miR-146a在白念珠菌引发的固有免疫应答中的调节作用

发布时间：2018-07-06 20:09

本文选题：白念珠菌 + 固有免疫应　；参考：《第二军医大学》2016年博士论文

【摘要】：白念珠菌是一种寄生于人体粘膜和皮肤表面的条件致病菌。正常情况下,其并不引起感染性疾病,但当机体免疫力下降或受到抑制时,白念珠菌趁虚而入,轻则引起各种癣病,重则引起系统性的念珠菌感染。系统性念珠菌感染病情凶险,治疗十分棘手,患者往往预后较差。白念珠菌穿过人体的皮肤和粘膜屏障后,首先被免疫系统的抗原递呈细胞(APC)所感知。常见的APC包括巨噬细胞、单核细胞、树突状细胞等,其表面表达高水平的模式识别受体(PRR),负责在第一时间感知入侵机体的白念珠菌。PRR感知白念珠菌的分子基础是识别器表面的病原体相关分子模式(PAMP)。根据分子结构和功能,PRR分为很多种,包括Toll样受体(TLR)、维甲酸诱导基因样受体(RLRs)和NOD样受体(NLR)。由于白念珠菌的体积较大,PAMP的种类较多,因此参与其免疫识别的PRR也较多。以前的研究表明TLR家族的TLR2、TLR4和TLR6均参与了念珠菌的免疫识别。此外,NLR家族的Dectin-1、Dectin-2等也参与了白念珠菌的免疫识别。当白念珠菌与APC表面的PRR结合后,胞内多条信号通路被迅速活化,最终导致APC在感染部位迅速释放各种炎症因子,比如白细胞介素1(IL-1),肿瘤坏死因子α(TNF-α)和I型干扰素等,在第一时间促进白念珠菌的免疫清除。另一方面,APC将白念珠菌吞噬,对其抗原进行消化处理,并将抗原片段递交给T细胞,同时分泌各种细胞因子,如IL-12,转化生长因子β(TGF-β)等,在局部形成特定的细胞因子格局,指导后续的获得性免疫应答过程。针对白念珠菌的免疫应答必须处在精细的调控之中。一方面,炎症反应过度会导致机体的免疫病理损伤。另一方面,如果炎症反应强度不足或持续时间过短,则无法清除入侵机体的病原菌,最终导致病原菌扩散。microRNA是一类长度在18-25个碱基的短链非编码RNA。以往的研究表明其广泛地参与了免疫细胞的分化发育以及免疫应答的调控过程。多个研究表明miR-146a是一个免疫调节功能十分活跃的分子,广泛参与了TLR信号通路、RIG-I信号通路的负向调节过程。在前期研究中,我们采用microRNA芯片分析了热灭活白念珠菌刺激人单核源性树突状细胞(Mo-DCs)的microRNA表达谱,发现miR-146a表达上调,但该结果尚未得到PCR的证实,且miR-146a表达增高的生物学意义仍不明确。在本研究中,我们对miR-146a在白念珠菌引发的固有免疫应答中的作用进行了探讨。第一部分热灭活白念珠菌通过NF-κB依赖性的方式上调细胞内miR-146a的表达我们首先以热灭活白念珠菌刺激THP-1细胞、RAW 264.3,单核源性树突状细胞和PMA诱导的巨噬细胞,然后采用RT-PCR检测miR-146a的表达变化情况,发现白念珠菌刺激可以上调细胞内miR-146a的表达。我们采用NF-κB的抑制剂PDTC预处理细胞,然后再以白念珠菌进行刺激,观察NF-κB被抑制后,miR-146a的表达变化情况,结果发现PDTC可以部分下调miR-146a的表达,说明白念珠菌上调miR-146a的表达在一定程度上依赖于NF-κB。此外,由于此前的研究表明Dectin-1是参与白念珠菌免疫识别的PRR之一,因此我们采用昆布多糖封闭Dectin-1,再用白念珠菌进行刺激,观察miR-146a的表达变化情况。结果发现昆布多糖对miR-146a的表达无影响。我们还采用Dectin-1激动剂Curdlan活化THP-1细胞,也发现细胞内miR-146a的表达丰度不受影响。这些结果说明,白念珠菌可以通过NF-κB上调细胞内miR-146a的表达,但这种上调miR-146a表达的功能与Dectin-1无关。第二部分miR-146a负向调节热灭活白念珠菌诱导THP-1细胞释放干扰素β的能力之后,我们进一步分析了白念珠菌上调miR-146a表达的生物意义。我们用热灭活白念珠菌刺激THP-1细胞,采用ELISA法检测培养基内干扰素β(IFN-β)的表达水平,用RT-PCR法检测IFN-β、干扰素刺激基因(ISG)基因2-5-寡腺苷酸合成酶(OAS-1)和抗粘病毒蛋白1(MX1)的表达。结果发现热灭活白念珠菌可以促进THP-1细胞释放IFN-β,上调OAS-1和MX1的表达。我们将miR-146a mimic和inhibitor转染入THP-1细胞,再用热灭活白念珠菌进行刺激,并用ELISA计策培养基内的IFN-β水平,以RT-PCR法检测OAS-1和MX1 m RNA的表达。发现miR-146a mimic可以抑制白念珠菌诱导THP-1细胞释放IFN-β的能力,而miR-146a inhibitor则可以增强白念珠菌诱导THP-1细胞释放IFN-β的能力,说明miR-146a是IFN-β的负向调节因子。此外,我们还发现,miR-146a mimic可以抑制白念珠菌上调OAS-1和MX1的能力,而miR-146a inhibitor则发挥了相反的作用。这些结果表明,在热灭活白念珠菌刺激的THP-1细胞中,miR-146a表达上调的生物学意义可能在于负向调节IFN-β的释放,并由此负向调控了ISG的表达。第三部分miR-146a作用靶点分析由于此前有研究表明白念珠菌刺激单核源性树突状细胞表达IFN-β与干扰素调节因子5(IRF5)有关,而生物信息学预测又发现IRF5是miR-146a的作用靶点。因此我们拟进一步分析IRF5是否是miR-146a的作用靶点。我们采用热灭活白念珠菌刺激THP-1细胞后,发现IRF5在m RNA和蛋白水平的表达并没有明显的变化。将miR-146a mimic或inhibitor转染入THP-1细胞后,然后用热灭活白念珠菌进行刺激,IRF5的表达也没有受到影响。因此我们确定IRF5并非miR-146a的作用靶点。接下来,我们用生物信息学软件预测到一个名为IFIT3的ISG是miR-146a的作用靶点。用热灭活白念珠菌刺激THP-1细胞后,我们发现IFIT3的m RNA和蛋白均呈现出先增高,后降低的趋势,提示miR-146a可以调节IFIT3的表达。我们将miR-146a mimic或inhibitor转染入THP-1细胞,并以外源性的IFN-β进行刺激,发现miR-146a的确可以下调IFIT3的表达。我们进一步采用报告基因技术验证miR-146a与IFIT3 m RNA的3’UTR是否有相互作用,却发现二者之间无相互作用。因此,miR-146a调节可能是通过间接作用调节IFIT3的表达。综上,我们认为在白念珠菌刺激的THP-1细胞中,miR-146a并不是通过IRF5调节IFN-β的表达,其虽然可以调节IFIT3的表达,但是并不是直接的相互作用,而是通过间接作用调节其表达。第四部分mi-146a通过IFIT3调节THP-1细胞的凋亡考虑到miR-146a可以调节IFIT3的表达,而以往的研究表明IFIT3与细胞凋亡有关。因此,我们接下来研究了miR-146a是否调节了THP-1细胞的凋亡,同时也分析了miR-146a调节THP-1细胞的凋亡是否与IFIT3有关。我们首先将miR-146ainhibitor或mimic转染入THP-1细胞,然后用白念珠菌对其进行刺激,采用Annexin V-PI法检测细胞的凋亡状况。结果我们发现:miR-146a mimic可以促进细胞的凋亡,而miR-146a inhibitor对细胞凋亡影响不显著。这些结果表明miR-146a是促进细胞凋亡的因素。随后,我们采用siRNA技术抑制IFIT3的表达,然后再用热灭活白念珠菌对THP-1细胞进行刺激。结果发现IFIT3表达下调后,细胞的凋亡增加。这说明IFIT3具有抗凋亡作用。综上,这部分结果说明了miR-146a具有促凋亡作用,其促凋亡的作用可能是通过下调IFIT3的表达来实现的。结论综上,本研究的主要发现是:白念珠菌可以通过活化NF-κB的方式上调THP-1细胞内miR-146a的表达,这种上调作用与参与白念珠菌识别的模式识别受体Dectin-1无关。MiR-146表达上调具有两个生物学意义:第一,负向调节IFN-β的产生,并由此调节了多个ISG的表达,且我们的研究证实miR-146a的这种生物学功能与其理论靶点IRF5无关;第二,miR-146a通过IFN-β→IFIT3轴调节细胞的凋亡。
