因表达异常,钙黏附蛋白家族cdh1、padh18a mRNA水平降低。结论 zdhhc17通过调节钙黏附蛋白家族cdh1、padh18a基因的表达,进而影响细胞黏附运动,最终参与调控斑马鱼胚胎集中延伸运动。     

小鼠发育期卵巢成纤维细胞生长因子10的表达

目的 研究小鼠卵巢发育期成纤维细胞生长因子10(FGF10)的表达. 方法 分离孕E14.5、E16.5、E18.5、生后第1天和3周龄小鼠卵巢,每组实验动物6只,应用免疫组织化学技术研究FGF10蛋白在卵巢中的表达;3周龄小鼠腹腔注射孕马血清促性腺激素(PMSG)48h后,再注射人绒毛膜促性腺激素(hCG),hCG注射后3h提取总mRNA,应用RT-PCR技术研究卵巢中FGF10、黄体生成素受体(LHR)、成纤维细胞生长因子7(FGF7)和成纤维细胞生长因子受体2Ⅲb(FGFR2Ⅲb)基因的表达变化. 结果 FGF10蛋白表达于卵母细胞,并且FGF10在生后第1天小鼠的卵母细胞上表达最强;两种促性腺激素处理后,FGF10 mRNA 与对照组相比表达下降,LHR mRNA表达升高,FGF7和FGFR2Ⅲb mRNA表达不变. 结论 FGF10蛋白特异表达于小鼠卵母细胞膜,促性腺激素影响卵巢FGF10 mRNA的表达,推测FGF10可能参与调控小鼠卵母细胞的生长和卵泡发育.     

鱼藤素对斑马鱼胚胎中细胞周期蛋白D1基因下调的作用:整胚原位杂交检测

背景:斑马鱼胚胎发育早期,细胞分裂增殖快速,低浓度的鱼藤素可以延缓斑马鱼胚胎发育。目的:观察鱼藤素对斑马鱼胚胎中细胞周期蛋白D1基因的表达影响。方法:收集正常受精后发育至2-cell期(受精后约0.75 h)以及shield期(受精后6 h)斑马鱼胚胎于12孔板,分别用100,200,400 nmol/L鱼藤素在28.5℃的培养箱中避光处理至shield期及受精后24 h。同时用1%的二甲基亚砜溶液作为对照组,于相同条件下培养。后收集对照组及实验组胚胎,制备细胞周期蛋白D1 RNA探针进行整胚原位杂交,于正置荧光显微镜下拍照观察染色情况以检测鱼藤素对胚胎中细胞周期蛋白D1基因的表达影响。结果与结论:鱼藤素对斑马鱼胚胎中细胞周期蛋白D1基因的表达有明显的负调控作用。Shield期对照组胚胎细胞周期蛋白D1在外包细胞中泛表达,随着给药浓度的增加,细胞周期蛋白D1的表达显著减少,400 nmol/L鱼藤素处理组几乎不表达。受精后24 h的胚胎中细胞周期蛋白D1在脑部、中枢神经系统、发育未成熟的眼部、体节、躯干、尾部等有表达,100 nmol/L鱼藤素处理组表达明显下调,在增殖性区域几乎不表达。200,400 nmol/L处理组胚胎发育明显延缓,细胞周期蛋白D1主要表达于背侧外胚层细胞。     

长春碱增加班马鱼肿瘤耐药基因abcb4的表达

目的通过研究长春碱(VBL)对斑马鱼早期发育过程中abcb4肿瘤耐药基因表达的影响,为进一步建立抗肿瘤药物多药耐药筛选的斑马鱼模型奠定重要的实验基础。方法将长春碱稀释成不同浓度梯度的药液,在受精后0.5~1.5 h的斑马鱼胚胎上完成药物暴露实验,计算出长春碱IC_(50)的数值。设置长春碱为实验组,胚胎培养液为空白对照组,分别作用于受精后0.5~1.5 h的斑马鱼胚胎,观察受精后24~120 h的斑马鱼胚胎发育情况,记录死亡、孵化及畸形的胚胎数目;收集不同组别的斑马鱼胚胎,通过实时荧光定量PCR和胚胎整体原位杂交的方法,检测斑马鱼胚胎中abcb4基因的表达量。结果计算出长春碱在斑马鱼胚胎中的IC_(50)为3.08μmol/L;与对照组比较,实验组的斑马鱼胚胎abcb4基因的mRNA水平明显升高(P0.05);通过胚胎整体原位杂交的方法,在受精后120 h斑马鱼胚胎的小肠部位发现有abcb4基因阳性杂交信号,且实验组斑马鱼胚胎在脑和心脏部位发现abcb4基因阳性杂交信号。结论长春碱能明显增加斑马鱼早期胚胎中abcb4肿瘤耐药基因的表达水平,提示使用斑马鱼可以复制肿瘤耐药模型。     

