蛋白激酶Stk38在抗病毒固有免疫反應(yīng)中的作用機制研究
發(fā)布時間：2018-07-31 18:03
【摘要】：固有免疫(innate immunity)又稱天然免疫(natural immunity),是機體本身具有的抵抗外來病原體入侵的防御能力。固有免疫應(yīng)答由模式識別受體(Pattern recognition receptors,PRRs)對病原體中的相對保守成分的識別而啟動。這些病原體中相對保守的成分被稱為病原相關(guān)分子模式(Pathogen associated molecular Patterns,PAMPs),在病原體生存或致病過程中所必須。模式識別受體識別相應(yīng)的配體之后,能夠通過激活下游的信號分子,經(jīng)一系列信號轉(zhuǎn)導(dǎo)過程,引起炎癥細(xì)胞因子和Ⅰ型干擾素等免疫介質(zhì)的分泌,促進(jìn)機體進(jìn)行免疫應(yīng)答反應(yīng)。對病毒成分的識別和應(yīng)答是固有免疫反應(yīng)的重要組成部分。對病毒核酸成分的識別主要通過TLRs、RLRs和病毒雙鏈DNA識別分子進(jìn)行。TLRs是一類膜受體家族,其中TLR3、TLR7和TLR8存在于細(xì)胞內(nèi)體膜上,可以識別內(nèi)體中的病毒成分。病毒被吞噬之后,其核酸成分就會暴露在吞噬細(xì)胞的內(nèi)體中,與相應(yīng)配體結(jié)合后TLRs能夠分別通過接頭蛋白TRIF或MyD88,經(jīng)下游信號轉(zhuǎn)導(dǎo)分子,活化IRF3、IRF7和NF-κB,促進(jìn)Ⅰ型干擾素和炎癥細(xì)胞因子的分泌。RLRs主要負(fù)責(zé)對胞漿中病毒RNA成分的識別,包括RIG-I和MDA-5兩種受體。病毒感染細(xì)胞后核酸成分進(jìn)入胞漿,被RLRs識別。RLRs完成識別后活化,并招募接頭分子MAVS,進(jìn)一步激活下游的信號通路,磷酸化IRF3、IRF7和NF-κB,誘導(dǎo)Ⅰ型干擾素和炎癥細(xì)胞因子產(chǎn)生。已發(fā)現(xiàn)的病毒雙鏈DNA識別分子眾多,主要通過接頭蛋白STING激活下游的信號通路,磷酸化IRF3、IRF7和NF-κB,誘導(dǎo)Ⅰ型干擾素和炎癥細(xì)胞因子產(chǎn)生。細(xì)胞分泌的Ⅰ型干擾素可以通過自分泌或旁分泌的方式與細(xì)胞膜上的Ⅰ型干擾素受體結(jié)合,活化與之偶聯(lián)的酪氨酸蛋白激酶Jak1(Janus kinase 1)和Tyk2(tyrosine kinase 2),經(jīng)Jak-STAT信號通路,激活下游大量干擾素誘導(dǎo)基因(IFN-stimulated genes,ISGs)的轉(zhuǎn)錄。從而增強機體抗病毒能力,同時還可以再作用于模式識別信號通路,進(jìn)一步促進(jìn)Ⅰ型干擾素的產(chǎn)生,通過正反饋作用使細(xì)胞短時間產(chǎn)生足量Ⅰ型干擾素。而其中STAT1分子在該通路的活化中有重要作用。除受到上游干擾素信號的激活外,STAT1還可以在RIG-I活化后,經(jīng)MAVS非依賴的信號直接磷酸化激活,發(fā)揮作用。由以上Ⅰ型干擾素分泌的信號網(wǎng)絡(luò)的復(fù)雜性可以看出,對Ⅰ型干擾素分泌的調(diào)控也是一個十分復(fù)雜的體系。Ⅰ型干擾素分泌的調(diào)控機制一直是固有免疫領(lǐng)域研究的熱點問題,對其深入研究也有利于我們更好地了解抗病毒固有免疫應(yīng)答機制,并尋找感染性疾病的潛在治療策略。絲氨酸/蘇氨酸蛋白激酶38(Serine/threoninekinase38,Stk38),是NDR蛋白激酶家族的成員。其作為一種新型的Hippo信號通路蛋白激酶,在細(xì)胞生命活動中有廣泛的生物學(xué)功能。我們通過前期研究發(fā)現(xiàn),Stk38分子可以促進(jìn)Smurf1介導(dǎo)的MEKK2的泛素化降解,負(fù)向調(diào)控TLR9介導(dǎo)的炎癥反應(yīng),參與固有免疫應(yīng)答。但其在抗病毒固有免疫領(lǐng)域是否有調(diào)控作用尚未見報道。本研究通過以下三個部分的內(nèi)容,對以Stk38為代表的Hippo信號通路分子在抗病毒固有免疫應(yīng)答中的調(diào)控機制展開研究。一、蛋白激酶Stk38對VSV誘導(dǎo)的巨噬細(xì)胞中IFN-β的表達(dá)調(diào)控作用本部分我們主要通過實驗探究Stk38分子對抗病毒固有免疫反應(yīng)是否有調(diào)控作用。利用小干擾RNA技術(shù),在小鼠原代腹腔巨噬細(xì)胞中轉(zhuǎn)染特異性靶向Stk38分子的siRNA后,通過實時熒光定量pcr檢測IFN-βmRNA表達(dá)水平,ELISA檢測細(xì)胞上清中IFN-β的含量,我們發(fā)現(xiàn)VSV感染誘導(dǎo)的IFN-β的產(chǎn)生能力明顯下降。類似地,在stk38基因敲除小鼠腹腔巨噬細(xì)胞中也發(fā)現(xiàn),VSV感染后其IFN-β的產(chǎn)生能力嚴(yán)重受損。另一方面,我們通過藥物篩選獲得穩(wěn)定表達(dá)外源性stk38分子的raw264.7細(xì)胞株,與對照細(xì)胞株相比,VSV感染后,stk38高表達(dá)細(xì)胞株IFN-β的分泌水平相應(yīng)升高。另外,我們還利用實時熒光定量PCR技術(shù)檢測了VSV感染后Stk38基因敲除小鼠腹腔巨噬細(xì)胞中VSV病毒的核酸成分。結(jié)果發(fā)現(xiàn),與對照細(xì)胞相比,stk38缺陷細(xì)胞中病毒的核酸拷貝數(shù)增高,復(fù)制能力更強。我們還在stk38缺陷小鼠體內(nèi)建立了VSV急性感染疾病模型。通過檢測小鼠血清中IFN-β的分泌水平我們發(fā)現(xiàn),急性VSV感染后,缺陷小鼠血清中IFN-β的分泌明顯減少。同時,Stk38缺陷小鼠的生存率也低于同窩對照野生型小鼠。通過以上實驗結(jié)果我們證實,Stk38對VSV誘導(dǎo)的巨噬細(xì)胞IFN-β的分泌具有正向調(diào)控作用,并能夠抑制病毒的增殖,同時在體內(nèi)對病毒感染小鼠也具有保護(hù)作用。二、Hippo信號效應(yīng)分子YAP對VSV誘導(dǎo)的巨噬細(xì)胞中IFN-β的表達(dá)調(diào)控作用由于Stk38是Hippo信號通路的調(diào)節(jié)分子,因此本部分我們?yōu)榱税l(fā)現(xiàn)Stk38對IFN-β分泌的調(diào)控機制,設(shè)計實驗探究Hippo通路的效應(yīng)分子yap是否參與Stk38對IFN-β的表達(dá)調(diào)控作用。在小鼠腹腔巨噬細(xì)胞中stk38缺失導(dǎo)致YAP表達(dá)水平增高。但是轉(zhuǎn)染yap分子的siRNA對細(xì)胞表達(dá)IFN-β的能力沒有影響,顯示YAP對IFN-β不存在調(diào)控作用。以上實驗結(jié)果表明,Stk38分子對IFN-β的調(diào)控是通過YAP非依賴的方式進(jìn)行的。三、Stk38分子調(diào)控VSV誘導(dǎo)的IFN-β表達(dá)的分子機制本部分主要內(nèi)容為Stk38調(diào)控IFN-β表達(dá)的具體分子機制相關(guān)的實驗結(jié)果。我們通過對干擾素相關(guān)信號通路活化情況的篩選發(fā)現(xiàn),VSV感染誘導(dǎo)的Stk38缺陷細(xì)胞中干擾素相關(guān)通路信號分子STAT1的磷酸化水平降低,提示Stk38可能通過調(diào)節(jié)STAT1的活化水平調(diào)控IFN-β的產(chǎn)生。通過文獻(xiàn)回顧尋找線索,我們發(fā)現(xiàn)糖原合酶激酶3(GSK3)分子對STAT1的活化具有正向調(diào)控作用。