本文選題：手足口病 + 腸道病毒71　； 參考：《中山大學(xué)》2016年博士論文

【摘要】：腸道病毒71(Enterovirus 71,EV71)的許多結(jié)構(gòu)特征與小核糖核酸病毒相符合,同時(shí)EV71也是腸道病毒家族的一個(gè)成員。目前,EV71被認(rèn)為是手足口病(hand foot and mouth disease,HFMD)的主要致病原。由于EV71感染引起HFMD的臨床特點(diǎn)不同于其它腸道病毒,如Cox A16和Cox B3,因此EV71感染引起的HFMD吸引了越來越多的關(guān)注。一些發(fā)生在中國臺灣和阜陽地區(qū)的流行性嚴(yán)重HFMD病例表明EV71感染可引起神經(jīng)系統(tǒng)病變,導(dǎo)致一些感染者心肺功能衰竭甚至死亡。然而,目前并沒有有效的EV71疫苗或抗病毒藥物。從2008年開始,我國有部分醫(yī)院開始使用糖皮質(zhì)激素來對癥治療伴有神經(jīng)系統(tǒng)癥狀和神經(jīng)源性肺水腫的重癥HFMD。雖然大量治療結(jié)果表明及時(shí)使用合適劑量的糖皮質(zhì)激素治療對改善重癥HFMD具有顯著的效果,但也有部分研究者認(rèn)為大劑量激素沖擊治療重癥HFMD不能改善HFMD的進(jìn)展和預(yù)后。后續(xù)的研究又認(rèn)為,激素治療的效果與使用的時(shí)間點(diǎn)有關(guān),只有及早使用才能取得良好的效果。總之,目前對應(yīng)用激素治療重癥HFMD還存在爭論,其主要原因是激素治療重癥HFMD的機(jī)制不清,這導(dǎo)致了糖皮質(zhì)激素治療重癥HFMD的有效性受到質(zhì)疑,也嚴(yán)重阻礙了使用推廣。針對激素治療的效果可能與使用的時(shí)間點(diǎn)有關(guān),本文收集了2008-2012年廣西與廣東地區(qū)EV-71感染死亡病例的臨床演變特點(diǎn)與其尸解特征,探討EV71神經(jīng)損傷途徑與合理的臨床分期。本論文和前人的研究都表明:腦干為EV71攻擊的主要靶位,腦干功能衰竭導(dǎo)致的神經(jīng)源性肺水腫和肺出血為患者的主要死因。因此,我們選擇使用體外培養(yǎng)的大鼠腦干神經(jīng)元作為研究對象。近期,有研究表明神經(jīng)元細(xì)胞膜表面鈣網(wǎng)蛋白(cell surface exposed Calreticulin,Ecto-CRT)表達(dá)上調(diào)可以增加細(xì)胞的免疫原性,并誘導(dǎo)小膠質(zhì)細(xì)胞吞噬仍然存活的神經(jīng)元�；谝堰M(jìn)行的激素治療的有效性以及EV71可以引起廣泛CNS炎癥的報(bào)道,我們推測EV71是通過增加神經(jīng)元細(xì)胞的免疫原性、誘導(dǎo)激活炎癥反應(yīng)來介導(dǎo)神經(jīng)元損傷的。材料和方法1.病原學(xué)檢測標(biāo)本采集:所有病例均用棉簽采集咽拭子、糞便或肛拭子,迅速將棉簽放入裝有3-5ml保存液的配套采樣管中,旋緊管蓋并密封待檢測。樣本檢測:樣品檢測采用美國ABI 7500實(shí)時(shí)熒光定量PCR儀進(jìn)行實(shí)時(shí)熒光RT-PCR方法(Real-timeRT-PCR)法進(jìn)行檢測,檢測試劑盒為Rneasy Mini Kit(德國QIAGEN公司),按照試劑盒說明書操作。結(jié)果判定標(biāo)準(zhǔn):檢測樣本Ct值30為陰性;檢測樣本Ct值≤30,則判樣本為陽性結(jié)果。2.病理檢查對人體組織的使用已經(jīng)得到廣州市婦女兒童醫(yī)療中心倫理委員會的許可(許可證號:2015090825)。我們對EV71導(dǎo)致死亡的14例HFMD病人的腦組織使用10%的福爾馬林進(jìn)行固定,固定3周后,進(jìn)行切片染色,切片厚度為5μm,使用HE染色。3.原代大鼠腦干神經(jīng)元的培養(yǎng)從出生3天的SD大鼠獲得腦干并體外培養(yǎng)腦干神經(jīng)元。步驟如下:麻醉處死新生3天的SD大鼠(Nembutal i.p.50 mg/kg),在鏡下分離腦干。使用組織切片機(jī)初步切碎腦組織;然后使用胰酶消化15min;加入DAN酶清除損傷細(xì)胞釋放出的DNA;1500RPM,離心5min;重懸細(xì)胞;1500RPM,離心5min;稀釋細(xì)胞至1.5×106 cells/ml;將細(xì)胞種入培養(yǎng)基[Neurobasal TM-A medium containing2%B27 supplement(Thermo Fisher Scientific Inc.),L-glutamine(0.25 m M,Invitrogen),Gluta Max-I(0.25 m M,Thermo Fisher Scientific Inc.),1%penicillin(100U/ml,Invitrogen),1%streptomycin(100μg/ml,Invitrogen)],置入5%二氧化碳培養(yǎng)箱37℃培養(yǎng)。4.細(xì)胞表面免疫熒光檢測需要進(jìn)行細(xì)胞表面免疫熒光檢測的腦干神經(jīng)元培養(yǎng)在直徑為13mm厚度為17um的coverslip上。去除細(xì)胞培養(yǎng)基,加入濃度為4%的多聚甲醛,4℃處理30 min;吸去多聚甲醛,用TBS沖洗兩遍,每次5 min;封閉液室溫封閉1 h;一級抗體在4℃孵育過夜;室溫下用TBS沖洗兩遍,每次5 min;用熒光二抗室溫孵育1 h;室溫下用TBS沖洗兩遍,每次10 min;用5μg/ml Hoechst染細(xì)胞核15 min;PBS洗一遍,最后每孔加入200ul等滲的磷酸鹽緩沖液,熒光顯微鏡拍照。需要進(jìn)行激光共聚焦拍照是,用厚度為170μm的Coverslip培養(yǎng)相應(yīng)的細(xì)胞。5.