目录
摘要Ⅱ
关键词Ⅱ
AbstractⅢ
引言
引言1
1 材料与方法2
1.1材料与仪器 2
1.1.1实验材料 2
1.1.2实验试剂及耗材1
1.2实验方法2
1.2.1过表达载体的构建2
1.2.2树突状细胞表型的流式细胞仪检测6
1.2.3生物信息学方法预测miRNA的靶基因和信号通路7
2结果与分析7
2.1过表达载体构建结果7
2.1.1目的基因PCR结果7
2.1.2菌落PCR结果8
2.1.3测序结果9
2.2过表达miRNA对树突状细胞表型的影响 9
2.3抑制miRNA片段对树突状细胞表型的影响9
2.4 miR21的靶基因及信号通路预测 11
3讨论15
致谢 16
参考文献16
ggamiR21调控树突状细胞免疫应答的研究
摘 要
microRNAs是一类长约22nt的单链小RNA，经细胞核到细胞质空间由多种酶和辅助蛋白协调完成加工成熟，在转录后水平调节基因的表达。在免疫细胞中，microRNA采取复杂的网络化方式进行调节，参与免疫细胞发育分化，细胞因子的分泌等发挥多种生物学效应；同时microRNA还参与抑制病原体入侵。本课题前期研究表明禽传染性支气管炎病毒（infectious bronchitis virus，IBV）侵染树突状细胞（dendritic cells，DCs）后，细胞中的ggamiR21表达量上调，抑制病毒侵染。为探究ggamiR21对树突状细胞表型发育的影响及信号通路的调控，本研究首先通过PCR的方法从鸡的基因组中扩增出含有ggamiR21目的基因的DNA片段，连接到表达载体pSilencer4.1上构建重组质粒，测序结果表明真核表达载体pSilencerggamiR21构建成功。其次，本研究通过流式细胞术实验验证ggamiR21对树突状细胞发育成熟有促进作用。最后，本研究采用生物信息学方法，预测了ggamiR21潜在的靶 *51今日免费论文网|www.51jrft.com +Q: #351916072# 
基因及调控的信号通路。初步预测出ggamiR21在MAPK信号通路、NOD样受体信号通路、细胞因子细胞因子受体相互作用、RNA降解、粘着连接方面具有调控作用。
 REGULATION THE IMMUNE RESPONSE OF DENDRITIC CELLS STIMULATED BY GGAMIR21
ABSTRACT
MicroRNAs are single stranded small RNAs with a length of about 22 nt. It was processed by many kinds of enzymes and auxiliary proteins from nucleus to cytoplasm, and regulate gene expression at posttranscription level. In immune cells, microRNA regulates various biological effect in a intricate network way, participating in the differentiation and cytokine secretion process of immune cells, which might advantageous for host to inhibits pathogen invasion. Previous studies have shown that the expression of ggamir21 in dendritic cells (DC) is upregulated when the cells are infected by infectious bronchitis virus (IBV) virus. In order to research the effect of ggamiR21 on the phenotype development and the regulation of signal pathway of dendritic cells, we first amplified the DNA fragments containing the target gene of ggamiR21 from chicken genome by PCR, and then connected it to the expression vector pSilencer4.1 to construct the recombinant vector. Sequence result confirm that this vector constructed correctly. Second, flow cytometry was used to verify the effect of ggamir21 on the development and maturation of dendritic cell. Result proved that the overexpression of ggamir21did decreased the MHCII of avian DCs. Last but not the least, , bioinformatics analysis indicated the potential target genes and signal pathways of ggamir21 that ggamir21 can regulate MAPK signaling, nod like receptor signaling, cytokinecytokine receptor interaction, RNA degradation and adherens junction.

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