目的:利用生物信息分析方法对骨关节炎软骨下骨全基因表达谱进行转录因子预测及分析.方法:下载基因芯片实验数据(GSE30322),使用软件包limma packagein R(版本:3.3.1)筛选差异表达基因,筛选差异基因的标准以基因表达上调或下调的倍数≥2为标准(P＜0.05),进一步使用Cytoscape软件(版本:3.4.0)的iRegulon插件预测分析调控这些差异表达基因的转录因子,并分析预测的转录因子所调控的差异表达基因.结果:发现上调的差异表达基因可能相关15个转录因子及其相对应的靶基因:FOXN4,NANOS1,E2F6,RAD21,MECOM,ETS1,MEF2A,POU2F3, BRCA1,GATA3,ZNF706,ZBTB33,SUZ12,DBP,SETDB1等.发现下调的差异表达基因可能相关12个转录因子及其相对应的靶基因:ARID3A,YY1,RDBP,ATF1,CRX,TAF1,XBP1,SOX3,E2F4,PGR,TIMM8A,HOXA2等.结论:预测分析的转录因子可能在骨关节炎软骨下骨致病机制调控中起了重要作用,其有可能成为新的防治骨关节炎的靶点.

目的:研究新型铜胺纳米材料(Cu-Cy-Nanos)对不同肿瘤细胞生长的影响,为后续研究提供新的理论基础和实验依据.方法:采用RNO脱色实验检测Cu-Cy-Nanos单线态氧的产量;MTT实验对比不同肿瘤细胞对Cu-Cy-Nanos的敏感性.结果:Cu Cy-Nanos受到紫外光的激发后可以产生单线态氧;MTT结果显示,Cu-Cy-Nanos对3种不同肿瘤细胞的生长均具有抑制作用(均P＜0.05),但不同癌细胞对Cu-Cy-Nanos的敏感性不同.Cu-Cy-Nanos对肝癌细胞的抑制作用最为明显(P＜0.05).结论:Cu-Cy-Nanos是具有潜在肝癌治疗效应的新型光敏剂.

原始生殖细胞(primordial germ cells,PGCs)的起源和迁移已在多种鱼类进行了研究,但多倍体鲫鲤原始生殖细胞的标记和迁移尚未见报道.本实验室前期通过远缘杂交方法成功获得了一个倍性明确、亲缘关系清晰的多倍体杂交鱼研究体系,该体系由异源四倍体鲫鲤及其二倍体父母本和三倍体杂交后代组成.本文利用RNA定点表达(localized RNA expression,LRE)技术,将来自于斑马鱼的nanos 1-3’-UTR与GFP融合后生成的mRNA注射入多倍体鱼受精卵中,首次对多倍体鲫鲤原始生殖细胞进行标记,并观察了其迁移途径.结果显示,多倍体鲫鲤PGCs能被斑马鱼GFP-nanos 1-3’-UTR标记,并且多倍体鲫鲤PGCs的迁移与斑马鱼类似.本实验结果为多倍体鲫鲤原始生殖细胞的产生和迁移的研究提供了一些基础数据.

为了标记团头鲂(Megalobrama amblycephala)的原始生殖细胞(Primordial Germ Cells,PGCs),首次克隆并鉴定了团头鲂nanos3基因(mananos3).mananos3全长1027 bp,包括48 bp 5′UTR(5′untranslated Region),490 bp 3′UTR和489 bp开放阅读框(Open Reading Frame,ORF).该基因编码162个氨基酸.通过序列比对发现Mananos3蛋白和其他物种Nanos蛋白一样,存在一个保守的RNA结合功能域,该功能域包含一个锌指基序(Motif).系统发育树结果显示,Mananos3与鲤(Cyprinus carpio)的Nanos3最为相近.半定量和定量PCR结果表明,mananos3具有较高的母源表达,并在胚胎发育早期高量表达,而在1000细胞期之后表达量逐渐降低.在成体组织中,mananos3仅在卵巢中检测到表达.mananos3和斑马鱼(Danio rerio)nanos3(zfnanos3)的3′UTR均可以介导绿色荧光蛋白特异标记团头鲂和斑马鱼胚胎发育早期的PGCs,但是mananos3的3′UTR能够更特异地标记团头鲂的PGCs.通过比对mananos3和zfnanos3的3′UTR发现,mananos3的3′UTR中有一个非经典的miR430识别位点(GCACTA).通过对该位点的突变研究证实其有利于nanos3在非PGCs组织中的降解.综上所述,团头鲂mananos3的3′UTR序列中的非经典miR430识别位点(GCACTA)可能与介导报告基因在PGCs中特异表达相关.

