發(fā)布時(shí)間：2018-05-10 18:29

  本文選題：腸道病毒71型 + 腦微血管內(nèi)皮細(xì)胞�。� 參考：《南方醫(yī)科大學(xué)》2012年博士論文

【摘要】：一、研究背景和目的 腸道病毒71型(enterovirus71, EV71)屬于小核糖核酸病毒科腸道病毒屬的成員,歸屬于人類腸道病毒A。目前已知EV71的感染可以導(dǎo)致手足口病、皰疹性咽峽炎、無菌性腦膜炎、腦炎和脊髓灰質(zhì)炎樣的麻痹性疾病等多種與神經(jīng)系統(tǒng)相關(guān)的疾病,嚴(yán)重病理癥狀通常在兒童中發(fā)生。自1974年Schmidt等人首次報(bào)道從美國加利福尼亞暴發(fā)的表現(xiàn)為神經(jīng)系統(tǒng)癥狀疾病(1969-1973年)的患者中分離到EV71,隨后,世界上許多國家相繼報(bào)道了EV71病毒在不同地區(qū)的流行情況,EV71病毒已在世界范圍內(nèi)引起多次暴發(fā)與流行。盡管EV71的基因組和生命周期相似于小核糖核酸病毒科的其它成員,但相應(yīng)引起的病理癥狀明顯不同；至今尚無特異性的治療方法及有效的針對性的疫苗預(yù)防EV71的感染,因此明確EV71的致病機(jī)制對控制嬰幼兒手足口病的流行與有效治療該疾病具有十分重要的意義。 相關(guān)研究證實(shí)在EV71患者中樞神經(jīng)系統(tǒng)(central nervous system, CNS)的腦干、神經(jīng)元等部位都已檢測到EV71基因及抗原的存在,證明EV71能進(jìn)入CNS。目前,對于EV71進(jìn)入CNS的途徑有兩種理論,如同脊髓灰質(zhì)炎：病毒入血穿血腦屏障或通過末梢神經(jīng)沿逆軸突運(yùn)輸感染CNS。有關(guān)研究證實(shí)新生小鼠口服EV71后,早期可引起持續(xù)性病毒血癥和血腦脊液屏障通透性增加,推測EV71可通過血運(yùn)途徑傳播,但腦組織低水平的病毒數(shù)量提示血源性途徑并非累及CNS的主要途徑,所以EV71穿血腦屏障(blood brain barrier, BBB)機(jī)制還未明確。眾所周知,BBB是循環(huán)和CNS之間一道主要的屏障,它限制著不同物質(zhì)在兩個(gè)部位之間的自由運(yùn)輸,對維持CNS的體內(nèi)平衡起著一個(gè)關(guān)鍵的角色,故明確EV71穿BBB的機(jī)制對于防治EV71所致的CNS感染有重要意義,這亦是我們想探討的內(nèi)容。我們知道,病毒感染過程的第一步是病毒通過與位于細(xì)胞表面的特異性受體結(jié)合而吸附于被感染的細(xì)胞的表面,然后利用細(xì)胞的內(nèi)吞作用進(jìn)入細(xì)胞或?qū)⒉《镜暮怂後尫胚M(jìn)細(xì)胞。所以我們推測構(gòu)成BBB的主要成分之一內(nèi)皮細(xì)胞可能是EV71的易感細(xì)胞,EV71與其結(jié)合而導(dǎo)致BBB結(jié)構(gòu)改變和功能障礙,從而感染中樞神經(jīng)系統(tǒng)。本研究采用分離鑒定的來自于重癥手足口病患者的EV71毒株感染HBMEC,形態(tài)學(xué)觀察HBMEC的細(xì)胞病變、透射電鏡檢測HBMEC中EV71的超微結(jié)構(gòu)與Real-Time PCR方法以檢測HBMEC細(xì)胞培養(yǎng)上清液中EV71拷貝數(shù)變化情況,以探討EV71是否能感染HBMEC并能在其中復(fù)制；且進(jìn)一步檢測EV71是否可以誘導(dǎo)HBMEC細(xì)胞骨架的改變并誘導(dǎo)其凋亡。最后,因?yàn)椴《境晒?shí)現(xiàn)對宿主細(xì)胞的感染并在細(xì)胞內(nèi)復(fù)制需要克服細(xì)胞對病毒感染產(chǎn)生的各種免疫防衛(wèi)反應(yīng),阻斷或影響宿主細(xì)胞的正常循環(huán)機(jī)制,利用宿主細(xì)胞的物質(zhì)和能量合成病毒自身物質(zhì),這種相互作用最終會導(dǎo)致宿主細(xì)胞蛋白表達(dá)模式的改變,這種改變影響著宿主細(xì)胞的正常生理功能,決定和反映著病毒感染致病的進(jìn)程和結(jié)果。為了研究EV71感染HBMEC后宿主細(xì)胞蛋白表達(dá)模式的改變,為揭示病毒與HBMEC的相互作用機(jī)制、病毒的分子致病機(jī)制、尋找病毒的作用靶標(biāo)等研究提供有意義的信息。本研究采用2-D技術(shù)分析HBMEC感染EV71后1h、16h、24h時(shí)間點(diǎn)與正常HBMEC的培養(yǎng)上清和細(xì)胞內(nèi)的蛋白表達(dá)差異點(diǎn),然后對差異蛋白點(diǎn)進(jìn)行酶切,提取肽片段,最后用MALDI-TOF/TOF-MS質(zhì)譜技術(shù)獲得PMF MSMS圖,并利用Internet上的蛋白質(zhì)數(shù)據(jù)庫對肽質(zhì)量進(jìn)行檢索,尋找具有相似肽質(zhì)量指紋圖的蛋白質(zhì),鑒定了差異率2.5的蛋白。并采用免疫印跡方法進(jìn)一步對其中波形蛋白(vimentin)差異蛋白的變化進(jìn)行了驗(yàn)證。 二、方法 1、分離與鑒定來自于臨床診斷為重癥手足口病患者的EV71毒株 采用來自于臨床診斷為重癥手足口病患者的糞便或肛拭子,經(jīng)腸道病毒通用型引物及EV71特異性引物檢測初步診斷為EV71感染。再經(jīng)RD細(xì)胞分離增殖病毒,并采用EV71(226bp)引物與EV71VP1全基因序列(1086bp)引物進(jìn)行RT-PCR再次鑒定,并測序與blast比對證實(shí)。 