肠道病毒71型感染的神经损伤特点和神经元损伤机制研究

发布时间：2018-06-07 07:46

  本文选题：手足口病 + 肠道病毒71　； 参考：《中山大学》2016年博士论文

【摘要】：肠道病毒71(Enterovirus 71,EV71)的许多结构特征与小核糖核酸病毒相符合,同时EV71也是肠道病毒家族的一个成员。目前,EV71被认为是手足口病(hand foot and mouth disease,HFMD)的主要致病原。由于EV71感染引起HFMD的临床特点不同于其它肠道病毒,如Cox A16和Cox B3,因此EV71感染引起的HFMD吸引了越来越多的关注。一些发生在中国台湾和阜阳地区的流行性严重HFMD病例表明EV71感染可引起神经系统病变,导致一些感染者心肺功能衰竭甚至死亡。然而,目前并没有有效的EV71疫苗或抗病毒药物。从2008年开始,我国有部分医院开始使用糖皮质激素来对症治疗伴有神经系统症状和神经源性肺水肿的重症HFMD。虽然大量治疗结果表明及时使用合适剂量的糖皮质激素治疗对改善重症HFMD具有显著的效果,但也有部分研究者认为大剂量激素冲击治疗重症HFMD不能改善HFMD的进展和预后。后续的研究又认为,激素治疗的效果与使用的时间点有关,只有及早使用才能取得良好的效果。总之,目前对应用激素治疗重症HFMD还存在争论,其主要原因是激素治疗重症HFMD的机制不清,这导致了糖皮质激素治疗重症HFMD的有效性受到质疑,也严重阻碍了使用推广。针对激素治疗的效果可能与使用的时间点有关,本文收集了2008-2012年广西与广东地区EV-71感染死亡病例的临床演变特点与其尸解特征,探讨EV71神经损伤途径与合理的临床分期。本论文和前人的研究都表明:脑干为EV71攻击的主要靶位,脑干功能衰竭导致的神经源性肺水肿和肺出血为患者的主要死因。因此,我们选择使用体外培养的大鼠脑干神经元作为研究对象。近期,有研究表明神经元细胞膜表面钙网蛋白(cell surface exposed Calreticulin,Ecto-CRT)表达上调可以增加细胞的免疫原性,并诱导小胶质细胞吞噬仍然存活的神经元。基于已进行的激素治疗的有效性以及EV71可以引起广泛CNS炎症的报道,我们推测EV71是通过增加神经元细胞的免疫原性、诱导激活炎症反应来介导神经元损伤的。材料和方法1.病原学检测标本采集:所有病例均用棉签采集咽拭子、粪便或肛拭子,迅速将棉签放入装有3-5ml保存液的配套采样管中,旋紧管盖并密封待检测。样本检测:样品检测采用美国ABI 7500实时荧光定量PCR仪进行实时荧光RT-PCR方法(Real-timeRT-PCR)法进行检测,检测试剂盒为Rneasy Mini Kit(德国QIAGEN公司),按照试剂盒说明书操作。结果判定标准:检测样本Ct值30为阴性;检测样本Ct值≤30,则判样本为阳性结果。2.病理检查对人体组织的使用已经得到广州市妇女儿童医疗中心伦理委员会的许可(许可证号:2015090825)。我们对EV71导致死亡的14例HFMD病人的脑组织使用10%的福尔马林进行固定,固定3周后,进行切片染色,切片厚度为5μm,使用HE染色。3.原代大鼠脑干神经元的培养从出生3天的SD大鼠获得脑干并体外培养脑干神经元。步骤如下:麻醉处死新生3天的SD大鼠(Nembutal i.p.50 mg/kg),在镜下分离脑干。使用组织切片机初步切碎脑组织;然后使用胰酶消化15min;加入DAN酶清除损伤细胞释放出的DNA;1500RPM,离心5min;重悬细胞;1500RPM,离心5min;稀释细胞至1.5×106 cells/ml;将细胞种入培养基[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%二氧化碳培养箱37℃培养。4.细胞表面免疫荧光检测需要进行细胞表面免疫荧光检测的脑干神经元培养在直径为13mm厚度为17um的coverslip上。去除细胞培养基,加入浓度为4%的多聚甲醛,4℃处理30 min;吸去多聚甲醛,用TBS冲洗两遍,每次5 min;封闭液室温封闭1 h;一级抗体在4℃孵育过夜;室温下用TBS冲洗两遍,每次5 min;用荧光二抗室温孵育1 h;室温下用TBS冲洗两遍,每次10 min;用5μg/ml Hoechst染细胞核15 min;PBS洗一遍,最后每孔加入200ul等渗的磷酸盐缓冲液,荧光显微镜拍照。需要进行激光共聚焦拍照是,用厚度为170μm的Coverslip培养相应的细胞。5.生物素酰化法检测细胞表面蛋白细胞处理:冰上用PBS-Ca2+-Mg2+(PBS with 0.1 m M Ca Cl2 and 1 m M Mg Cl2)处理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)]冰上处理细胞,摇床30min;冲洗细胞表面过剩的生物素(PBS-Ca2+-Mg2+plus glycine 100 m M)两遍,每次20min,4°C;刮下并重悬细胞(PBS-Ca2+-Mg2+);离心,2,000 rpm,4°C;重悬并裂解细胞[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;离心14,000 g for 10 min at 4°C;取上清;将上清与streptavidin-agarose beads混合孵育过夜,4°C;离心,5000RPM,3min;洗脱(100°C,5 min in SDS-PAGE裂解液);10%SDS-PAGE胶电泳检测蛋白。