生物素酰化法檢測細(xì)胞表面蛋白細(xì)胞處理:冰上用PBS-Ca2+-Mg2+(PBS with 0.1 m M Ca Cl2 and 1 m M Mg Cl2)處理10min;使用NHS-SS-biotin 1.25 mg/ml(Pierce)[biotinylation buffer(10 m M triethanolamine,2 m M Ca Cl2,150 m M Na Cl,p H 7.5)]冰上處理細(xì)胞,搖床30min;沖洗細(xì)胞表面過剩的生物素(PBS-Ca2+-Mg2+plus glycine 100 m M)兩遍,每次20min,4°C;刮下并重懸細(xì)胞(PBS-Ca2+-Mg2+);離心,2,000 rpm,4°C;重懸并裂解細(xì)胞[lysis buffer(1%Triton X-100,150 m M Na Cl,5 m M EDTA,50 m M Tris,p H 7.5)containing protease inhibitors],4°C,45min;離心14,000 g for 10 min at 4°C;取上清;將上清與streptavidin-agarose beads混合孵育過夜,4°C;離心,5000RPM,3min;洗脫(100°C,5 min in SDS-PAGE裂解液);10%SDS-PAGE膠電泳檢測蛋白。結(jié)果1.EV-71感染引起腦干神經(jīng)元的炎癥反應(yīng),并促使膠質(zhì)細(xì)胞吞噬神經(jīng)元,從而導(dǎo)致腦干功能障礙本文收集2008-2012年廣西與廣東地區(qū)EV71感染死亡病例的臨床演變特點(diǎn)與其尸解特征,對EV71感染致神經(jīng)系統(tǒng)損傷進(jìn)行了定位與癥狀分析。臨床研究表明EV71致中樞神經(jīng)系統(tǒng)感染的患兒可有典型的腦干腦炎癥狀;中腦、基底節(jié)、丘腦、腦干及自主神經(jīng)功能紊亂癥狀。本組病例出現(xiàn)神經(jīng)系統(tǒng)癥狀至死亡平均時(shí)間接近2天,按腦干腦炎分級均經(jīng)歷III級,尸解所見腦干篩狀壞死,部分病例累及延髓,可見血管周圍有較多淋巴細(xì)胞、膠質(zhì)細(xì)胞浸潤,噬神經(jīng)細(xì)胞現(xiàn)象,證實(shí)病毒嗜神經(jīng)學(xué)說,腦干為EV71攻擊的主要靶位,EV-71感染引起腦干神經(jīng)元的炎癥反應(yīng),并促使膠質(zhì)細(xì)胞吞噬神經(jīng)元,最終導(dǎo)致腦干功能衰竭。2.EV71殼蛋白不直接引起神經(jīng)元死亡我們選擇使用體外培養(yǎng)的SD大鼠腦干神經(jīng)元作為研究對象。出生3天的大鼠腦干神經(jīng)元在體外分別培養(yǎng)5天、10天、15天和20天后我們發(fā)現(xiàn),體外培養(yǎng)5天的大鼠腦干神經(jīng)元胞體已經(jīng)充分長大、軸突粗壯和突觸連接已充分建立,而體外培養(yǎng)10天、15天和20天的神經(jīng)元有聚集和衰老死亡的趨勢。因此,我們選擇將腦干神經(jīng)元在體外培養(yǎng)5天后就開始使用。有研究表明,神經(jīng)毒性病毒的殼蛋白常常在其引起的神經(jīng)病理過程中發(fā)揮重要作用。EV71的外殼由四個(gè)蛋白組成,分別命名為VP1、VP2、VP3和VP4。為了檢測這些殼蛋白對神經(jīng)元的影響,我們構(gòu)建了這些蛋白的過表達(dá)質(zhì)粒。但是在神經(jīng)元細(xì)胞中,我們發(fā)現(xiàn),單獨(dú)或同時(shí)過表達(dá)VP1、VP2、VP3、VP4均不會誘導(dǎo)腦干神經(jīng)元凋亡,也不會增加腦干神經(jīng)元的總死亡率。3.EV71殼蛋白VP1級聯(lián)激活神經(jīng)元內(nèi)質(zhì)網(wǎng)應(yīng)激及細(xì)胞自噬,從而誘導(dǎo)CRT在神經(jīng)元表面上調(diào),增強(qiáng)神經(jīng)元的免疫原性研究顯示,EV71患者體內(nèi)有免疫因子的變化,但EV71激活固有免疫系統(tǒng)的機(jī)制不清。細(xì)胞表面鈣網(wǎng)蛋白(cell surface exposed Calreticulin,Ecto-CRT)屬于損傷相關(guān)模式分子,其可以增強(qiáng)細(xì)胞的免疫原性。有報(bào)道表明,神經(jīng)元細(xì)胞Ecto-CRT上調(diào)可以介導(dǎo)小膠質(zhì)細(xì)胞吞噬仍然存活的神經(jīng)元。我們發(fā)現(xiàn)VP1過表達(dá)的腦干神經(jīng)元Ecto-CRT表達(dá)增高,而VP2,VP3或VP4的過表達(dá)并沒有引起類似的現(xiàn)象進(jìn)一步,我們觀察到VP1過表達(dá)的腦干神經(jīng)元內(nèi)e IF2αSer51的磷酸化水平顯著增高,VP1可以激活腦干神經(jīng)元內(nèi)質(zhì)網(wǎng)(ER)產(chǎn)生應(yīng)激。使用內(nèi)質(zhì)網(wǎng)應(yīng)激抑制劑Salubrinal來阻斷內(nèi)質(zhì)網(wǎng)應(yīng)激的激活導(dǎo)致Ecto-CRT的水平明顯下降。同時(shí),在腦干神經(jīng)元內(nèi)使用si RNA阻斷ERp57的表達(dá)也可以消除VP1誘導(dǎo)的CRT轉(zhuǎn)位。但是,我們使用內(nèi)質(zhì)網(wǎng)應(yīng)激誘導(dǎo)劑THAPS直接激活內(nèi)質(zhì)網(wǎng)應(yīng)激卻沒有觸發(fā)CRT向細(xì)胞表面轉(zhuǎn)位。這說明,內(nèi)質(zhì)網(wǎng)應(yīng)激的激活是VP1誘導(dǎo)CRT向細(xì)胞膜轉(zhuǎn)位的必要而非充分條件。有研究表明,EV71感染會誘導(dǎo)細(xì)胞自噬活性增高。使用熒光顯微鏡檢測細(xì)胞內(nèi)過表達(dá)GFP-LC3的聚集情況表明VP1轉(zhuǎn)染細(xì)胞內(nèi)點(diǎn)狀GFP-LC3明顯增多,這提示VP1的過表達(dá)成功激活了神經(jīng)細(xì)胞自噬。用si RNA干擾細(xì)胞內(nèi)源性Atg5的表達(dá)后,VP1過表達(dá)引起的Ecto-CRT上調(diào)受到明顯抑制。同時(shí),自噬抑制劑3-Methyladenine(3-MA)在阻斷自噬激活的同時(shí)顯著抑制了VP1過表達(dá)引起的Ecto-CRT上調(diào)。但是單獨(dú)使用自噬激活誘導(dǎo)劑Rapamycin或Metformin都無法引發(fā)CRT的細(xì)胞膜轉(zhuǎn)位。