【作者】 张姗姗；

【导师】 华进联；

【作者基本信息】 西北农林科技大学 ， 细胞生物学， 2011， 硕士

【摘要】 胚胎干细胞(embryonic stem cells,ESCs)作为一种高度未分化细胞,可被诱导分化为包括生殖细胞在内的机体几乎所有的细胞类型。生殖细胞是高等哺乳动物体内非常特殊的一类成体细胞,承担着传宗接代和种族延续的重任。国内外已有多篇有关小鼠胚胎干细胞向雄性生殖细胞成功诱导分化的报道,但诱导效率均很低,关键原因在于对雄性哺乳动物生殖细胞发生的确切机理仍不清楚。多项研究显示,作为一类新发现的小RNA分子,miRNA可能在哺乳动物雄性生殖细胞发生过程中发挥着重要作用。本研究旨在通过定量PCR等手段筛选确定与哺乳动物雄性生殖细胞发生相关的特异miRNA分子,合成相应模拟物以及构建该miRNA的表达载体,转染小鼠胚胎干细胞系(mESCs),并结合RA诱导,观察该miRNA超表达对mESCs向雄性生殖细胞诱导效率的影响;进一步通过生物信息学手段预测该miRNA作用的靶基因,构建含有该基因的双荧光素酶载体,通过载体转染试验对预测结果进行验证,初步揭示该miRNA发挥作用的可能靶基因。1依据文献初步筛选与哺乳动物生殖发生可能相关的4个miRNA分子,分别为mmu-miR-34c、mmu-miR-449a、mmu-let-7e、mml-miR-122a。设计相应茎环引物,定量PCR检测其在小鼠各个不同组织器官的表达。结果发现,miR-34c在成年小鼠睾丸中表达量最高,与其他各个组织表达量差异均显著;检测其在不同发育阶段小鼠睾丸及成年小鼠睾丸生精细胞和间质细胞的表达,结果显示,miR-34c在性成熟小鼠(出生后1个月)睾丸中表达量最高,且只在生精细胞中高表达,其次为体成熟睾丸,胚胎期及出生后至性成熟之前的小鼠睾丸表达较少,而该miRNA分子在小鼠卵巢组织的表达较低;以上结果表明该miRNA为睾丸生精细胞特异且高度表达。2合成miR-34c模拟物,同时构建含该miRNA原转录本序列的真核表达载体(pMiR-34c-GFP质粒),转染mESCs并结合1μmol/L维甲酸(RA)诱导,通过形态学、碱性磷酸酶(AP)染色、多能性及生殖标记基因的定量PCR以及免疫荧光染色等手段,观察该miRNA对小鼠胚胎干细胞向雄性生殖细胞诱导分化的影响。结果显示,与对照组相比,转染miR-34c的mESCs,经RA诱导48 h后,AP阳性克隆数减少,精原细胞样的大圆细胞数明显增多;定量PCR和免疫荧光染色的结果显示,多能性相关基因如Oct4、c-Myc显著下降,而生殖相关基因如Vasa、Scp3表达上调。初步证明,miR-34c在小鼠胚胎干细胞向生殖细胞诱导分化过程中发挥着重要作用,且能提高向生殖细胞诱导分化的效率。3采用RNA22、PicTar等软件预测该miRNA分子作用的与生殖发生相关基因的靶位点,选定RARg基因,构建含该作用位点的双荧光素酶表达载体。将载体转染Hela细胞,结合荧光素发光强弱等检测手段对生物信息学预测结果进行验证。结果证明,miR-34c可作用于RARg (RA的γ亚型受体)基因的3’UTR,进而在雄性生殖细胞发生尤其是减数分裂过程中发挥着可能的调控作用。综上,本研究初步找出与哺乳动物雄性生殖发生密切相关的miRNA—miR-34c,并证明它在mESCs向雄性生殖细胞分化过程中发挥着重要作用;此外,我们寻找出一个miR-34c发挥生殖调控作用的靶基因RARg,并借此提出了该miRNA发挥调控作用的可能模式。更多还原

【Abstract】 Embryonic stem cell (ESC), as a kind of pluripotent cell line, can be differentiated into nearly all sorts of cell types, including germ cells. Germ cells, as special adult cells in mammals, take the responsibility of transferring genetic materials to the next generation. By now, there are many researches on induction of mESCs into male germ cells, but with a low efficiency, the basic reason is that the regulating mechanism of male germ cell development in mammals is still unclear. Studies show that miRNA, as a kind of newly found small RNAs, might play an important role in spermatogenesis in mammals.In this study, several miRNAs, which might be related to gametogenesis, were initially selected and detected in all the mouse organs by semi- and real-time PCR to find a testis-specific miRNA. Specific miRNA mimics were synthesized and pri-miRNA-GFP plasmid vector was constructed, and they were transfected into mESCs. To study the effect on mESCs differentiation into male germ cells, specific miRNA was overexpressed, combined with retinoic acid (RA) induction, meanwhile. Genes targeted by the specific miRNA were then predicted by bioinformatics and dual-luciferase reporter vector was constructed. By miRNA mimics and vector co-transfection experiment, the predicted target gene was confirmed, and by this, the probable pathway regulated by this specific miRNA was initially studied.1. According to the published papers, several miRNAs (mmu-miR-34c, mmu-miR-449a, mmu-let-7e, mml-miR-122a), which might be related to gametogenesis, were initially chosen. The corresponding stem-loop primers were designed and these four miRNAs were detected in all the mouse organs by semi- and real-time PCR. Data showed that, in adult mouse testis, only miR-34c was specifically and highly expressed, with high but not specific expression of miR-449a; miR-122a was liver-specific and let-7e was widely expressed in nearly all the organs. Then the expression of miR-34c in mouse testes of different developmental stages and two sorts of testis cells was detected by real-time PCR. Data showed that, miR-34c was most highly expressed in testis of sexually matured mouse, exactly in spermatogenic cells; followed by mature mouse testis, with a little expression in testis from embryo to sexual maturity. There’s no such phenomenon in mouse ovary.2. miR-34c mimics were symthesized by GenePharma, and pMiR-34c-GFP plasmid was constructed. it was overexpressed in mESCs, combined with 1μmol/L RA induction for 48 h, and the effect of miR-34c on induction efficiency of mESC differentiation into male germ cells was evaluated by cell morphology, Alkaline phosphatase (AP) staining, real-time PCR and immunofluorescence staining. Data showed that, after 48 h induction, compared with control, there were fewer AP positive clones and more spermatogonia-like cells in the transfected group, and the expression of Oct4 and c-Myc was down-regulated, with Vasa and Scp3 up-regulated. By this, we initially inferred that, miR-34c could improve the differentiation efficiency of mESCs into germ cells in vitro, and may play an important part during this process.3. Gametogenesis-related genes targeted by miR-34c were predicted by bioinformatics (such as RNA22, PicTar softwares), then RARg gene was selected and the recombined dual-luciferase reporter vector was constructed. By miRNA mimics and vector co-transfection experiment, the predicted target gene was confirmed, and by this, the probable pathway regulated by this specific miRNA was initially studied.In conclusion, we found a male germ cell specific miRNA--miR-34c, and data showed that it might be pivotal in mESCs differentiation into male germ cells in vitro; we also found a gene (RARg) targeted by miR-34c when it functions in regulating spermatogenesis, and proposed a mode hypothesis that miR-34c regulated male germ cell differentiation in mammals.更多还原