鱼藤素可影响斑马鱼胚胎Wnt信号通路相关基因的表达

背景：前期研究发现,在斑马鱼早期发育的不同时期,经过一定浓度鱼藤素处理的胚胎表现出不同程度的发育延缓和表型异常。 目的：观察斑马鱼早期胚胎发育过程中鱼藤素对wnt信号通路相关基因表达的影响。 方法：收集孵育至shield期的斑马鱼胚胎于6孔板中,加入0.6 μmol/L浓度的鱼藤素溶液,避光处理,28 ℃培养箱中孵养。用0.1%二甲基亚砜溶液作为对照组,相同条件培养。在受精后6,8,10,12,14,24 h时用正置荧光显微镜拍照观察鱼藤素处理后斑马鱼胚胎发育表型。实时荧光定量核酸扩增检测鱼藤素作用前后目的基因表达水平的变化。 结果与结论：shield期处理的斑马鱼胚胎在bud期停止生长;wnt信号通路上的wnt9a,wnt10b和axin2以及与wnt信号具有密切联系的snail1b,Jnk2表达发生显著变化,其中wnt9a,wnt10b,axin2,snail1b表达下调,Jnk2表达上调。结果提示鱼藤素对斑马鱼体内wnt信号具有负调节作用。     

NGF和bFGF及其受体在金黄地鼠神经管的定位

用免疫组织化学和免疫电镜方法研究了神经生长因子 (NGF)和碱性成纤维细胞因子 (b FGF)及其受体 (Trk A、FGFR)在金黄地鼠神经管发育中的定位分布。结果显示 :NGF和 b FGF的表达时序不同 ,但与各自受体的表达时序基本一致 ,其定位相似。受精后第 8d即出现 b FGF阳性细胞 ,NGF于第 9d出现 ,随着胚龄增加 ,免疫阳性着色逐渐增强 ,二者均定位于神经细胞胞质、神经管内界膜、外界膜 ,脊索和脊神经节细胞胞质中 ,b FGF还分布于神经细胞核。第 8d可检测到少量散在的 FGFR金标颗粒 ,Trk A于第 9d出现 ,随着胚胎的发育 ,金标颗粒逐渐增多 ,主要定位于细胞膜、胞质的基质、内质网、核膜及胞核中。上述结果表明 ,NGF、b FGF及其受体在金黄地鼠胚神经管形成和分化中按着一定的时序出现于细胞的一定部位     

急性免疫性血小板减少症患儿IgG-Fc受体表达变化

目的 探讨小儿急性免疫性血小板减少症(aITP)外周血单核细胞(MC) IgG-Fc受体(FcγR)表达变化.方法 急性ITP患儿组27例,同龄健康对照组25例,流式细胞术检测单核细胞表面活化型受体FcγR Ⅰ及FcγRⅢ表达;荧光定量PCR( real-time PCR)检测MC活化型FcγRⅡa及抑制型FcγRⅡb mRNA表达;酶联免疫吸附法(ELISA)检测血浆IFN-γ、IL-4和IL-10浓度.结果 (1)急性ITP患儿组MC FcγR Ⅰ和FcγRⅢ表达明显高于对照组(P＜0.05);FcγRⅡa mRNA及FcγRⅡa/FcγRⅡb mRNA比值显著高于对照组及治疗后,经地塞米松治疗后,活化型FcγR Ⅰ、FcγRⅢ、FcγRⅡa mRNA表达下降,抑制型FcγRⅡb mRNA表达增加,FcγR活化型/抑制型平衡逐渐恢复.(2)血浆炎症细胞因子IFN-γ浓度较对照组显著增高;抗炎因子IL-4显著低于对照组.IFN-γ浓度水平与MC FcγRⅡa mRNA表达显著正相关(r=0.79,P＜0.05),IL-4与FcγRⅡb mRNA表达正相关(r=0.84,P＜0.05).结论 单核细胞活化型FcγRs过度表达,抑制型FcγRⅡb表达降低,活化型/抑制型FcγRs表达失衡可能参与儿童急性ITP免疫发病机制,MC FcγRs异常表达可能与细胞因子IFN-γ、IL-4的改变有关,地塞米松治疗可恢复FcγRs活化型/抑制型平衡表达.     

