FAPα酶激活式靶向抗肿瘤前药: Z-GP-Dox对斑马鱼的毒性评价

目的:考察新设计合成的一种FAPα酶激活式靶向抗肿瘤新药甘脯酰阿霉素(Z-GP-Dox)对斑马鱼的毒性作用。方法:以阿霉素作为对照,用不同浓度的Z-GP-Dox处理4月龄的成年斑马鱼及其受精后24h(24hpf)的胚胎,观测其死亡率,并通过显微镜观察Z-GP-Dox对斑马鱼胚胎发育的影响,从形态学和电生理学方面评价其对斑马鱼心脏的毒性作用。结果:Dox对照组的斑马鱼死亡率具有明显的浓度依赖性,而经酰化修饰的前药Z-GP-Dox处理组的斑马鱼死亡率相对较低。Dox可导致斑马鱼胚胎发育严重畸形,心脏功能受损;而相同浓度的前药Z-GP-Dox处理组的胚胎发育基本正常,幼鱼的心脏形态和心率与空白对照组差异不显著。然而,当Z-GP-Dox被FAPα酶解后,其毒性则明显增强,与Dox对照组的毒性相当。结论:与Dox相比,经结构改造的前药Z-GP-Dox对斑马鱼的毒性显著降低,且具有FAPα酶激活式靶向释放特性。     

温度刺激对斑马鱼仔鱼基因转录表达的影响

许多优良鱼类养殖品种不耐低温或高温的特点给水产养殖业带来诸多限制和困难,这些鱼类在胚胎和仔鱼等早期阶段的抗寒和抗热能力比成体更差,育苗过程中很容易受到温度突然变化的影响。虽然目前利用基因芯片技术已研究了温度刺激对几种鱼类成体组织中基因表达的影响,但温度刺激对仔鱼基因转录表达的影响还未见报道。研究以斑马鱼受精后96h的出膜仔鱼为实验材料,分别在低温(16℃)和高温(34℃)条件下处理12h和24h,用基因芯片技术检测温度刺激对其基因表达的影响。与培养在28℃的对照相比,低温和高温处理后共有3633个基因发生差异表达,其中低温处理后差异表达基因数目多于高温处理,而且低温抑制基因数目多于诱导表达基因的数目。生物信息学分析结果表明,低温诱导基因主要参与RNA加工和核糖体生物发生等生物学过程,高温诱导基因则主要参与应激反应和未折叠蛋白结合。低温抑制基因主要参与蛋白质水解、视觉感知以及铁离子结合等生物学功能,高温抑制基因参与的生物学功能包括DNA复制、神经系统过程和类固醇激素生物合成等。除了已报道的温度刺激响应基因外,研究鉴定出了大量尚未报道与温度刺激相关的基因,如参与RNA加工的rnmtl1a和pus3基因,以及参与转录调控的twistnb和aebp2基因等。研究结果为进一步揭示鱼类冷或热适应的分子机理和培养耐寒或耐热的养殖新品种提供理论基础。     

Global analysis of hematopoietic and vascular endothelial gene expression by tissue specific microarray profiling in zebrafish

In this study, we utilize fluorescent activated cell sorting (FACS) of cells from transgenic zebrafish coupled with microarray analysis to globally analyze expression of cell type specific genes. We find that it is possible to isolate cell populations from Tg(fli1:egfp)(y1) zebrafish embryos that are enriched in vascular, hematopoietic and pharyngeal arch cell types. Microarray analysis of GFP+ versus GFP- cells isolated from Tg(fli1:egfp)(y1) embryos identifies genes expressed in hematopoietic, vascular and pharyngeal arch tissue, consistent with the expression of the fli1:egfp transgene in these cell types. Comparison of expression profiles from GFP+ cells isolated from embryos at two different time points reveals that genes expressed in different fli1+ cell types display distinct temporal expression profiles. We also demonstrate the utility of this approach for gene discovery by identifying numerous previously uncharacterized genes that we find are expressed in fli1:egfp-positive cells, including new markers of blood, endothelial and pharyngeal arch cell types. In parallel, we have developed a database to allow easy access to both our microarray and in situ results. Our results demonstrate that this is a robust approach for identification of cell type specific genes as well as for global analysis of cell type specific gene expression in zebrafish embryos.     

双酚A对斑马鱼胚胎的毒性作用

Bisphenol A(BPA)is a chemical estrogen-like sub-stance with properties that are of environmental concern.It is widely used in the chemical industry to manufactureepoxy-and polyester-styrene resins.It has been reportedthat BPA ranges between0and33μg in each plasticcup[1].After atwo-weekexposureto0.5%bisphenol Aithas beenreportedthat disattachments betweensertoli cellsand spermatogonia were observed while spermatogoniawere arrangedin disorder and displacement of spermatogo-nia away fromthe basement membrance ...     

