目录
摘要Ⅲ
关键词Ⅲ
AbstractⅣ
引言
引言
文献综述Ⅵ
方案论证Ⅷ
1 材料与方法1
1.1主要材料与仪器 1
1.2方法 1
1.2.1miRNA的选择和miRNA靶基因预测1
1.2.2 DF1细胞培养和传代1
1.2.3基因组DNA 抽提1
1.2.4质粒酶切2
1.2.5鸡骨髓细胞的培养2
1.2.6 miRNA靶基因验证2
1.2.7蛋白质印迹法分析3
1.2.8统计学方法3
2结果与分析4
2.1miR6675和miR1635表达质粒构建及验证4
2.2miR6675和miR1635靶基因预测6
2.3miR6675和miR1635靶基因验证8
2.4miR6675和miR1635 抑制DF1细胞中DICER蛋白表达9
3讨论 10
3.1miRNA直接靶向Wnt和Ca信号通路中的靶基因10
3.2miRNA通过DICER间接调控Wnt信号通路11 致谢12
参考文献13
鸡源microRNA对鸡树突状细胞Wnt和Ca信号通路调控的初探
摘 要
树突状细胞是机体专职的抗原递呈细胞，是宿主识别病原启动免疫应答的关键细胞。最近的研究发现Wnt和Ca信号通路的激活直接调控树突状细胞（DCs）的发育和成熟。微RNA（microRNA，miRNA）是存在于所有多细胞生物中的一大类调节分子。本项目初步探索了鸡源miRNA对DCs的Wnt和Ca信号通路的调控。首先,我们筛选并通过荧光定量试验验证了miR1635及miR6675的靶基因中与Wnt或Ca信号通路有关的6个靶基因。结果发现这些靶基因的mRNA含量在miR1635或miR6675过表达的细胞中均显著降低。相反，当miR1635或miR6675被抑制剂抑制后，这6个靶基因的表达显著增强。其次,我们通过westernblot实验发现调控Wnt信号通路的Dicer蛋白在m *51今日免费论文网|www.51jrft.com +Q: ￥351916072$ 
iR1635或miR6675过表达后表达水平显著降低。这一结果表明,miR1635和miR6675既可以通过直接靶向Wnt和Ca信号通路中的靶基因来调控Wnt和Ca信号通路,也可以调控Dicer酶来间接调控Wnt信号通路，进而影响树突状细胞的发育和成熟。总体而言,我们的数据揭示了鸡源miRNA调控树突状细胞Wnt和Ca信号通路的分子机制,这种新颖的调节途径为DCs分化过程中miRNA与信号分子的相互作用提供了更多见解。
REGULATION OF miRNAs ON Wnt AND Ca sIGNALING pATHWAYs IN DENDRITIC CELLs
ABSTRACT
DCs are specialized antigenpresenting cells with the ability to induce both innate and acquired immunity. Wnt and Ca signaling pathways are recently found particularly important for the development and maturation of dendritic cells (DCs). MicroRNA (miRNA) is a large class of regulatory molecules found in all multicellular organisms. Specific miRNA species have been reported to affect the function of DCs. Especially in the posttranscriptional regulation, which plays a vital role in the control of dendritic celll differentiation. Hence, our study try to figure out how microRNA regulate the Wnt and Ca signal to influence the function of DCs. Firstly, we screened and verified 6 target genes of miR1635 and miR6675 related to Wnt or Ca signaling pathway by fluorescence realtime quantitative PCR. The content of these target mRNAs significantly reduced in miR1635 or miR6675 overexpression groups. In contrast, silenced the expression of miR1635 or miR6675 enhanced the mRNA expression of these target genes. Secondly, westernblot experiments found that the protein expression level of Dicer that regulates the Wnt signaling pathway decreased in miR1635 or miR6675 overexpression groups. Thses results hint us that miR1635 and miR6675 might regulate Wnt and Ca signaling pathways by directly targeting the target genes in Wnt and Ca signaling pathways, or indirectly regulating Wnt signaling pathways by influence dicer, which in turn affect development and maturation of dendritic cells. Overall, our data reveals the molecular mechanism of microRNAs regulating Wnt and Ca signaling pathways. This novel regulatory circuit provides additional insight into how microRNAs interact with signaling molecules during dendritic cell differentiation.

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