這些結(jié)果表明,VP1引起的細(xì)胞自噬是CRT轉(zhuǎn)位所必需的,但是自噬也是CRT轉(zhuǎn)位的必要而非充分條件。深入實(shí)驗(yàn)表明,聯(lián)合使用THAPS和Rapamycin可以有效的增加腦干神經(jīng)元Ecto-CRT的水平。在VP1轉(zhuǎn)染神經(jīng)元細(xì)胞內(nèi),我們發(fā)現(xiàn)Salubrinal可以抑制自噬的激活。反過來,3-MA對VP1誘導(dǎo)e IF2α活化無影響。這說明,VP1誘導(dǎo)的內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng)介導(dǎo)了后續(xù)的自噬激活和最終的Ecto-CRT上調(diào)。4.糖皮質(zhì)激素抑制內(nèi)質(zhì)網(wǎng)應(yīng)激,阻斷VP1促Ecto-CRT上調(diào)的作用糖皮質(zhì)激素已被用于治療伴有中樞神經(jīng)系統(tǒng)并發(fā)癥的重癥HFMD患者。我們發(fā)現(xiàn)地塞米松(DEX)抑制了VP1誘導(dǎo)的Ecto-CRT的表達(dá)。但DEX可以抑制VP1過表達(dá)或THAPS誘導(dǎo)的內(nèi)質(zhì)網(wǎng)應(yīng)激卻不能阻斷Rapamycin激活細(xì)胞自噬。這些結(jié)果提示,糖皮質(zhì)激素不僅可以作為免疫抑制劑減少炎癥因子的表達(dá)水平來抑制炎癥,還可以減輕VP1誘導(dǎo)的內(nèi)質(zhì)網(wǎng)應(yīng)激從而直接保護(hù)神經(jīng)元。結(jié)論本論文的結(jié)果證實(shí)了EV-71感染引起腦干神經(jīng)元的炎癥反應(yīng),并促使膠質(zhì)細(xì)胞吞噬神經(jīng)元,從而導(dǎo)致腦干功能障礙。通過使用原代培養(yǎng)的腦干神經(jīng)元,我們闡明了EV71殼蛋白VP1通過激活腦干神經(jīng)元內(nèi)質(zhì)網(wǎng)應(yīng)激和細(xì)胞自噬,從而誘導(dǎo)Ecto-CRT上調(diào)來介導(dǎo)神經(jīng)元損傷的新機(jī)制。同時(shí),我們發(fā)現(xiàn)糖皮質(zhì)激素可以減輕VP1誘導(dǎo)的內(nèi)質(zhì)網(wǎng)應(yīng)激和Ecto-CRT上調(diào)從而直接保護(hù)神經(jīng)元,為使用糖皮質(zhì)激素來治療重癥手足口病提供了理論依據(jù)。
[Abstract]:Many structural features of enterovirus 71 (Enterovirus 71, EV71) conforms to small ribonucleic viruses, and EV71 is also a member of the enterovirus family. Currently, EV71 is considered to be the main cause of hand foot and mouth disease (hand foot and mouth disease, HFMD). The clinical characteristics of HFMD are different from other enterovirus because of EV71 infection. Such as Cox A16 and Cox B3, HFMD caused by EV71 infection has attracted more and more attention. Some cases of severe HFMD in Taiwan and Fuyang, China, indicate that EV71 infection can cause nervous system lesions, and cause cardiac failure and even death of some infected people. However, there is no effective EV71 vaccine or antiviral activity at present. Drugs. Since 2008, some hospitals in our country have begun to use glucocorticoids to treat severe HFMD. with neurogenic symptoms and neurogenic pulmonary edema, although a large number of treatment results show that timely use of appropriate dose of glucocorticoid therapy has a significant effect on improving severe HFMD, but some researchers believe that The impact of large dose hormone impact therapy on severe HFMD does not improve the progress and prognosis of HFMD. Subsequent studies also suggest that the effect of hormone therapy is related to the time points used, only early use can achieve good results. In a word, there is still a debate on the application of hormone therapy for severe HFMD, the main reason is that hormone therapy for severe HFMD is the main reason. The effectiveness of glucocorticoid in the treatment of severe HFMD has been questioned, and