花生四烯酸应用及研究进展

为探究脂肪酸延长酶4b (elovl4b)基因在淡水鱼长链多不饱和脂肪酸(LC-PUFAs)合成中的作用机制, 研究首先使用CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats /CRISPR-associated systems)系统成功构建elovl4b 基因第2外显子上缺失2个碱基的基因突变纯合子模型(elovl4b^–/–), 并分析elovl4b基因敲除后斑马鱼肝脏的脂肪酸组成变化。分析发现, 相比于野生型(WT)斑马鱼, elovl4b^–/–斑马鱼肝脏总脂肪酸占比、C22PUFA占比显著上升, 多种LC-PUFAs组成占比出现显著变化。进一步进行斑马鱼肝脏的转录组测序, 创建基因文库, 筛选出差异表达基因进行富集分析和数据库功能注释分析, 用qPCR技术对KEGG 通路中的差异表达基因(P<0.05)进行验证。结果显示, 基因表达谱变化显著, 共有809个基因表达出现显著性差异(485 个下调, 324 个上调), 包括磷酸戊糖途径在内的多条通路显著富集。脂肪酸合成等通路显著上调, 花生四烯酸代谢、初级胆汁酸合成等通路显著下调。随机选取12条基因通过qPCR (Real-time Quantitative PCR)进行验证, 基因的表达趋势与转录组测序所得结果基本一致。上述结果说明elovl4b缺失后, 机体内各类LC-PUFAs的组成及包括花生四烯酸代谢、脂肪酸代谢、脂肪生成和代谢在内的许多重要的机体生命过程发生了相应性的变化, 进一步揭示了elovl4b在LC-PUFAs合成过程中的作用, 能为相关的心血管、慢性肥胖等疾病的治疗提供理论依据。

     

多氯联苯(PCB1254)对斑马鱼(Brachydanio rerio)毒性及其组织结构的影响

本文报道了多氯联苯(PCB₁₂₅₄)对斑马鱼(Brachydanio rerio)的亚急性毒性试验及其组织结构的影响。结果表明,PCB₁₂₅₄对斑马鱼存活最大无影响浓度(NOEC)为0.40μg·L^-1,最低有影响浓度(LOEC)为2.0μg·L^-1, PCB₁₂₅₄对斑马鱼30d半致死剂量为5.09μg·L^-1,95% 可信区间范围为3.79～6.52μg·L^-1。在不同浓度的PCB₁₂₅₄作用30d后,斑马鱼的组织结构发生明显变化, PCB₁₂₅₄浓度大于2μg·L^-1时,对斑马鱼鳃组织呼吸上皮细胞结构产生损伤而影响其呼吸代谢;高浓度PCB₁₂₅₄(50μg·L^-1)对斑马鱼肝脏细胞结构产生明显损害,肝脏细胞结构表现出异常,肝细胞核变形萎缩,并有大量脂褐素沉积。     

Morpholino artifacts provide pitfalls and reveal a novel role for pro-apoptotic genes in hindbrain boundary development

Morpholino antisense oligonucleotides (MOs) are widely used as a tool to achieve loss of gene function, but many have off-target effects mediated by activation of Tp53 and associated apoptosis. Here, we re-examine our previous MO-based loss-of-function studies that had suggested that Wnt1 expressed at hindbrain boundaries in zebrafish promotes neurogenesis and inhibits boundary marker gene expression in the adjacent para-boundary regions. We find that Tp53 is highly activated and apoptosis is frequently induced by the MOs used in these studies. Co-knockdown of Tp53 rescues the decrease in proneural and neuronal marker expression, which is thus an off-target effect of MOs. While loss of gene expression can be attributed to cell loss through apoptotic cell death, surprisingly we find that the ectopic expression of hindbrain boundary markers is also dependent on Tp53 activity and its downstream apoptotic effectors. We examine whether this non-specific activation of hindbrain boundary gene expression provides insight into the endogenous mechanisms underlying boundary cell specification. We find that the pro-apoptotic Bcl genes puma and bax-a are required for hindbrain boundary marker expression, and that gain of function of the Bcl-caspase pathway leads to ectopic boundary marker expression. These data reveal a non-apoptotic role for pro-apoptotic genes in the regulation of gene expression at hindbrain boundaries. In light of these findings, we discuss the precautions needed in performing morpholino knockdowns and in interpreting the data derived from their use.     

A spectrophotometric assay for feruloyl esterases

We have developed a spectrophotometric assay for the quantitative determination of feruloyl esterase activity based on release of 4-nitrophenol from a novel substrate, 4-nitrophenyl ferulate in an emulsion of Triton X-100 in aqueous buffer solution. The release of 4-nitrophenol was linear with reaction time at an early stage of the reaction with various esterase preparations. The method proposed here is accurate, rapid, and easy to perform.     

不同生存条件下东方田鼠肝脏基因表达谱分析

目的比较实验室条件下饲养的东方田鼠和野外捕捉东方田鼠肝脏的基因表达差异,寻找可能参与肝脏病变的关键基因。方法以实验室条件饲养的东方田鼠和野外捕捉东方田鼠为研究对象,分别抽提RNA,逆转录成cDNA,体外转录为cRNA并进行片段化;利用表达谱芯片分别进行杂交,扫描后筛选差异基因,并应用real-time PCR方法对部分基因的表达水平进行进一步测定,验证芯片数据的结果。结果实验室饲养东方田鼠肝组织与野外捕捉东方田鼠相比,共有99个基因和41个EST差异表达。其中参与机体代谢的基因占主导,约占35.4%;其次为参与信号通路的基因,约占24.2%;参与细胞周期和免疫的基因分别占6.1%和3.0%。结论利用基因表达谱芯片初步筛选了可能参与东方田鼠脂肪肝形成过程的基因,发现机体代谢通路的基因占主导,肝脏中细胞色素家族基因表达差异明显。