因此我們利用GSK3分子的小干擾RNA或特異性抑制劑CHIR-99021處理細(xì)胞,發(fā)現(xiàn)對GSK3分子的抑制確實能夠降低VSV誘導(dǎo)的STAT1的活化和IFN-β的表達(dá)。同時,在HEK293細(xì)胞中轉(zhuǎn)染相關(guān)分子的高表達(dá)載體,我們發(fā)現(xiàn)Stk38分子可以與GSK3分子結(jié)合,通過阻斷Akt對GSK3磷酸化修飾的機制,對GSK3的活性起到保護(hù)作用。最后,CHIR-99021能夠阻斷Stk38分子對STAT1活化和IFN-β表達(dá)的促進(jìn)作用。我們通過本部分實驗,初步證明Stk38可以通過調(diào)節(jié)GSK3-STAT1信號,促進(jìn)VSV感染誘導(dǎo)的IFN-β的合成和分泌。綜上所述,我們通過實驗證實,Hippo信號通路新型蛋白激酶Stk38可以通過GSK3-STAT1信號途徑,促進(jìn)VSV感染誘導(dǎo)的IFN-β的合成和分泌。我們的研究首次發(fā)現(xiàn)Stk38分子在抗病毒固有免疫應(yīng)答中具有調(diào)控作用,并初步探討了其作用機制,提出Stk38分子能夠通過與GSK3分子的相互作用,并經(jīng)GSK3-STAT1信號促進(jìn)IFN-β分泌,增強機體抗病毒的能力。對Stk38調(diào)控IFN-β分泌機制的發(fā)現(xiàn),一方面進(jìn)一步豐富了Stk38的生物學(xué)作用,使對該分子的功能有了新的認(rèn)識;另一方面也對抗病毒固有免疫應(yīng)答調(diào)控的信號網(wǎng)絡(luò)有了部分拓展,為IFN-β分泌的調(diào)控機制研究提供了新的思路;另外,為病毒感染性疾病的治療提供了一個潛在的新靶點。
[Abstract]:Innate immunity, also known as the natural immune (natural immunity), is the body's own defense ability to resist the invasion of foreign pathogens. The intrinsic immune response is initiated by the recognition of the Pattern recognition receptors (PRRs) on the relative conservatives in the pathogen. These pathogens are relatively conservative. Pathogen associated molecular Patterns (PAMPs), which is known as the pathogen associated molecular model (PAMPs), is necessary in the survival or pathogenesis of the pathogen. After the pattern recognition receptor recognizes the corresponding ligand, it can activate the downstream signal molecules through a series of signal transduction pathways, causing inflammatory cytokines and type I interferon and other immunization. The secretion of the medium promotes the body's immune response. Identification and response to the virus components are an important part of the inherent immune response. The identification of viral nucleic acids is mainly through the TLRs, RLRs and viral double stranded DNA recognition molecules that are a class of membrane receptor families, and TLR3, TLR7 and TLR8 exist on the intracellular membrane of the virus. To identify the virus components in the internal body. After the virus is phagocyted, its nucleic acid components will be exposed to the phagocytic inner body. After binding to the corresponding ligand, TLRs can transmit molecules through the joint protein TRIF or MyD88 respectively through the downstream signal transduction molecules, activating IRF3, IRF7 and NF- kappa B, promoting the secretion of.RLRs mainly by type I interferon and inflammatory cytokines. Responsible for identifying the RNA components of the cytoplasm of the cytoplasm, including two receptors of RIG-I and MDA-5. After the virus infected cells, the nucleic acid components entered the cytoplasm, activated by the RLRs recognition.RLRs, and recruited the joint molecule MAVS, further activating the downstream signal pathway, phosphorylated IRF3, IRF7, and NF- kappa B, inducing type I interferon and inflammatory cytokine production. The virus double stranded DNA recognition molecules are numerous, mainly through the joint protein STING activating the downstream signal pathway, phosphorylated IRF3, IRF7 and NF- kappa B, inducing type I interferon and inflammatory cytokine production. Activation and activation of tyrosine protein kinase Jak1 (Janus kinase 1) and Tyk2 (tyrosine kinase 2) activate the transcription of a large number of interferon induced genes (IFN-stimulated genes, ISGs) downstream by Jak-STAT signaling pathway, thereby enhancing the