本文選題：白念珠菌 + 固有免疫應　； 參考：《第二軍醫(yī)大學》2016年博士論文

【摘要】：白念珠菌是一種寄生于人體粘膜和皮膚表面的條件致病菌。正常情況下,其并不引起感染性疾病,但當機體免疫力下降或受到抑制時,白念珠菌趁虛而入,輕則引起各種癬病,重則引起系統性的念珠菌感染。系統性念珠菌感染病情兇險,治療十分棘手,患者往往預后較差。白念珠菌穿過人體的皮膚和粘膜屏障后,首先被免疫系統的抗原遞呈細胞(APC)所感知。常見的APC包括巨噬細胞、單核細胞、樹突狀細胞等,其表面表達高水平的模式識別受體(PRR),負責在第一時間感知入侵機體的白念珠菌。PRR感知白念珠菌的分子基礎是識別器表面的病原體相關分子模式(PAMP)。根據分子結構和功能,PRR分為很多種,包括Toll樣受體(TLR)、維甲酸誘導基因樣受體(RLRs)和NOD樣受體(NLR)。由于白念珠菌的體積較大,PAMP的種類較多,因此參與其免疫識別的PRR也較多。以前的研究表明TLR家族的TLR2、TLR4和TLR6均參與了念珠菌的免疫識別。此外,NLR家族的Dectin-1、Dectin-2等也參與了白念珠菌的免疫識別。當白念珠菌與APC表面的PRR結合后,胞內多條信號通路被迅速活化,最終導致APC在感染部位迅速釋放各種炎癥因子,比如白細胞介素1(IL-1),腫瘤壞死因子α(TNF-α)和I型干擾素等,在第一時間促進白念珠菌的免疫清除。另一方面,APC將白念珠菌吞噬,對其抗原進行消化處理,并將抗原片段遞交給T細胞,同時分泌各種細胞因子,如IL-12,轉化生長因子β(TGF-β)等,在局部形成特定的細胞因子格局,指導后續(xù)的獲得性免疫應答過程。針對白念珠菌的免疫應答必須處在精細的調控之中。一方面,炎癥反應過度會導致機體的免疫病理損傷。另一方面,如果炎癥反應強度不足或持續(xù)時間過短,則無法清除入侵機體的病原菌,最終導致病原菌擴散。microRNA是一類長度在18-25個堿基的短鏈非編碼RNA。以往的研究表明其廣泛地參與了免疫細胞的分化發(fā)育以及免疫應答的調控過程。多個研究表明miR-146a是一個免疫調節(jié)功能十分活躍的分子,廣泛參與了TLR信號通路、RIG-I信號通路的負向調節(jié)過程。在前期研究中,我們采用microRNA芯片分析了熱滅活白念珠菌刺激人單核源性樹突狀細胞(Mo-DCs)的microRNA表達譜,發(fā)現miR-146a表達上調,但該結果尚未得到PCR的證實,且miR-146a表達增高的生物學意義仍不明確。在本研究中,我們對miR-146a在白念珠菌引發(fā)的固有免疫應答中的作用進行了探討。第一部分熱滅活白念珠菌通過NF-κB依賴性的方式上調細胞內miR-146a的表達我們首先以熱滅活白念珠菌刺激THP-1細胞、RAW 264.3,單核源性樹突狀細胞和PMA誘導的巨噬細胞,然后采用RT-PCR檢測miR-146a的表達變化情況,發(fā)現白念珠菌刺激可以上調細胞內miR-146a的表達。我們采用NF-κB的抑制劑PDTC預處理細胞,然后再以白念珠菌進行刺激,觀察NF-κB被抑制后,miR-146a的表達變化情況,結果發(fā)現PDTC可以部分下調miR-146a的表達,說明白念珠菌上調miR-146a的表達在一定程度上依賴于NF-κB。此外,由于此前的研究表明Dectin-1是參與白念珠菌免疫識別的PRR之一,因此我們采用昆布多糖封閉Dectin-1,再用白念珠菌進行刺激,觀察miR-146a的表達變化情況。結果發(fā)現昆布多糖對miR-146a的表達無影響。我們還采用Dectin-1激動劑Curdlan活化THP-1細胞,也發(fā)現細胞內miR-146a的表達豐度不受影響。這些結果說明,白念珠菌可以通過NF-κB上調細胞內miR-146a的表達,但這種上調miR-146a表達的功能與Dectin-1無關。第二部分miR-146a負向調節(jié)熱滅活白念珠菌誘導THP-1細胞釋放干擾素β的能力之后,我們進一步分析了白念珠菌上調miR-146a表達的生物意義。我們用熱滅活白念珠菌刺激THP-1細胞,采用ELISA法檢測培養(yǎng)基內干擾素β(IFN-β)的表達水平,用RT-PCR法檢測IFN-β、干擾素刺激基因(ISG)基因2-5-寡腺苷酸合成酶(OAS-1)和抗粘病毒蛋白1(MX1)的表達。結果發(fā)現熱滅活白念珠菌可以促進THP-1細胞釋放IFN-β,上調OAS-1和MX1的表達。我們將miR-146a mimic和inhibitor轉染入THP-1細胞,再用熱滅活白念珠菌進行刺激,并用ELISA計策培養(yǎng)基內的IFN-β水平,以RT-PCR法檢測OAS-1和MX1 m RNA的表達。