结果1.EV-71感染引起脑干神经元的炎症反应,并促使胶质细胞吞噬神经元,从而导致脑干功能障碍本文收集2008-2012年广西与广东地区EV71感染死亡病例的临床演变特点与其尸解特征,对EV71感染致神经系统损伤进行了定位与症状分析。临床研究表明EV71致中枢神经系统感染的患儿可有典型的脑干脑炎症状;中脑、基底节、丘脑、脑干及自主神经功能紊乱症状。本组病例出现神经系统症状至死亡平均时间接近2天,按脑干脑炎分级均经历III级,尸解所见脑干筛状坏死,部分病例累及延髓,可见血管周围有较多淋巴细胞、胶质细胞浸润,噬神经细胞现象,证实病毒嗜神经学说,脑干为EV71攻击的主要靶位,EV-71感染引起脑干神经元的炎症反应,并促使胶质细胞吞噬神经元,最终导致脑干功能衰竭。2.EV71壳蛋白不直接引起神经元死亡我们选择使用体外培养的SD大鼠脑干神经元作为研究对象。出生3天的大鼠脑干神经元在体外分别培养5天、10天、15天和20天后我们发现,体外培养5天的大鼠脑干神经元胞体已经充分长大、轴突粗壮和突触连接已充分建立,而体外培养10天、15天和20天的神经元有聚集和衰老死亡的趋势。因此,我们选择将脑干神经元在体外培养5天后就开始使用。有研究表明,神经毒性病毒的壳蛋白常常在其引起的神经病理过程中发挥重要作用。EV71的外壳由四个蛋白组成,分别命名为VP1、VP2、VP3和VP4。为了检测这些壳蛋白对神经元的影响,我们构建了这些蛋白的过表达质粒。但是在神经元细胞中,我们发现,单独或同时过表达VP1、VP2、VP3、VP4均不会诱导脑干神经元凋亡,也不会增加脑干神经元的总死亡率。3.EV71壳蛋白VP1级联激活神经元内质网应激及细胞自噬,从而诱导CRT在神经元表面上调,增强神经元的免疫原性研究显示,EV71患者体内有免疫因子的变化,但EV71激活固有免疫系统的机制不清。细胞表面钙网蛋白(cell surface exposed Calreticulin,Ecto-CRT)属于损伤相关模式分子,其可以增强细胞的免疫原性。有报道表明,神经元细胞Ecto-CRT上调可以介导小胶质细胞吞噬仍然存活的神经元。我们发现VP1过表达的脑干神经元Ecto-CRT表达增高,而VP2,VP3或VP4的过表达并没有引起类似的现象进一步,我们观察到VP1过表达的脑干神经元内e IF2αSer51的磷酸化水平显著增高,VP1可以激活脑干神经元内质网(ER)产生应激。使用内质网应激抑制剂Salubrinal来阻断内质网应激的激活导致Ecto-CRT的水平明显下降。同时,在脑干神经元内使用si RNA阻断ERp57的表达也可以消除VP1诱导的CRT转位。但是,我们使用内质网应激诱导剂THAPS直接激活内质网应激却没有触发CRT向细胞表面转位。这说明,内质网应激的激活是VP1诱导CRT向细胞膜转位的必要而非充分条件。有研究表明,EV71感染会诱导细胞自噬活性增高。使用荧光显微镜检测细胞内过表达GFP-LC3的聚集情况表明VP1转染细胞内点状GFP-LC3明显增多,这提示VP1的过表达成功激活了神经细胞自噬。用si RNA干扰细胞内源性Atg5的表达后,VP1过表达引起的Ecto-CRT上调受到明显抑制。同时,自噬抑制剂3-Methyladenine(3-MA)在阻断自噬激活的同时显著抑制了VP1过表达引起的Ecto-CRT上调。但是单独使用自噬激活诱导剂Rapamycin或Metformin都无法引发CRT的细胞膜转位。这些结果表明,VP1引起的细胞自噬是CRT转位所必需的,但是自噬也是CRT转位的必要而非充分条件。深入实验表明,联合使用THAPS和Rapamycin可以有效的增加脑干神经元Ecto-CRT的水平。在VP1转染神经元细胞内,我们发现Salubrinal可以抑制自噬的激活。反过来,3-MA对VP1诱导e IF2α活化无影响。这说明,VP1诱导的内质网应激反应介导了后续的自噬激活和最终的Ecto-CRT上调。4.糖皮质激素抑制内质网应激,阻断VP1促Ecto-CRT上调的作用糖皮质激素已被用于治疗伴有中枢神经系统并发症的重症HFMD患者。我们发现地塞米松(DEX)抑制了VP1诱导的Ecto-CRT的表达。但DEX可以抑制VP1过表达或THAPS诱导的内质网应激却不能阻断Rapamycin激活细胞自噬。这些结果提示,糖皮质激素不仅可以作为免疫抑制剂减少炎症因子的表达水平来抑制炎症,还可以减轻VP1诱导的内质网应激从而直接保护神经元。结论本论文的结果证实了EV-71感染引起脑干神经元的炎症反应,并促使胶质细胞吞噬神经元,从而导致脑干功能障碍。通过使用原代培养的脑干神经元,我们阐明了EV71壳蛋白VP1通过激活脑干神经元内质网应激和细胞自噬,从而诱导Ecto-CRT上调来介导神经元损伤的新机制。同时,我们发现糖皮质激素可以减轻VP1诱导的内质网应激和Ecto-CRT上调从而直接保护神经元,为使用糖皮质激素来治疗重症手足口病提供了理论依据。
[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.
【学位授予单位】：中山大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R512.5
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