body's anti-virus ability and also reacting on the pattern recognition signaling pathway to further promote I The production of type IFN interferon produces a full amount of interferon I in short time by positive feedback, and the STAT1 molecule plays an important role in the activation of this pathway. In addition to the activation of the upstream interferon signal, STAT1 can also be activated directly by the MAVS non dependent signal after activation of RIG-I. The complexity of the interferon signaling network shows that the regulation of the type I interferon secretion is also a very complex system. The regulatory mechanism of type I interferon is a hot issue in the field of inherent immunity. It is also helpful for us to better understand the mechanism of antiviral inherent immune response and to find out the mechanism of antiviral innate immune response. The potential treatment strategy for finding infectious diseases. Serine / threonine protein kinase 38 (Serine/threoninekinase38, Stk38), a member of the NDR protein kinase family, is a new type of Hippo signaling protein kinase, which has extensive biological functions in cell life activities. We found that Stk38 molecules can be promoted through previous studies. The ubiquitination of Smurf1 mediated MEKK2, which negatively regulates the inflammatory response mediated by TLR9, participates in the inherent immune response. But it has not been reported in the field of antiviral innate immunity. The present study is about the intrinsic immune response of Hippo signaling molecules represented by Stk38 in the following three parts. The role of the regulatory mechanism in the study. 1, the role of protein kinase Stk38 in the regulation of the expression of IFN- beta in macrophages induced by VSV. This part is mainly through the experiment to explore the regulation of Stk38 molecules against the inherent immune response of the virus. By using small interference RNA technology, the specific target Stk38 in the mouse primary peritoneal macrophages is transferred to the specific target Stk38. After siRNA, the expression level of IFN- beta mRNA was detected by real-time fluorescence quantitative PCR, and the content of IFN- beta in cell supernatant was detected by ELISA. We found that the production ability of IFN- beta induced by VSV infection was significantly decreased. Similarly, it was found in the peritoneal macrophages of the stk38 knockout mice that the production capacity of IFN- beta was seriously damaged after the VSV infection. On the one hand, we obtained the RAW264.7 cell line that stably expressed the exogenous stk38 molecule by drug screening. Compared with the control cell line, the secretory level of the stk38 high expression cell line IFN- beta was raised correspondingly after VSV infection. In addition, we also detected the peritoneal macrophages in Stk38 knockout mice after VSV infection by real-time fluorescent quantitative PCR technique. The nucleic acid components of the VSV virus found that the number of DNA copies of the virus in the stk38 deficient cells increased and the replication ability was stronger compared with the control cells. We also established the VSV acute infection model in the stk38 deficient mice. By detecting the secretion of IFN- beta in the serum of the mice, we found that the acute VSV infection, the defective mice after the acute VSV infection. The secretion of IFN- beta in