[Abstract]:Candida albicans is a conditional pathogen parasitic on the surface of the mucous membrane and skin of the human body. Under normal circumstances, it does not cause infectious diseases, but when the body's immunity is reduced or suppressed, Candida albicans takes the advantage of the disease and causes systemic candidal infection. Systemic Candida infection is a dangerous and dangerous disease. The common APC includes macrophages, monocytes, dendritic cells and so on, which express the high level of pattern recognition receptor (PRR) on the surface, which is responsible for the first time to perceive the invasion of APC. The molecular basis of Candida albicans.PRR perceiving Candida albicans is the pathogen associated molecular pattern (PAMP) on the surface of the recognizer. According to the molecular structure and function, PRR is divided into many species, including Toll like receptor (TLR), retinoic acid induced gene like receptor (RLRs) and NOD like receptor (NLR). Because of the larger volume of Candida albicans, the variety of PAMP is more, and the cause is more. PRR is involved in its immune recognition. Previous studies have shown that the TLR2, TLR4 and TLR6 of the TLR family are involved in the immune recognition of Candida. In addition, the Dectin-1, Dectin-2, etc. of the NLR family are also involved in the immune recognition of Candida albicans. When Candida albicans are combined with PRR of the APC surface, multiple intracellular signaling pathways are rapidly activated and ultimately guided. APC rapidly releases various inflammatory factors at the infected site, such as interleukin 1 (IL-1), tumor necrosis factor alpha (TNF- alpha) and type I interferon and so on. In the first time, the immune clearance of Candida albicans is promoted. On the other hand, APC phagocytosis of Candida albicans, the antigen is eliminated, and the antigen fragments are delivered to T cells and secreted at the same time. The cytokine, such as IL-12, transforming growth factor beta (TGF- beta) and so on, forms a specific cytokine pattern in the region to guide the subsequent acquired immune response. The immune response to Candida albicans must be in fine control. On the one hand, the excessive inflammatory response can lead to the immune pathological damage of the body. On the other hand, if inflammation is affected. It is impossible to clear the pathogenic bacteria of the invading organism and eventually lead to the spread of.MicroRNA, a class of short chain non coded RNA. with 18-25 bases, which showed that it was widely involved in the differentiation and development of immune cells and the regulation of immune response. Multiple studies showed that miR-1 46a is a very active immunomodulatory molecule, which has been widely involved in the negative regulation of the TLR signaling pathway and the RIG-I signaling pathway. In the previous study, we used microRNA chips to analyze the microRNA expression profiles of thermally inactivated Candida albicans stimulated human mononuclear dendritic cells (Mo-DCs), and found that the expression of miR-146a was up-regulated, but the conclusion was that the expression of miR-146a was up-regulated. The fruit has not been confirmed by PCR, and the biological significance of the