目的 探讨nanos1基因在日本血吸虫的定位以及不同发育时期的表达情况.方法 在NCBI上搜索日本血吸虫nanos1基因,PCR扩增、测序并将其全长序列经BLAST软件分析,利用DNAMAN6.0和ClustalX2.0.9进行多重序列比对,利用MAGA 5.05构建进化树进行系统进化分析.体外转录地高辛(DIG)标记的RNA探针,并将其用于日本血吸虫感染后第18、24和42天3个发育时期的雌雄虫整体原位杂交,以确定nanos1基因在日本血吸虫的定位.结果 PCR扩增获得日本血吸虫nanos1基因,生物信息学分析结果表明其编码的蛋白与曼氏血吸虫nanos2蛋白同源性最高.体外转录成功的RNA探针用于日本血吸虫整体原位杂交,在感染后第24、42天雌虫的卵巢和卵黄腺以及18天雌虫的卵巢检测到阳性杂交信号,而各个发育阶段的雄虫均未观察到明显阳性杂交信号.结论 日本血吸虫nanos1基因高表达于感染后第18天雌虫的卵巢以及第24、42天雌虫的卵巢和卵黄腺.该研究初步揭示了日本血吸虫nanos1基因的表达模式,为研究nanos1基因的功能提供了重要线索.

Piwi-interacting RNAs (piRNAs) and PIWI proteins are essential in germ cells to repress transposons and regulate mRNAs.In Drosophila,piRNAs bound to the PIWI protein Aubergine (Aub) are transferred maternally to the embryo and regulate maternal mRNA stability through two opposite roles.They target mRNAs by incomplete base pairing,leading to their destabilization in the soma and stabilization in the germ plasm.Here,we report a function of Aub in translation.Aub is required for translational activation of nanos mRNA,a key determinant of the germ plasm.Aub physically interacts with the poly(A)-binding protein (PABP)and the translation initiation factor elF3.Polysome gradient profiling reveals the role of Aub at the initiation step of translation.In the germ plasm,PABP and elF3d assemble in foci that surround Aub-containing germ granules,and Aub acts with elF3d to promote nanos translation.These results identify translational activation as a new mode of mRNA regulation by Aub,highlighting the versatility of PIWI proteins in mRNA regulation.

A large proportion of patients with idiopathic spermatogenic failure(SPGF;oligozoospermia or nonobstructive azoospermia[NOA])do not receive a diagnosis despite an extensive diagnostic workup.Recent evidence has shown that the etiology remains undefined in up to 75％of these patients.A number of genes involved in germ-cell proliferation,spermatocyte meiotic divisions,and spermatid development have been called into play in the pathogenesis of idiopathic oligozoospermia or NOA.However,this evidence mainly comes from case reports.Therefore,this study was undertaken to identify the molecular causes of SPGF.To accomplish this,15 genes(USP9Y,NR5A1,KLHL10,ZMYND15,PLK4,TEX15,TEX11,MEIOB,SOHLH1,HSF2,SYCP3,TAF4B,NANOS1,SYCE1,and RHOXF2)involved in idiopathic SPGF were simultaneously analyzed in a cohort of 25 patients with idiopathic oligozoospermia or NOA,accurately selected after a thorough diagnostic workup.After next-generation sequencing(NGS)analysis,we identified the presence of rare variants in the NR5A1 and TEX11 genes with a pathogenic role in 3/25(12.0％)patients.Seventeen other different variants were identified,and among them,13 have never been reported before.Eleven out of 17 variants were likely pathogenic and deserve functional or segregation studies.The genes most frequently mutated were MEIOB,followed by USP9Y,KLHL10,NR5A1,and SOHLH1.No alterations were found in the SYCP3,TAF4B,NANOS1,SYCE1,or RHOXF2 genes.In conclusion,NGS technology,by screening a specific custom-made panel of genes,could help increase the diagnostic rate in patients with idiopathic oligozoospermia or NOA.