the serum decreased significantly. At the same time, the survival rate of Stk38 deficient mice was also lower than that in the same nest control wild type mice. Through the experimental results, we confirmed that Stk38 has a positive regulatory effect on the secretion of IFN- beta in macrophages induced by VSV, and can inhibit the proliferation of the virus and also protect the virus infected mice in the body. Protection. Two, Hippo signal effect molecule YAP regulates the expression of IFN- beta in VSV induced macrophages because Stk38 is a regulator of Hippo signaling pathway. Therefore, in order to discover the regulation mechanism of Stk38 on the secretion of IFN- beta, we designed the experiment to explore whether the Hippo pathway's response molecule Yap participates in Stk38 on the expression of IFN- beta. Control. Stk38 deletion in mouse peritoneal macrophages increased the level of YAP expression. But siRNA transfected with Yap molecules had no effect on the ability to express IFN- beta, indicating that YAP had no regulatory effect on IFN- beta. The above results showed that the regulation of Stk38 molecules on IFN- beta was carried out through non dependence of YAP. Three, Stk38 score. The molecular mechanism of the expression of IFN- beta induced by VSV is the main content of the specific molecular mechanism related to the regulation of the expression of IFN- beta by Stk38. Through screening of the activation of interferon related signaling pathway, we found that the phosphorylated water of the interferon related pathway signal molecule STAT1 in the Stk38 deficient cells induced by VSV infection Level reduction suggests that Stk38 may regulate the production of IFN- beta by regulating the activation level of STAT1. Through literature review, we found that glycogen synthase kinase 3 (GSK3) molecules have a positive regulatory effect on the activation of STAT1. Therefore, we use small interference RNA or specific inhibitor CHIR-99021 to treat cells, and find out GSK3 on GSK3. Inhibition of molecules can indeed reduce the activation of VSV induced STAT1 and the expression of IFN- beta. At the same time, the high expression vector transfected with related molecules in HEK293 cells, we found that the Stk38 molecule can bind to the GSK3 molecule and protect the activity of GSK3 by blocking the mechanism of the phosphorylation of GSK3 by the Akt. Finally, CHIR-99021 can be blocked. We have proved that Stk38 can promote the synthesis and secretion of IFN- beta induced by VSV infection by adjusting the GSK3-STAT1 signal to promote the synthesis and secretion of IFN- beta by regulating the GSK3-STAT1 signal. In summary, we have proved that the new protein kinase Stk38 of the Hippo signaling pathway can be passed through the GSK3-STAT1 signal path through the experiment. Diameter, promoting the synthesis and secretion of IFN- beta induced by VSV infection. Our study first found that Stk38 molecules play a regulatory role in the antiviral inherent immune response, and preliminarily discussed its mechanism of action. It is suggested that Stk38 molecules can interact with GSK3 molecules and promote IFN- beta secretion through GSK3-STAT1 signals to enhance the body's antiviral activity. The discovery of the regulation of the secretion of IFN- beta by Stk38 further enriches the biological function of Stk38 to make a new understanding of the function of the molecule; on the other hand, it has also developed a part of the signal network against the regulation of the inherent immune response of the virus, and provides a new way of thinking for the study of the regulation mechanism of IFN- beta secretion. The treatment of viral infectious diseases provides a potential new target.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R392
