Dicer酶对巨噬细胞功能极化的影响

发布时间：2018-01-15 01:02

本文关键词：Dicer酶对巨噬细胞功能极化的影响　出处：《第三军医大学》2012年硕士论文　论文类型：学位论文

【摘要】：研究目的：观察巨噬细胞在M1，M2不同功能极化状态下Dicer酶的表达情况；构建针对Dicer酶的干扰载体，建立Dicer酶表达抑制的巨噬细胞株；检测Dicer酶表达抑制巨噬细胞功能和M1、M2功能极化的改变，明确小分子RNA合成通路障碍对巨噬细胞功能极化的影响。研究方法：(1)通过Real time PCR和Western blot检测原代骨髓来源巨噬细胞、腹腔巨噬细胞以及巨噬细胞系RAW264.7在M1、M2不同功能状态下Dicer酶的表达情况。(2)将Dicer基因mRNA作为RNA干扰的靶区，设计特异的Dicer-siRNA干扰序列，插入含U6和H1双启动子的慢病毒载体Double-promoter pFIV-H1/U6siRNAExpression vector中，构建成抑制Dicer基因表达的RNA干扰表达载体，并转染小鼠巨噬细胞株RAW264.7中，通过Real time PCR和Western blot观察干扰载体对Dicer酶表达的抑制效果；通过microRNA real time PCR检测对mir-155、mir-223的抑制效果；通过CCK8检测对细胞增殖的影响；通过凋亡试剂盒检测对细胞死亡凋亡的影响；通过Transwell小室检测细胞迁移功能；通过FITC-dextran吞噬实验检测巨噬细胞的吞噬功能。(3)通过Real time PCR和体外细胞杀菌实验检测抑制Dicer酶表达后巨噬细胞M1\M2功能状态的变化。研究结果：(1) Dicer酶在巨噬细胞M1功能状态下表达降低，耐受时表达升高；在巨噬细胞M2功能状态下表达增高。(2)成功构建Dicer-siRNA慢病毒表达载体及筛选出有效干扰Dicer酶表达的稳定巨噬细胞株；CCK8及凋亡实验显示，当Dicer酶受抑制后，细胞增殖未受影响，凋亡/死亡率有所增高；迁移实验证实Dicer酶表达受抑后细胞迁移能力没有明显改变；吞噬实验证明Dicer酶抑制未影响细胞的吞噬活力。(3) Dicer酶表达下调抑制了巨噬细胞向M2功能方向极化，促进巨噬细胞向M1功能方向极化，并可增强巨噬细胞的杀菌能力。结论：（1）巨噬细胞不同功能状态下Dicer酶的表达水平各异，，表明Dicer表达水平在不同极化条件下受到不同调控；（2）在巨噬细胞中抑制Dicer酶表达可增加细胞死亡和凋亡，其增殖、吞噬、迁移等基本生物行为未受明显影响（3）巨噬细胞Dicer酶表达受抑制导致细胞向M2型极化减弱，向M1型极化及杀菌作用增强。
[Abstract]:Objective: to observe the expression of Dicer enzyme in macrophages under different functional polarization of M1M 2. The interference vector for Dicer enzyme was constructed and the macrophage cell line with inhibition of Dicer enzyme expression was established. The expression of Dicer enzyme inhibited the changes of macrophage function and M1M2-2 functional polarization, and the effect of RNA synthesis pathway obstacle on macrophage functional polarization was determined. Methods the primary bone marrow-derived macrophages were detected by Real time PCR and Western blot. Peritoneal macrophages and macrophage cell line RAW264.7 were found in M1. The expression of Dicer enzyme in different function of M2) mRNA of Dicer gene was used as the target region of RNA interference and specific Dicer-siRNA interference sequence was designed. Inserted into the lentivirus vector Double-promoter pFIV-H1/U6siRNAExpression vector containing U6 and H1 double promoters. RNA interference expression vector was constructed to inhibit the expression of Dicer gene and transfected into mouse macrophage cell line RAW264.7. Real time PCR and Western blot were used to observe the inhibitory effect of interference vector on Dicer expression. The inhibitory effect of microRNA real time PCR on mir-155 mir-223 was detected. The effect of CCK8 on cell proliferation; The effect of apoptosis on cell death was detected by apoptosis kit. Cell migration function was detected by Transwell chamber. Detection of phagocytic function of macrophages by FITC-dextran phagocytosis assay. The functional changes of macrophages M1\ M2 were detected by Real time PCR and bactericidal assay in vitro. The results showed that the expression of Dicer enzyme decreased in macrophage M1 functional state, but increased during tolerance. Dicer-siRNA lentivirus expression vector was successfully constructed and stable macrophage cell lines which could interfere with the expression of Dicer enzyme were screened. CCK8 and apoptosis assay showed that when Dicer enzyme was inhibited, cell proliferation was not affected, and apoptosis / mortality was increased. Migration assay confirmed that the ability of cell migration did not change after the inhibition of Dicer enzyme expression. Phagocytosis test showed that Dicer inhibited the phagocytic activity of macrophages. The down-regulation of the expression of Dicer enzyme inhibited macrophages from polarization to M2 function. It can promote macrophage polarization to M1 function, and enhance the bactericidal ability of macrophage. Conclusion the expression of Dicer enzyme in macrophages is different in different functional states, indicating that the expression of Dicer is regulated by different polarization conditions. (2) inhibiting the expression of Dicer in macrophages can increase cell death and apoptosis, proliferation and phagocytosis. Migration and other basic biological behaviors were not significantly affected. (3) the inhibition of Dicer enzyme expression in macrophages resulted in the decrease of M2 polarization, and the enhancement of M1 polarization and bactericidal effect.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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