本文關(guān)鍵詞：Dicer酶對(duì)巨噬細(xì)胞功能極化的影響　出處：《第三軍醫(yī)大學(xué)》2012年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： MicroRNAs Dicer 巨噬細(xì)胞 巨噬細(xì)胞極化

【摘要】：研究目的：觀(guān)察巨噬細(xì)胞在M1，M2不同功能極化狀態(tài)下Dicer酶的表達(dá)情況；構(gòu)建針對(duì)Dicer酶的干擾載體，建立Dicer酶表達(dá)抑制的巨噬細(xì)胞株；檢測(cè)Dicer酶表達(dá)抑制巨噬細(xì)胞功能和M1、M2功能極化的改變，明確小分子RNA合成通路障礙對(duì)巨噬細(xì)胞功能極化的影響。 研究方法：(1)通過(guò)Real time PCR和Western blot檢測(cè)原代骨髓來(lái)源巨噬細(xì)胞、腹腔巨噬細(xì)胞以及巨噬細(xì)胞系RAW264.7在M1、M2不同功能狀態(tài)下Dicer酶的表達(dá)情況。(2)將Dicer基因mRNA作為RNA干擾的靶區(qū)，設(shè)計(jì)特異的Dicer-siRNA干擾序列，插入含U6和H1雙啟動(dòng)子的慢病毒載體Double-promoter pFIV-H1/U6siRNAExpression vector中，構(gòu)建成抑制Dicer基因表達(dá)的RNA干擾表達(dá)載體，并轉(zhuǎn)染小鼠巨噬細(xì)胞株RAW264.7中，通過(guò)Real time PCR和Western blot觀(guān)察干擾載體對(duì)Dicer酶表達(dá)的抑制效果；通過(guò)microRNA real time PCR檢測(cè)對(duì)mir-155、mir-223的抑制效果；通過(guò)CCK8檢測(cè)對(duì)細(xì)胞增殖的影響；通過(guò)凋亡試劑盒檢測(cè)對(duì)細(xì)胞死亡凋亡的影響；通過(guò)Transwell小室檢測(cè)細(xì)胞遷移功能；通過(guò)FITC-dextran吞噬實(shí)驗(yàn)檢測(cè)巨噬細(xì)胞的吞噬功能。(3)通過(guò)Real time PCR和體外細(xì)胞殺菌實(shí)驗(yàn)檢測(cè)抑制Dicer酶表達(dá)后巨噬細(xì)胞M1\M2功能狀態(tài)的變化。 研究結(jié)果：(1) Dicer酶在巨噬細(xì)胞M1功能狀態(tài)下表達(dá)降低，耐受時(shí)表達(dá)升高；在巨噬細(xì)胞M2功能狀態(tài)下表達(dá)增高。(2)成功構(gòu)建Dicer-siRNA慢病毒表達(dá)載體及篩選出有效干擾Dicer酶表達(dá)的穩(wěn)定巨噬細(xì)胞株；CCK8及凋亡實(shí)驗(yàn)顯示，當(dāng)Dicer酶受抑制后，細(xì)胞增殖未受影響，凋亡/死亡率有所增高；遷移實(shí)驗(yàn)證實(shí)Dicer酶表達(dá)受抑后細(xì)胞遷移能力沒(méi)有明顯改變；吞噬實(shí)驗(yàn)證明Dicer酶抑制未影響細(xì)胞的吞噬活力。(3) Dicer酶表達(dá)下調(diào)抑制了巨噬細(xì)胞向M2功能方向極化，促進(jìn)巨噬細(xì)胞向M1功能方向極化，并可增強(qiáng)巨噬細(xì)胞的殺菌能力。 結(jié)論：（1）巨噬細(xì)胞不同功能狀態(tài)下Dicer酶的表達(dá)水平各異，，表明Dicer表達(dá)水平在不同極化條件下受到不同調(diào)控；（2）在巨噬細(xì)胞中抑制Dicer酶表達(dá)可增加細(xì)胞死亡和凋亡，其增殖、吞噬、遷移等基本生物行為未受明顯影響（3）巨噬細(xì)胞Dicer酶表達(dá)受抑制導(dǎo)致細(xì)胞向M2型極化減弱，向M1型極化及殺菌作用增強(qiáng)。
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R363

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 張記;彭?xiàng)?何海洋;申子剛;吳玉章;李晉濤;;細(xì)胞編碼microRNA在輪狀病毒復(fù)制中的作用及其表達(dá)譜分析[J];免疫學(xué)雜志;2011年06期

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