本文編號：2156396
[Abstract]:Innate immunity, also known as the natural immune (natural immunity), is the body's own defense ability to resist the invasion of foreign pathogens. The intrinsic immune response is initiated by the recognition of the Pattern recognition receptors (PRRs) on the relative conservatives in the pathogen. These pathogens are relatively conservative. Pathogen associated molecular Patterns (PAMPs), which is known as the pathogen associated molecular model (PAMPs), is necessary in the survival or pathogenesis of the pathogen. After the pattern recognition receptor recognizes the corresponding ligand, it can activate the downstream signal molecules through a series of signal transduction pathways, causing inflammatory cytokines and type I interferon and other immunization. The secretion of the medium promotes the body's immune response. Identification and response to the virus components are an important part of the inherent immune response. The identification of viral nucleic acids is mainly through the TLRs, RLRs and viral double stranded DNA recognition molecules that are a class of membrane receptor families, and TLR3, TLR7 and TLR8 exist on the intracellular membrane of the virus. To identify the virus components in the internal body. After the virus is phagocyted, its nucleic acid components will be exposed to the phagocytic inner body. After binding to the corresponding ligand, TLRs can transmit molecules through the joint protein TRIF or MyD88 respectively through the downstream signal transduction molecules, activating IRF3, IRF7 and NF- kappa B, promoting the secretion of.RLRs mainly by type I interferon and inflammatory cytokines. Responsible for identifying the RNA components of the cytoplasm of the cytoplasm, including two receptors of RIG-I and MDA-5. After the virus infected cells, the nucleic acid components entered the cytoplasm, activated by the RLRs recognition.RLRs, and recruited the joint molecule MAVS, further activating the downstream signal pathway, phosphorylated IRF3, IRF7, and NF- kappa B, inducing type I interferon and inflammatory cytokine production. The virus double stranded DNA recognition molecules are numerous, mainly through the joint protein STING activating the downstream signal pathway, phosphorylated IRF3, IRF7 and NF- kappa B, inducing type I interferon and inflammatory cytokine production. Activation and activation of tyrosine protein kinase Jak1 (Janus kinase 1) and Tyk2 (tyrosine kinase 2) activate the transcription of a large number of interferon induced genes (IFN-stimulated genes, ISGs) downstream by Jak-STAT signaling pathway, thereby enhancing the body's anti-virus ability and also reacting on the pattern recognition signaling pathway to further promote I The production of type IFN interferon produces a full amount of interferon I in short time by positive feedback, and the STAT1 molecule plays an important role in the activation of this pathway. In addition to the activation of the upstream interferon signal, STAT1 can also be activated directly by the