increased expression of miR-146a is still not clear. In this study, we explored the role of miR-146a in the intrinsic immune response induced by Candida albicans. The first part of the heat inactivated Candida albicans, through the NF- kappa B dependent manner, the expression of miR-146a in the cell modulation, we first heat the cells. Inactivated Candida albicans stimulated THP-1 cells, RAW 264.3, mononuclear dendritic cells and PMA induced macrophages. Then RT-PCR was used to detect the changes in the expression of miR-146a. It was found that Candida albicans stimulated the expression of miR-146a in cells. We used NF- kappa B inhibitor PDTC to pretreat the cells and then use Candida albicans for Candida albicans. Stimulated, the expression changes of miR-146a after the inhibition of NF- kappa B were observed. The results showed that PDTC could partly down regulate the expression of miR-146a. It was suggested that the expression of miR-146a was partly dependent on NF- kappa B.. Since previous studies showed that Dectin-1 was one of the PRR of Candida albicans immune recognition, we used Kunming. When the polysaccharide was closed to Dectin-1, the expression of miR-146a was observed with Candida albicans. The results showed that the expression of miR-146a had no effect on the expression of miR-146a. We also used the Dectin-1 agonist Curdlan to activate THP-1 cells, and also found that the expression abundance of miR-146a in the cells was not affected. These results suggest that Candida albicans can pass through NF- kappa B up-regulated the expression of miR-146a in the cell, but the function of the up regulation of miR-146a was not related to Dectin-1. Second part miR-146a negatively regulated the ability of Candida albicans to induce the release of interferon beta in THP-1 cells. We further analyzed the biological significance of albicans albicans up regulation of miR-146a surface. We use heat to inactivate Candida albicans. ELISA assay was used to detect the expression level of interferon beta (IFN- beta) in cultured THP-1 cells and the expression of IFN- beta, IFN- beta, ISG gene (ISG) gene 2-5- oligoadenylate synthetase (OAS-1) and anti visco protein 1 (MX1). The results showed that thermally inactivated Candida albicans could promote the release of IFN- beta in THP-1 cells, up regulation of OAS-1 and impurities. 1, we transfected miR-146a mimic and inhibitor into THP-1 cells, stimulated the Candida albicans with heat inactivation, and cultured the IFN- beta level in the base with ELISA strategy. The expression of OAS-1 and MX1 m RNA was detected by RT-PCR method. Or can enhance the ability of Candida albicans to induce the release of IFN- beta in THP-1 cells, indicating that miR-146a is a negative regulator of IFN- beta. In addition, we also found that miR-146a mimic inhibits the ability of Candida albicans to increase OAS-1 and MX1, while miR-146a inhibitor plays the opposite role. These results suggest that the activation of Candida albicans in heat inactivated Candida albicans. In the THP-1 cells, the biological significance of the up regulation of miR-146a expression may lie in the negative regulation of the release of IFN- beta, and thus negatively regulates the expression of ISG. The third part of the target analysis of miR-146a is due to the previous study that the expression of IFN- beta in Monal derived dendritic cells expressed by Candida albicans was related