Small RNAs, including miRNAs, siRNAs, and PIWI-interacting RNAs (piRNAs), are small noncoding RNAs that are 21-30 nucleotides in length and play important roles in diverse biological processes.The zebrafish (Danio rerio) is a model organism, and small RNAs have been explored in its embryos, sexual glands,adult tissues, ovarian follicular cells, and spermatozoa.However,little is known about small RNAs in zebrafish primordial germ cells (PGCs) because of the difficulties of cell isolation and rare cell numbers.For zebrafish, PGCs are formed by means of preformation and can determine the fate of reproduction.By means of transgenic zebrafish carrying a kop-egfp-nanos3-3'UTR inserted fragment [1]and microscale small RNA high-throughput sequencing p2], the profile of small RNAs in early zebrafish PGCs was characterized in thisstudy.Three developmental stages were included in this study: 6 h post-fertilization (hpf;shield stage), 11 hpf (3-somite stage), and 24 hpf (prim-5 stage).

旨在探讨军曹鱼nanos1基因(Rcnanos1)的序列特征,及其在胚胎和性腺发育过程中的功能,利用cDNA末端快速扩增技术(RACE)克隆获得Rcnanos1基因的cDNA序列,全长1290 bp,其中5'非编码区139 bp,3'非编码区470 bp,开放阅读框(ORF)681 bp,共编码226个氨基酸.序列多重比对结果显示,RcNanos1氨基酸序列包含两个连续的锌指功能结构域,与尖吻鲈(Lates calcarifer)Nanos1的一致性最高(99.6％).系统进化分析表明军曹鱼Nanos1与尖吻鲈(Lates calcarifer)和斜带石斑鱼(Epinephelus coioides)的同源蛋白亲缘关系最近.实时荧光定量PCR(qRT-PCR)结果显示,Rcnanos1在150 dph(days post hatching)军曹鱼的13种组织中均有表达,但在性腺中的表达量显著高于其它组织.胚胎发育过程中,Rcnanos1在早期卵裂阶段的表达水平较低,16细胞期时表达量开始显著升高,并在1 dph仔鱼中的表达量上升至最大值.Rcnanos1在精巢和卵巢首周年发育过程中的表达模式相似,在精巢中(Ⅱ-Ⅴ期),Rcnanos1的表达水平呈逐渐升高趋势,360 dph(Ⅴ期)时的表达量最高;在卵巢中(Ⅰ-Ⅲ期),Rcnanos1的表达量同样在360 dph(Ⅲ期)时达到峰值.由此推测,Rcnanos1基因可能在胚胎和性腺发育过程发挥一定的调控作用,可为揭示军曹鱼生殖细胞发生发育的分子机理提供理论基础.

目前,水牛精原干细胞(spermatogonial stem cells,SSCs)体外培养系统因尚无法支持水牛(Bubalus bubalis)SSCs体外长久培养而限制了水牛雄性遗传资源的高效利用.通过添加合适小分子化合物优化水牛SSCs体外培养系统是一个可选方案.本研究推测GSK3β特异性抑制剂CHIR99021能促进水牛精原细胞的体外增殖和自我更新.为了验证此假说,本研究探索了添加不同浓度CHIR99021对水牛精原细胞体外培养效果的影响.分别将不同浓度CHIR99021(0,5,10,20 μmol/L)添加到水牛精原细胞体外培养细胞培养液中,设置4个试验组,体外培养7 d后收集细胞,进行实时定量PCR(real-time quantitative PCR,RT-qPCR)和免疫荧光染色细胞计数.RT-qPCR结果表明,添加10、20 μmol/L的CHIR99021显著提高了 SSCs特异性表达基因(PLZF,Nanos2)和增殖相关基因(CCND1,CCNE1)的表达水平;显著降低了分化相关基因(DMRT1,DAZL)和氧化相关基因(CAT,SOD3)的表达水平.通过免疫荧光染色进行DDX4+和PGP9.5+细胞计数,发现添加10 μmol/L CHIR99021显著增加了精原细胞数量.以上结果说明,在体外培养系统中添加CHIR99021能有效维持水牛精原细胞的SSCs分子特征,促进其体外增殖,显著改善了水牛精原细胞体外培养条件.本研究能为利用小分子化合物优化水牛SSCs体外培养系统提供重要参考.