發(fā)現miR-146a mimic可以抑制白念珠菌誘導THP-1細胞釋放IFN-β的能力,而miR-146a inhibitor則可以增強白念珠菌誘導THP-1細胞釋放IFN-β的能力,說明miR-146a是IFN-β的負向調節(jié)因子。此外,我們還發(fā)現,miR-146a mimic可以抑制白念珠菌上調OAS-1和MX1的能力,而miR-146a inhibitor則發(fā)揮了相反的作用。這些結果表明,在熱滅活白念珠菌刺激的THP-1細胞中,miR-146a表達上調的生物學意義可能在于負向調節(jié)IFN-β的釋放,并由此負向調控了ISG的表達。第三部分miR-146a作用靶點分析由于此前有研究表明白念珠菌刺激單核源性樹突狀細胞表達IFN-β與干擾素調節(jié)因子5(IRF5)有關,而生物信息學預測又發(fā)現IRF5是miR-146a的作用靶點。因此我們擬進一步分析IRF5是否是miR-146a的作用靶點。我們采用熱滅活白念珠菌刺激THP-1細胞后,發(fā)現IRF5在m RNA和蛋白水平的表達并沒有明顯的變化。將miR-146a mimic或inhibitor轉染入THP-1細胞后,然后用熱滅活白念珠菌進行刺激,IRF5的表達也沒有受到影響。因此我們確定IRF5并非miR-146a的作用靶點。接下來,我們用生物信息學軟件預測到一個名為IFIT3的ISG是miR-146a的作用靶點。用熱滅活白念珠菌刺激THP-1細胞后,我們發(fā)現IFIT3的m RNA和蛋白均呈現出先增高,后降低的趨勢,提示miR-146a可以調節(jié)IFIT3的表達。我們將miR-146a mimic或inhibitor轉染入THP-1細胞,并以外源性的IFN-β進行刺激,發(fā)現miR-146a的確可以下調IFIT3的表達。我們進一步采用報告基因技術驗證miR-146a與IFIT3 m RNA的3’UTR是否有相互作用,卻發(fā)現二者之間無相互作用。因此,miR-146a調節(jié)可能是通過間接作用調節(jié)IFIT3的表達。綜上,我們認為在白念珠菌刺激的THP-1細胞中,miR-146a并不是通過IRF5調節(jié)IFN-β的表達,其雖然可以調節(jié)IFIT3的表達,但是并不是直接的相互作用,而是通過間接作用調節(jié)其表達。第四部分mi-146a通過IFIT3調節(jié)THP-1細胞的凋亡考慮到miR-146a可以調節(jié)IFIT3的表達,而以往的研究表明IFIT3與細胞凋亡有關。因此,我們接下來研究了miR-146a是否調節(jié)了THP-1細胞的凋亡,同時也分析了miR-146a調節(jié)THP-1細胞的凋亡是否與IFIT3有關。我們首先將miR-146ainhibitor或mimic轉染入THP-1細胞,然后用白念珠菌對其進行刺激,采用Annexin V-PI法檢測細胞的凋亡狀況。結果我們發(fā)現:miR-146a mimic可以促進細胞的凋亡,而miR-146a inhibitor對細胞凋亡影響不顯著。這些結果表明miR-146a是促進細胞凋亡的因素。隨后,我們采用siRNA技術抑制IFIT3的表達,然后再用熱滅活白念珠菌對THP-1細胞進行刺激。結果發(fā)現IFIT3表達下調后,細胞的凋亡增加。這說明IFIT3具有抗凋亡作用。綜上,這部分結果說明了miR-146a具有促凋亡作用,其促凋亡的作用可能是通過下調IFIT3的表達來實現的。結論綜上,本研究的主要發(fā)現是:白念珠菌可以通過活化NF-κB的方式上調THP-1細胞內miR-146a的表達,這種上調作用與參與白念珠菌識別的模式識別受體Dectin-1無關。MiR-146表達上調具有兩個生物學意義:第一,負向調節(jié)IFN-β的產生,并由此調節(jié)了多個ISG的表達,且我們的研究證實miR-146a的這種生物學功能與其理論靶點IRF5無關;第二,miR-146a通過IFN-β→IFIT3軸調節(jié)細胞的凋亡。
[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.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R519.3

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8 曾躍斌;;白念珠菌全基因組表達譜芯片在抗真菌藥物研究中的應用[A];中國藥理學會第十一屆全國化療藥理學術研討會論文集[C];2012年

9 周萬青;沈瀚;張之烽;張葵;;白念珠菌臨床分離調查及基因分型研究[A];中華醫(yī)學會第七次全國中青年檢驗醫(yī)學學術會議論文匯編[C];2012年

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