2、EV71對人腦微血管內(nèi)皮細(xì)胞的感染 采用已分離鑒定的EV71毒株感染HBMEC并設(shè)置空白對照組：觀察HBMEC感染EV71后不同時(shí)間點(diǎn)的形態(tài)改變,并采用兔抗-EV71多克隆抗體檢測HBMEC內(nèi)EV71抗原的存在,透射電鏡直接觀察EV71作用HBMEC內(nèi)是否存在EV71病毒顆粒,并采用(226bp)EV71特異性引物對EV71感染HBMEC和RD細(xì)胞不同時(shí)間點(diǎn)的培養(yǎng)上清液行定量Real-Time PCR,以檢測HBMEC和RD細(xì)胞感染EV71不同時(shí)間點(diǎn)分泌的EV71拷貝數(shù)變化以得出EV71在HBMEC的復(fù)制趨勢圖。 3、采用Jc-1線粒體早期凋亡試劑盒檢測EV71感染HBMEC8h時(shí)能否導(dǎo)致HBMEC的早期線粒體膜電位降低；采用Annexin-V FITC/PI kit檢測EV71能否導(dǎo)致HBMEC的凋亡；采用羅丹明-鬼筆環(huán)肽染色以探討EV71感染HBMEC能否導(dǎo)致HBMEC細(xì)胞骨架的重排。 4、EV71感染HBMEC差異蛋白質(zhì)組學(xué)的研究 采用分離EV71毒株在不同時(shí)間點(diǎn)感染HBMEC,收集裂解細(xì)胞,采用Bradford染色液定量蛋白,一向等電聚焦,二向聚丙烯酰胺凝膠電泳(SDS-PAGE),并進(jìn)行銀染和考染,銀染膠用來分析、考染膠進(jìn)行質(zhì)譜鑒定。采用ImageScanner掃描儀對膠進(jìn)行掃描,凝膠圖像用ImageMaster7.0進(jìn)行分析。設(shè)置差異點(diǎn)倍數(shù)為2.5倍，對差異點(diǎn)進(jìn)行確認(rèn)，，剔除單塊膠個(gè)別豐度過高或低的點(diǎn)。挖取差異點(diǎn)進(jìn)行酶切，通過基質(zhì)輔助激光解析電離飛行時(shí)間質(zhì)譜和蛋白信息數(shù)據(jù)庫建立差異蛋白質(zhì)譜。并對鑒定出的蛋白進(jìn)行功能分析,并采用免疫印跡技術(shù)進(jìn)一步驗(yàn)證鑒定出的差異蛋白vimentin在HBMEC感染EV71不同時(shí)間點(diǎn)及正常HBMEC中的表達(dá)。 三、統(tǒng)計(jì)分析 數(shù)據(jù)均用均數(shù)±標(biāo)準(zhǔn)差表示,采用SPSS13.0進(jìn)行統(tǒng)計(jì)分析。不同時(shí)間點(diǎn)的凋亡率以及HBMEC與RD細(xì)胞兩組間的EV71拷貝數(shù)比較采用兩個(gè)重復(fù)測量因素的方差分析,線粒體凋亡采用獨(dú)立樣本t檢驗(yàn)分析。P0.05認(rèn)為有統(tǒng)計(jì)學(xué)意義,相關(guān)統(tǒng)計(jì)圖采用Excel或Origin繪圖軟件制作 四、結(jié)果 1、分離鑒定出一株重癥EV71毒株。 2、從形態(tài)學(xué)觀察,EV71可以導(dǎo)致HBMEC的細(xì)胞病變,隨著感染時(shí)間延長,病變程度愈嚴(yán)重；通過透射電鏡可以發(fā)現(xiàn)EV71處理HBMEC內(nèi)存在直徑20-30nm的病毒顆粒；采用抗EV71多克隆抗體進(jìn)一步證實(shí)EV71組HBMEC內(nèi)EV71抗原的存在；而采用EV71特異性引物行實(shí)時(shí)熒光定量PCR表明EV71能感染HBMEC,并且可在HBMEC內(nèi)復(fù)制,其拷貝數(shù)在感染第3天達(dá)到最高峰,其復(fù)制最高峰出現(xiàn)時(shí)間比EV71易感細(xì)胞RD細(xì)胞早,但最終拷貝數(shù)低于RD細(xì)胞(P0.01)；通過采用羅丹明-鬼筆環(huán)肽染色證實(shí)EV71感染HBMEC能導(dǎo)致HBMEC細(xì)胞骨架的重排；另外采用JC-1試劑盒證實(shí)EV71能在8h誘導(dǎo)HBMEC早期凋亡；采用annexin-V FITC/PI凋亡檢測試劑盒檢測到EV71能誘導(dǎo)HBMEC的凋亡,而隨著感染時(shí)間的延長,主要以細(xì)胞壞死為主,感染時(shí)間對EV71感染所誘導(dǎo)的凋亡和壞死有顯著影響(P0.01)。 3、通過雙向凝膠電泳與質(zhì)譜鑒定,初步鑒定了HBMEC感染EV71在0、1、16、24h的28個(gè)差異表達(dá)蛋白。 4、通過免疫印跡對EV71感染HBMEC與HBMEC的差異表達(dá)蛋白進(jìn)一步驗(yàn)證,證實(shí)EV71感染HBMEC可以誘導(dǎo)Vimentin的表達(dá)下調(diào),與蛋白質(zhì)組學(xué)結(jié)果一致。 五、結(jié)論 1、我們分離鑒定了來自于重癥手足口病患者的EV71野生毒株,體外模擬EV71感染HBMEC模型。 2、通過形態(tài)學(xué)觀察及免疫熒光和透射電鏡方法證實(shí)EV71能感染HBMEC;采用Real-time PCR方法進(jìn)一步證實(shí)EV71能在HBMEC內(nèi)復(fù)制,這說明HBMEC是EV71的易感細(xì)胞,但復(fù)制效率低于RD細(xì)胞。HBMEC上可能存在EV71的特異性受體,EV71與BBB內(nèi)皮細(xì)胞上特異性受體結(jié)合進(jìn)而通過內(nèi)吞作用進(jìn)入細(xì)胞里面復(fù)制,導(dǎo)致其形態(tài)與功能改變而穿BBB入腦。 3、采用JC-1線粒體凋亡檢測試劑盒證實(shí)EV71感染HBMEC能誘導(dǎo)其線粒體膜電位降低,并且采用Annex-V FITC/PI染色方法證實(shí)EV71能誘導(dǎo)HBMEC的凋亡,隨著感染時(shí)間延長,細(xì)胞主要以壞死為主。說明EV71可誘導(dǎo)HBMEC線粒體的凋亡,而線粒體結(jié)構(gòu)和功能障礙是各種刺激因素誘導(dǎo)細(xì)胞凋亡的中心事件,并由此導(dǎo)致線粒體內(nèi)凋亡誘發(fā)因子的釋放,參與對細(xì)胞凋亡的調(diào)控,而引起了HBMEC的凋亡。 4、EV71感染HBMEC能導(dǎo)致HBMEC細(xì)胞微絲結(jié)構(gòu)的重排,而微絲結(jié)構(gòu)的改變可能對于病毒的復(fù)制和活化也起到一定的輔助作用。 5、蛋白質(zhì)組學(xué)研究共發(fā)現(xiàn)EV71感染HBMEC進(jìn)程中28個(gè)差異表達(dá)蛋白,按功能分為9類。結(jié)合差異譜中表達(dá)量發(fā)生改變的蛋白的功能與EV71感染和未感染HBMEC蛋白表達(dá)量變化分析,發(fā)現(xiàn)了一些在感染中有重要意義的分子靶標(biāo)。 6、Vimentin差異表達(dá)蛋白可能在EV71感染HBMEC事件中起著一定的作用。