it is a serious hindrance to the use. The effect of hormone therapy may be related to the time points used. This paper collected the characteristics of the evolution of the death cases of EV-71 infected cases in Guangxi and Guangdong for 2008-2012 years, and discussed the EV71 nerve. This paper and previous studies have shown that brainstem is the main target of EV71 attack, neurogenic pulmonary edema and pulmonary hemorrhage caused by brainstem failure are the main causes of death. Therefore, we choose to use in vitro cultured rat brain stem Shen Jing Yuan as the research object. The up-regulated expression of cell surface exposed Calreticulin (Ecto-CRT) on the surface of the cell membrane can increase the immunogenicity of the cells and induce the microglia to phagocyt the still alive neurons. Based on the effectiveness of the hormone therapy and the reports that EV71 can cause extensive CNS inflammation, we speculate that EV71 is increased by the increase. Immunogenicity of neuron cells, inducing activation of inflammatory response to mediate neuron damage. Materials and methods 1. pathogenic detection specimens were collected: all cases were collected by cotton swabs to collect swabs, feces or anal swabs, quickly put the cotton swabs into a supporting sampling tube containing 3-5ml preservative, tighten the lid and seal to be detected. Sample samples were detected. The test sample was detected by real time fluorescence RT-PCR method (Real-timeRT-PCR) method of real time fluorescence quantitative PCR with ABI 7500. The test kit was Rneasy Mini Kit (German QIAGEN company), and the test sample was operated according to the kit instruction. The result was that the test sample Ct value was 30 negative, and the detection sample Ct value was less than 30, then the sample was positive. .2. pathology examination of human tissues has been licensed by the ethics committee of Guangzhou women's and children's Medical Center (license number: 2015090825). We immobilized the brain tissue of 14 cases of HFMD patients resulting from EV71 death. After 3 weeks of fixation, the slice was stained with a slice thickness of 5 micron m, and HE was used to stain.3. original. The brainstem neurons of the rat were cultured from the SD rats born 3 days to obtain brain stem and in vitro culture of brain stem neurons. The following steps were as follows: the SD rats (Nembutal i.p.50 mg/kg) were killed by the anaesthesia and the brain stem was separated under the microscope. The brain tissue was chopped by the tissue microtome; then the trypsin was used to digest 15min, and the DAN enzyme was added to remove the damaged cells. DNA; 1500RPM, centrifuge 5min; suspended cells; 1500RPM, centrifuge 5min; diluted cells to 1.5 * 106 cells/ml; the