to the interferon regulator 5 (IRF5). It is also found that IRF5 is the target of miR-146a. Therefore, we will further analyze whether IRF5 is the target of miR-146a. We found that the expression of IRF5 at m RNA and protein levels has not changed significantly after the use of thermally inactivated Candida albicans to stimulate the expression of M RNA and protein. The expression of IRF5 was not affected by the activation of Candida albicans with heat inactivation. So we determined that IRF5 was not the target of miR-146a. Then, we predicted that a ISG named IFIT3 was the target of miR-146a with bioinformatics software. We found m RNA of IFIT3 by using the thermal inactivated Candida albicans to stimulate the THP-1 cells. And the protein showed a tendency to increase first and then decrease, suggesting that miR-146a could regulate the expression of IFIT3. We transfected miR-146a mimic or inhibitor into THP-1 cells and stimulated by exogenous IFN- beta. It was found that miR-146a could downregulate the expression of IFIT3. We further used the report gene technique to verify miR-146a and IFIT3 M. If 3 'UTR has interaction, it is found that there is no interaction between the two. Therefore, miR-146a regulation may regulate the expression of IFIT3 by indirect action. In conclusion, we think that miR-146a does not regulate the expression of IFN- beta through IRF5 in the THP-1 cells stimulated by Candida albicans, although it can regulate the expression of IFIT3, but it is not direct. The fourth part mi-146a regulates the apoptosis of THP-1 cells through IFIT3, which regulates the apoptosis of THP-1 cells through IFIT3, considering that miR-146a can regulate the expression of IFIT3, while previous studies have shown that IFIT3 is associated with apoptosis. The analysis of whether the apoptosis of THP-1 cells by miR-146a is related to IFIT3. We first transfect miR-146ainhibitor or mimic into THP-1 cells, then use Candida albicans to stimulate them, and detect the cell apoptosis using Annexin V-PI method. We found that miR-146a mimic can promote cell apoptosis and miR-146a inhibitor is fine. The effect of apoptosis is not significant. These results suggest that miR-146a is a factor promoting apoptosis. Then, we use siRNA technique to inhibit the expression of IFIT3, and then use heat inactivated Candida albicans to stimulate the THP-1 cells. The results showed that the apoptosis of the cells increased after the down regulation of IFIT3 expression. This shows that IFIT3 has anti apoptosis effect. The results show that miR-146a has the role of promoting apoptosis, and its role in promoting apoptosis may be achieved by down regulating the expression of IFIT3. Conclusion the main discovery of this study is that Candida albicans can increase the expression of miR-146a in THP-1 cells by activating NF- kappa B, the up regulation and the pattern recognition of Candida albicans. The up-regulated expression of the other receptor Dectin-1 has two biological significance: first, the negative regulation of the production of IFN- beta and thereby regulating the expression of multiple ISG, and our study confirms that this biological function of miR-146a is independent of its theoretical target IRF5; second, miR-146a regulates cell apoptosis through the IFN- beta - IFIT3 axis.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R519.3

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