[Abstract]:First, research background and purpose
The enterovirus 71 (enterovirus71, EV71) is a member of the genus enterovirus of the family small ribonucleic virus, belonging to human enterovirus A.. The current known infection of EV71 can lead to hand foot and mouth disease, herpetic angina, aseptic meningitis, encephalitis, and poliomyelitis like paralysis diseases, and a variety of nervous system related diseases. Severe pathological symptoms usually occur in children. Since 1974, Schmidt and others first reported that EV71 was isolated from patients with neurological symptoms (1969-1973 years) outbreaks in California, United States. Subsequently, many countries in the world reported the prevalence of EV71 virus in different regions. The EV71 virus has been cited worldwide. Although the genome and life cycle of EV71 are similar to other members of the family of small ribonucleic virus, the corresponding pathological symptoms are obviously different. There are no specific treatment methods and effective targeted vaccines to prevent EV71 infection. Therefore, it is clear that the pathogenesis of EV71 is to control infants and feet. The prevalence of oral diseases is of great importance in the effective treatment of the disease.
Relevant studies have confirmed that the presence of EV71 genes and antigens has been detected in the brain stem and neurons of the central nervous system (central nervous system, CNS) in EV71 patients. It is proved that EV71 can enter CNS. currently. There are two theories for EV71 entering CNS, like spinal gray matter: the virus enters the blood barrier or passes through the end of the nerve. CNS. related studies of the trans axon transport infection confirmed that after the oral EV71 was taken by the newborn mice, the early onset of sustained viremia and the permeability of the blood cerebrospinal fluid barrier (CSF) barrier increased, and it was suggested that EV71 could be transmitted through the blood transport pathway, but the number of low levels of the virus in the brain suggests that the blood source pathway is not the main pathway involved in CNS, so EV71 is wearing the blood brain barrier (blood). The mechanism of brain barrier, BBB) is not yet clear. As we all know, BBB is a major barrier between circulation and CNS. It restricts the free transport of different substances between two parts and plays a key role in maintaining the balance of CNS in the body. Therefore, it is important