cells were seeded into the medium [Neurobasal TM-A medium containing2%B27 supplement. N (100U/ml, Invitrogen), 1%streptomycin (100 mu g/ml, Invitrogen)], the surface immunofluorescence of.4. cells cultured in the 5% carbon dioxide incubator at 37 C was detected on the brain stem neurons of the cell surface immunofluorescence on coverslip with the diameter of 13mm with the thickness of 17um, and the cell culture medium was removed, and the concentration of polyoxymethylene was 4%. Treatment at 4 centigrade 30 min; sucked out polyoxymethylene, rinsed two times with TBS, 5 min each time; closed solution was closed at room temperature 1 h; first class antibody was incubated at 4 centigrade; TBS was washed at room temperature for two times, 5 min at room temperature. 1 h was incubated with fluorescence two against room temperature; TBS was washed for two times at room temperature, 10 min each time; 5 micron g/ml Hoechst dye nuclear 15 min; wash again, and last pore each hole The 200ul isosotic phosphate buffer solution was photographed with a fluorescence microscope. The laser confocal photography was needed. The cell surface protein cell processing was detected by.5. biotinylation of the corresponding cell.5. with the thickness of 170 mu Coverslip: PBS-Ca2+-Mg2+ (PBS with 0.1 M M Ca Cl2 and 1); Tin 1.25 mg/ml (Pierce) [biotinylation buffer (10 m M triethanolamine, 2 m M Ca Cl2150 7.5) Split cell [lysis buffer (1%Triton X-100150 m M Na Cl, 5 m M EDTA, 50 m 14000). %SDS-PAGE gel electrophoresis detected the protein. Results 1.EV-71 infection caused the inflammatory response of brain stem neurons and promoted glial cells to phagocytate neurons, resulting in brain stem dysfunction. This paper collected the characteristics of the clinical evolution of EV71 infected cases in Guangxi and Guangdong and its autopsy characteristics for 2008-2012 years, and damage to the nervous system caused by EV71 infection. The clinical study showed that children with EV71 induced central nervous system infection could have typical symptoms of brainstem encephalitis, middle brain, basal ganglia, thalamus, brain stem and autonomic nervous dysfunction. The average time of nervous system symptoms to death was close to 2 days and III level was experienced in the brain stem encephalitis classification in this group. To see the necrosis of the brain stem sieved necrosis and the involvement of the medulla in some cases, there are many lymphocytes, glial cell infiltration, and phagocytosis around the blood vessels, confirming the theory of the virus eosinophilia and the brain stem as the main target for EV71 attack. EV-71 infection causes the inflammation of the brain stem neurons and promotes the glial cells to phagocytate the neurons, eventually