MAVS non dependent signal after activation of RIG-I. The complexity of the interferon signaling network shows that the regulation of the type I interferon secretion is also a very complex system. The regulatory mechanism of type I interferon is a hot issue in the field of inherent immunity. It is also helpful for us to better understand the mechanism of antiviral inherent immune response and to find out the mechanism of antiviral innate immune response. The potential treatment strategy for finding infectious diseases. Serine / threonine protein kinase 38 (Serine/threoninekinase38, Stk38), a member of the NDR protein kinase family, is a new type of Hippo signaling protein kinase, which has extensive biological functions in cell life activities. We found that Stk38 molecules can be promoted through previous studies. The ubiquitination of Smurf1 mediated MEKK2, which negatively regulates the inflammatory response mediated by TLR9, participates in the inherent immune response. But it has not been reported in the field of antiviral innate immunity. The present study is about the intrinsic immune response of Hippo signaling molecules represented by Stk38 in the following three parts. The role of the regulatory mechanism in the study. 1, the role of protein kinase Stk38 in the regulation of the expression of IFN- beta in macrophages induced by VSV. This part is mainly through the experiment to explore the regulation of Stk38 molecules against the inherent immune response of the virus. By using small interference RNA technology, the specific target Stk38 in the mouse primary peritoneal macrophages is transferred to the specific target Stk38. After siRNA, the expression level of IFN- beta mRNA was detected by real-time fluorescence quantitative PCR, and the content of IFN- beta in cell supernatant was detected by ELISA. We found that the production ability of IFN- beta induced by VSV infection was significantly decreased. Similarly, it was found in the peritoneal macrophages of the stk38 knockout mice that the production capacity of IFN- beta was seriously damaged after the VSV infection. On the one hand, we obtained the RAW264.7 cell line that stably expressed the exogenous stk38 molecule by drug screening. Compared with the control cell line, the secretory level of the stk38 high expression cell line IFN- beta was raised correspondingly after VSV infection. In addition, we also detected the peritoneal macrophages in Stk38 knockout mice after VSV infection by real-time fluorescent quantitative PCR technique. The nucleic acid components of the VSV virus found that the number of DNA copies of the virus in the stk38 deficient cells increased and the replication ability was stronger compared with the control cells. We also established the VSV acute infection model in the stk38 deficient mice. By detecting the secretion of IFN- beta in the serum of the mice, we found that the acute VSV infection, the defective mice after the acute VSV infection. The secretion of IFN- beta in the serum decreased significantly. At the same time, the survival rate of Stk38 deficient mice was also lower than that in the same nest control wild type mice. Through the experimental results, we confirmed that Stk38 has a positive regulatory effect on the secretion of IFN- beta in macrophages induced by VSV, and can inhibit the proliferation of the virus and also protect the virus infected mice in the