for the mechanism of EV71 to wear BBB to prevent CNS infection caused by EV71. We know that the first step of the virus infection process is that the virus is adsorbed on the surface of the infected cell by binding to the specific receptor located on the surface of the cell and then using the endocytosis of the cell to enter the cell or release the virus's nucleic acid into the cell. So we speculate that the main components of the BBB are the main components of the virus. An endothelial cell may be a susceptible cell of EV71. EV71 is associated with BBB structural changes and dysfunction and thus infects the central nervous system. In this study, the isolated EV71 strains from patients with severe hand foot and mouth disease infected HBMEC, morphological observation of HBMEC cytopathic lesions, and transmission electron microscopy to detect the ultrastructure of EV71 in HBMEC. Structure and Real-Time PCR method to detect the change of EV71 copy number in the supernatant of HBMEC cell culture, to explore whether EV71 can infect HBMEC and be able to copy it, and further detect whether EV71 can induce the change of HBMEC cytoskeleton and induce its apoptosis. Finally, the virus successfully infects the host cell and is in the cell Internal replication needs to overcome the various immune defense responses of the cell to virus infection, blocking or affecting the normal circulation mechanism of the host cells, using the substance and energy of the host cells to synthesize the virus itself. This interaction ultimately leads to the change in the protein expression pattern of the host cell, which affects the positive cell of the host. Normal physiological functions, determine and reflect the process and results of viral infection. In order to study the changes in the protein expression patterns of host cells after EV71 infection, the interaction mechanism of the virus and HBMEC, the molecular pathogenesis of the virus, and the target for the search for the action of the virus are provided. This study uses the 2-D technology. After HBMEC infection of EV71, 1H, 16h, 24h time point and normal HBMEC culture supernatant and protein expression in cells were different. Then, the difference protein points were cut by enzyme, and peptide fragments were extracted. Finally, the PMF MSMS map was obtained by MALDI-TOF/TOF-MS mass spectrometry, and the peptide quality was retrieved by using the egg white matter database on Internet to find the phase of the peptide. The protein of peptide mass fingerprinting was identified with a difference rate of 2.5, and the difference of vimentin (vimentin) protein was verified by immunoblotting.