基因芯片分析鱼藤素对斑马鱼胚胎发育的影响

背景：鱼藤素具有延缓斑马鱼胚胎发育的作用,但其具体作用机制尚未明确。 目的：分析鱼藤素处理斑马鱼胚胎的基因与信号传导通路表达差异。 方法：斑马鱼胚胎来自于根据Zebrafish book中描述的方法养的殖斑马鱼。用0.6 μmol/L鱼藤素处理2-细胞期斑马鱼胚胎,以二甲基亚砜孵化液处理2-细胞期斑马鱼胚胎为对照。4 hpf 时期提取二甲基亚砜组及鱼藤素组总RNA,利用基因芯片技术检测鱼藤素处理斑马鱼胚胎后基因的差异表达,采用实时荧光定量PCR验证基因芯片结果,结合聚类分析及通路分析来探索鱼藤素的作用机制。 结果与结论：芯片结果显示,鱼藤素处理斑马鱼胚胎后差异表达上调3倍以上的基因407个,下调的461个。PCR 验证结果与芯片结果相符。经KEGG 通路分析,涉及14条通路。鱼藤素可能通过干扰细胞的代谢生长及影响其分化起作用。     

胚胎期铅暴露对斑马鱼胚胎及幼鱼NMDA受体mRNA表达的影响

目的:了解胚胎期铅暴露对斑马鱼胚胎及幼鱼N-甲基-D-天冬氨酸(NMDA)受体mRNA表达的影响。方法:野生型AB品系斑马鱼胚胎醋酸铅暴露浓度分别为0、0.1、0.5、2.5和12.5 μmol/L,提取各组受精后24、48、72、96和120 h(hpf)斑马鱼胚胎或幼鱼总RNA,实时定量PCR检测NMDA受体亚基NR1.1、NR1.2和NR2B的mRNA表达量。结果:(1)对照组NR1.1和NR1.2及NR2B表达量在胚胎发育过程中逐渐升高,在孵化期(72 hpf)表达量增加明显,在幼鱼早期(96 hpf)时达到高峰(与24 hpf时比较, P<0.01),在120 hpf时仍处于较高水平。(2)随着铅暴露浓度增高,NR1.1表达量增加并有高峰前移的趋势,2.5 μmol/L和12.5 μmol/L铅暴露组NR1.1表达高峰期在72 hpf,并且显著高于对照组(P<0.05);铅暴露组NR1.2和NR2B动态表达也呈类似规律,但NR1.2表达高峰期呈平台化趋势,横跨72 hpf至120 hpf阶段,NR2B表达高峰期出现在72 hpf和120 hpf阶段。(3)NR1.1、NR1.2及NR2B mRNA表达量之间Pearson相关系数值分别为r_NR1.1-1.2 =0.681、r_NR1.1-2B=0.637和r_NR1.2-2B =0.514,均有统计学意义(P<0.01)。结论:在斑马鱼胚胎发育过程NR1.1、NR1.2及NR2B mRNA表达水平逐渐升高,在幼鱼早期达到高峰;胚胎和幼鱼阶段NR1.1、NR1.2及NR2B之间mRNA表达水平存在关联;铅有上调NR1.1、NR1.2和NR2B mRNA表达作用并使表达峰期前移,改变了正常的NMDA受体表达规律。     

Expression of TMEM16 paralogs during murine embryogenesis

The TMEM16 protein family has recently been identified through several different experimental strategies including bioinformatic and microarray-based approaches. In mice and humans, there exist 10 paralogs with each containing eight putative transmembrane domains and a conserved C-terminal domain of unknown function. Mutation of at least one member of this family is associated with a human disorder, and several members of this gene family are overexpressed in different types of cancer. Despite their apparent relevance to normal development and disease, little is known about the expression of TMEM16 paralogs during embryonic development. Here, we provide a phylogenetic analysis of mouse and human TMEM16 paralogs and report the expression of Tmem16a, Tmem16b, Tmem16c, Tmem16f, Tmem16h, Tmem16j, and Tmem16k during murine embryogenesis with an emphasis on the respiratory, digestive, skeletal, and integumentary systems. These data should encourage investigations into the functions of TMEM16 paralogs in vertebrate development.     