body. Protection. Two, Hippo signal effect molecule YAP regulates the expression of IFN- beta in VSV induced macrophages because Stk38 is a regulator of Hippo signaling pathway. Therefore, in order to discover the regulation mechanism of Stk38 on the secretion of IFN- beta, we designed the experiment to explore whether the Hippo pathway's response molecule Yap participates in Stk38 on the expression of IFN- beta. Control. Stk38 deletion in mouse peritoneal macrophages increased the level of YAP expression. But siRNA transfected with Yap molecules had no effect on the ability to express IFN- beta, indicating that YAP had no regulatory effect on IFN- beta. The above results showed that the regulation of Stk38 molecules on IFN- beta was carried out through non dependence of YAP. Three, Stk38 score. The molecular mechanism of the expression of IFN- beta induced by VSV is the main content of the specific molecular mechanism related to the regulation of the expression of IFN- beta by Stk38. Through screening of the activation of interferon related signaling pathway, we found that the phosphorylated water of the interferon related pathway signal molecule STAT1 in the Stk38 deficient cells induced by VSV infection Level reduction suggests that Stk38 may regulate the production of IFN- beta by regulating the activation level of STAT1. Through literature review, we found that glycogen synthase kinase 3 (GSK3) molecules have a positive regulatory effect on the activation of STAT1. Therefore, we use small interference RNA or specific inhibitor CHIR-99021 to treat cells, and find out GSK3 on GSK3. Inhibition of molecules can indeed reduce the activation of VSV induced STAT1 and the expression of IFN- beta. At the same time, the high expression vector transfected with related molecules in HEK293 cells, we found that the Stk38 molecule can bind to the GSK3 molecule and protect the activity of GSK3 by blocking the mechanism of the phosphorylation of GSK3 by the Akt. Finally, CHIR-99021 can be blocked. We have proved that Stk38 can promote the synthesis and secretion of IFN- beta induced by VSV infection by adjusting the GSK3-STAT1 signal to promote the synthesis and secretion of IFN- beta by regulating the GSK3-STAT1 signal. In summary, we have proved that the new protein kinase Stk38 of the Hippo signaling pathway can be passed through the GSK3-STAT1 signal path through the experiment. Diameter, promoting the synthesis and secretion of IFN- beta induced by VSV infection. Our study first found that Stk38 molecules play a regulatory role in the antiviral inherent immune response, and preliminarily discussed its mechanism of action. It is suggested that Stk38 molecules can interact with GSK3 molecules and promote IFN- beta secretion through GSK3-STAT1 signals to enhance the body's antiviral activity. The discovery of the regulation of the secretion of IFN- beta by Stk38 further enriches the biological function of Stk38 to make a new understanding of the function of the molecule; on the other hand, it has also developed a part of the signal network against the regulation of the inherent immune response of the virus, and provides a new way of thinking for the study of the regulation mechanism of IFN- beta secretion. The treatment of viral infectious diseases provides a potential new target.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R392
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