microRNA在胚胎血发生中的功能及调控研究

发布时间：2019-05-29 08:33

【摘要】：microRNA是近年来发现的一类长度约为23个核苷酸的单链非编码RNA,主要由RNA多聚酶Ⅱ转录,经核糖核酸酶Ⅲ (Drosha及Dicer)剪切形成。成熟microRNA通过碱基配对与靶基因mRNA的3'-UTR相结合,造成靶基因mRNA的降解或蛋白翻译水平上受抑制,从而对靶基因的表达进行负向调控。microRNA参与多种生物学过程,对器官形成及组织分化均有重要作用。据目前文献报道,至少有30余组microRNA在成人血细胞中有独特的表达,其中多组microRNA与造血干细胞的分化有关,并且与胚胎造血中的关键因子相互作用,但它们在胚胎造血中的功能仍有待于进一步的探讨。为了揭示microRNA在哺乳动物胚胎造血中的作用,本文以小鼠胚胎干细胞诱导分化形成的拟胚体作为胚胎发育的模型。第一步,在先前实验积累数据的基础上,初步确定了在胚胎血发生中有特异性表达的候选microRNA。其次,为了验证这些候选microRNA是否能够调控胚胎血发生,我们首先构建了逆转录病毒载体,通过反转录病毒在小鼠胚胎干细胞中过表达候选microRNA。再次,我们构建了能在条件诱导过表达候选microRNA的小鼠胚胎干细胞系,可在拟胚体发育的特定时间段利用四环素诱导高表达候选microRNA,再利用实时荧光定量和流式细胞分析等方式检测血发生及胚胎发育的变化情况,并对数据进行分析后得到了候选microRNA的可能调节因子及下游靶基因。这部分工作为下一步研究这些候选microRNA的作用途径并找出它们的直接作用位点奠定了基础。另外,为了方便研究microRNA及其他外源性基因在人类胚胎干细胞中的作用,本文尝试构建了条件诱导基因表达人类胚胎干细胞系。通过在Actb基因的3’UTR区域发生同源重组,导入了四环素调控启动子,潮霉素(Hygromycine)抗性基因,定点重组酶系统元件(LoxP, Frt)等序列。在成功整合之后,就能够更进一步地定点重组入外源基因并可在四环素衍生物强力霉素诱导下表达转入的外源基因或microRNA。这个细胞系为我们研究在人类胚胎干细胞中研究基因或microRNA的功能提供了一个相当有用的工具。
[Abstract]:MicroRNA is a class of single strand non-coding RNA, with a length of about 23 nucleotides found in recent years, which is mainly transcribed by RNA polymerase II and cut by ribonuclease III (Drosha and Dicer). Mature microRNA binds to the 3'-UTR of target gene mRNA through base pairing, resulting in the degradation of target gene mRNA or the inhibition of protein translation level, thus negatively regulating the expression of target gene. MicroRNA is involved in a variety of biological processes. It plays an important role in organ formation and tissue differentiation. According to the current literature, at least 30 groups of microRNA have unique expression in adult blood cells, among which many groups of microRNA are related to the differentiation of hematopoietic stem cells and interact with key factors in embryonic hematopoiesis. However, their function in embryonic hematopoiesis still needs to be further discussed. In order to reveal the role of microRNA in embryonic hematopoiesis in mammals, the embryoid induced by mouse embryonic stem cells was used as a model of embryonic development. In the first step, on the basis of the accumulation of data in previous experiments, the candidate microRNA. with specific expression in embryonic hematogenesis was preliminarily determined. Secondly, in order to verify whether these candidate microRNA can regulate embryonic hematogenesis, we first constructed retrovirus vector and overexpressed candidate microRNA. in mouse embryonic stem cells by retrovirus. Thirdly, we constructed a mouse embryonic stem cell line which can overexpress candidate microRNA in conditional induction, which can induce high expression candidate microRNA, by tetracycline at a specific time of embryoid development. The changes of hematogenesis and embryonic development were detected by real-time fluorescence quantitative analysis and flow cytometry, and the possible regulatory factors and downstream target genes of candidate microRNA were obtained after the data were analyzed. This part of the work lays a foundation for the next step to study the pathway of action of these candidate microRNA and find out their direct sites of action. In addition, in order to facilitate the study of the role of microRNA and other exogenous genes in human embryonic stem cells, a conditional induced gene expression human embryonic stem cell line was constructed. By homologous recombination in the 3'UTR region of Actb gene, tetracycline regulated promoter, hygromycin (Hygromycine) resistance gene and site-directed recombinant enzyme system element (LoxP, Frt) were introduced. After successful integration, the foreign gene can be further recombined into the foreign gene and the transferred foreign gene or microRNA. can be expressed under the induction of doxycycline derivative doxycycline. This cell line provides a useful tool for us to study the function of genes or microRNA in human embryonic stem cells.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R3411

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