leading to the brainstem work. We chose the SD rat brain stem neurons in vitro as the research object. The brain stem neurons of the 3 day old rats were cultured for 5 days in vitro, 10 days, 15 days and 20 days later, we found that the brain stem neurons of the rat brain stem were fully grown and the axon was thick for 5 days in vitro. The connections between the strong and the synapses have been fully established, and the neurons in the culture in vitro for 10 days, 15 days and 20 days have the trend of aggregation and aging death. Therefore, we choose to use the brainstem neurons for 5 days in vitro culture. 1 of the shell was made up of four proteins, named VP1, VP2, VP3, and VP4. to detect the effects of these shell proteins on neurons. We constructed the overexpressed plasmids of these proteins. But in neuronal cells, we found that both VP1, VP2, VP3, and VP4 do not induce apoptosis of brain stem neurons, nor increase the brain stem in neuron cells. The total death rate of the neuron.3.EV71 shell protein VP1 activates the endoplasmic reticulum stress and autophagy in the neuron, which induces the up regulation of CRT on the neuron surface, and the immunogenicity of the neurons in the EV71 patients shows that there is a change in the immune factors in the body, but the mechanism of EV71 activation of the inherent immune system is not clear. The cell surface calcium net protein (cell sur) Face exposed Calreticulin, Ecto-CRT) belongs to the damage related model molecules, which can enhance the immunogenicity of cells. It is reported that the up regulation of neuronal cells Ecto-CRT can mediate microglia phagocytosis still surviving neurons. We found that VP1 overexpressed brain stem nerve element Ecto-CRT expression is higher, and VP2, VP3 or VP4 overexpression. It did not cause a similar phenomenon further. We observed that the level of phosphorylation of E IF2 alpha Ser51 in the brain stem neurons with VP1 overexpression was significantly increased, and VP1 could activate the endoplasmic reticulum (ER) of brain stem neurons to produce stress. The activation of endoplasmic reticulum stress, using the endoplasmic reticulum stress inhibitor Salubrinal, resulted in a significant decrease in the level of Ecto-CRT. The use of Si RNA to block ERp57 expression in brain stem neurons also eliminates the VP1 induced CRT transposition. However, the use of endoplasmic reticulum stress inducer THAPS directly activates endoplasmic reticulum stress but does not trigger the transposition of the CRT to the cell surface. This suggests that the activation of endoplasmic reticulum stress is necessary and not sufficient for VP1 induced CRT to the cell membrane translocation. Conditions. Some studies have shown that EV71 infection induces