Expression of the Ku70 subunit (XRCC6) and protection from low dose ionizing radiation during zebrafish embryogenesis

The Ku70 protein, a product of the XRCC6 gene, is a component of the nonhomologous end-joining (NHEJ) pathway of DNA repair, which protects cells from the effects of radiation-induced DNA damage. Although the spatial expression of Ku70 during vertebrate embryogenesis has not been described, DNA repair proteins are generally considered to be "housekeeping" genes, which are required for radioprotection in all cells. Here, we report the cloning and characterization of the zebrafish Ku70 ortholog. In situ hybridization and RT-PCR analyses demonstrate that Ku70 mRNA is maternally provided and expressed uniformly among embryonic blastomeres. Later during embryogenesis, zygotically transcribed Ku70 mRNA specifically accumulates in neural tissue, including the retina and proliferative regions of the developing brain. In the absence of genotoxic stress, morpholino-mediated knockdown of Ku70 expression does not affect zebrafish embryogenesis. However, exposure of Ku70 morpholino-injected embryos to low doses of ionizing radiation leads to marked cell death throughout the developing brain, spinal cord, and tail. These results suggest that Ku70 protein plays a crucial role in protecting the developing nervous system from radiation-induced DNA damage during embryogenesis.     

Embryonic and larval expression of zebrafish voltage-gated sodium channel alpha-subunit genes.

Whereas it is known that voltage-gated calcium channels play important roles during development, potential embryonic roles of voltage-gated sodium channels have received much less attention. Voltage-gated sodium channels consist of pore-forming alpha-subunits (Na(v)1) and auxiliary beta-subunits. Here, we report the embryonic and larval expression patterns for all eight members of the gene family (scna) coding for zebrafish Na(v)1 proteins. We find that each scna gene displays a distinct expression pattern that is temporally and spatially dynamic during embryonic and larval stages. Overall, our findings indicate that scna gene expression occurs sufficiently early during embryogenesis to play developmental roles for both muscle and nervous tissues.     

TLR4、MyD88、NF-κBmRNA在实验性自身免疫性肌炎小鼠淋巴结中的表达及意义

目的:研究TLR4、MyD88、NF-κB mRNA在实验性自身免疫性肌炎小鼠淋巴结中的表达情况,探讨TLR4在肌炎发病中的作用。方法:将30只雌性BALB/c小鼠随机分为5组(每组6只):第1组为正常对照组,其它4组分别为肌炎模型1周处理组、2周处理组、3周处理组和4周处理组,后4组均应用肌球蛋白诱导实验性自身免疫性肌炎模型(EAM)。采用实时荧光定量PCR方法检测各组小鼠淋巴结TLR4、MyD88、NF-κB mRNA表达水平。结果:(1)与正常小鼠相比,肌炎各组小鼠淋巴结中TLR4、MyD88、NF-κB mRNA表达均有统计学差异,表现为不同程度升高(P<0.01),第3组升高最为显著(P<0.01),第4组较第5组升高(P<0.01);(2)各组淋巴结中TLR4 mRNA表达水平与MyD88 mRNA、NF-κBmRNA表达水平均呈正相关(r=0.906,r=0.967,P<0.01),MyD88 mRNA表达水平与NF-κB mRNA表达水平呈正相关(r=0.919,P<0.01)。结论:TLR4在自身免疫性肌炎发生发展过程中发挥重要作用,且以MyD88依赖型信号途径为主,并通过激活NF-κB促进炎性因子释放。     

Expression and phylogenetic analyses of three zebrafish FoxI class genes.

We have identified three novel members of the zebrafish forkhead class I gene family, which we have named foxi2, foxi3a, and foxi3b. We have reported previously the identification of zebrafish foxi1, which is required for otic placode and jaw development. Expression analysis shows that foxi2 is expressed within the chordamesoderm during early somitogenesis and the retina and pharyngeal arches during later stages. The foxi3a and foxi3b genes likely represent a recently duplicated pair, and they are similarly expressed in epidermal mucous cells throughout embryogenesis and early larval stages. None of these newly identified FoxI genes are expressed in otic precursor cells and, therefore, are unlikely to share functional overlap with foxi1 in the development of the inner ear. In addition to these zebrafish FoxI paralogs, we have identified 16 new FoxI sequences in species ranging from Ciona intestinalis to Homo sapiens. We present an extensive phylogenetic analysis of the FoxI class that includes these new sequences together with those previously reported. This analysis supports the existence of three subfamilies within the FoxI class, each containing at least one zebrafish member.