increased autophagy in cells. The aggregation of overexpressed GFP-LC3 in cells by fluorescence microscopy indicates that the dot like GFP-LC3 in VP1 transfected cells is significantly increased, which suggests that the overexpression of VP1 activates the autophagy of the neurons. Si RNA interferes with the expression of endogenous Atg5 in cells, and VP1 Overwatch At the same time, the autophagy inhibitor 3-Methyladenine (3-MA), which inhibited the activation of autophagy, inhibited the up regulation of Ecto-CRT induced by VP1 overexpression, but the use of autophagy activator Rapamycin or Metformin alone could not trigger the cell membrane translocation of CRT. These results suggest that VP1 caused by VP1. Autophagy is essential for CRT transposition, but autophagy is also a necessary and not sufficient condition for CRT transposition. Further experiments have shown that combined use of THAPS and Rapamycin can effectively increase the level of Ecto-CRT in brain stem neurons. In VP1 transfected neurons, we found that Salubrinal can inhibit the activation of autophagy. In turn, 3-MA is to VP1 lure. The activation of E IF2 alpha has no effect. This suggests that the VP1 induced endoplasmic reticulum stress response mediates subsequent activation of autophagy and the final Ecto-CRT up regulation of.4. glucocorticoid inhibits endoplasmic reticulum stress, blocking the role of VP1 to promote Ecto-CRT up-regulation, and the glucocorticoid has been used in the treatment of severe HFMD patients with complications of the central nervous system. Dexamethasone (DEX) inhibits the expression of Ecto-CRT induced by VP1, but DEX inhibits the overexpression of VP1 or THAPS induced endoplasmic reticulum stress but does not block the autophagy of Rapamycin activated cells. These results suggest that glucocorticoids can not only reduce the expression level of inflammatory factors as immunosuppressants to inhibit inflammation, but also reduce VP1 lure. The results of this paper confirm that EV-71 infection causes the inflammatory response of brain stem neurons and urges glial cells to phagocytate neurons and lead to brain stem dysfunction. By using primary cultured brain stem neurons, we clarify that EV71 shell protein VP1 is activated by brain stem neurons. Endoplasmic reticulum stress and autophagy induce a new mechanism for Ecto-CRT up-regulation to mediate neuronal damage. At the same time, we found that glucocorticoids can reduce VP1 induced endoplasmic reticulum stress and Ecto-CRT up-regulation to protect neurons directly, providing a theoretical basis for the use of glucocorticoids for the treatment of severe hand foot and mouth disease.
【學(xué)位授予單位】：中山大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R512.5
，

本文編號：1990402