Two, method
1, isolation and identification of EV71 strains from clinically diagnosed patients with severe hand foot mouth disease.
The fecal or anal swabs from patients with severe hand foot and mouth disease were preliminarily diagnosed as EV71 infection by common primers of enterovirus and EV71 specific primers, and then RD cells were isolated and proliferating, and EV71 (226bp) primers and EV71VP1 full gene sequence (1086bp) primers were used to reidentify RT-PCR, and sequencing and blas were sequenced and Blas. T alignment proved.
2, EV71 infection of human brain microvascular endothelial cells
The isolated EV71 strains were infected with HBMEC and the blank control group was set up. The morphological changes of HBMEC infected EV71 at different time points were observed and the existence of EV71 antigen in HBMEC was detected by Rabbit anti -EV71 polyclonal antibody. The presence of EV71 virus particles in HBMEC was observed directly by transmission electron microscopy, and (226bp) EV71 specificity was used. The culture supernatant of EV71 infected HBMEC and RD cells at different time points was used for quantitative Real-Time PCR to detect the EV71 copy number of HBMEC and RD cells infected with EV71 at different time points to obtain the replication trend of EV71 in HBMEC.
3, whether the early mitochondrial membrane potential of EV71 infection was detected by Jc-1 mitochondrial early apoptosis kit could lead to the decrease in the early mitochondrial membrane potential of HBMEC; the detection of EV71 could lead to the apoptosis of HBMEC by Annexin-V FITC/PI kit, and the Luo Danming phelic cyclic peptide staining was used to investigate whether EV71 infection HBMEC could lead to the rearrangement of the skeleton of the HBMEC cells.
4, EV71 infection HBMEC differential proteomics
The isolated EV71 strains were infected with HBMEC at different time points, collecting lysate cells, using Bradford staining liquid quantitative protein, always isoelectric focusing, two polyacrylamide gel electrophoresis (SDS-PAGE), silver staining and staining, silver staining gel used for analysis, test gel for qualitative identification. The gel was scanned with ImageScanner scanner and gel was used to scan the gel. The image was analyzed with ImageMaster7.0. The difference point multiplier was 2.5 times, the difference points were confirmed, the individual abundances were excessively high or low. The difference points were removed and the enzyme was cut. The differential protein mass spectrometry was built by the matrix assisted laser analytical ionization time of flight mass spectrometry and protein information database. Functional analysis and immunoblotting were used to further verify the expression of the differential protein vimentin in HBMEC infected EV71 at different time points and in normal HBMEC.
Three, statistical analysis
The data were expressed with mean standard deviation, and SPSS13.0 was used for statistical analysis. The rate of apoptosis at different time points and the number of EV71 copies between the two groups of HBMEC and RD cells were compared with the variance analysis of two repeated measurement factors. The mitochondrial apoptosis was analyzed by independent sample t test and.P0.05 recognition was statistically significant, and the correlation statistical map was Excel Or Origin drawing software
Four, the result
1, a severe EV71 strain was isolated and identified.
2, from morphological observation, EV71 can lead to the cytopathic disease of HBMEC. With the prolonged infection, the more serious the lesion is, EV71 can be found by transmission electron microscopy to deal with the virus particles in 20-30nm in the diameter of HBMEC, and the existence of EV71 antigen in the EV71 group HBMEC is further confirmed by anti EV71 polyclonal antibody; and EV71 specific induction is used. Real time fluorescence quantitative PCR shows that EV71 can infect HBMEC, and can be replicated in HBMEC, and its copy number reaches the peak at third days of infection. The peak time of its replication peak appears earlier than that of EV71 cell RD cells, but the final copy number is lower than that of RD cells (P0.01); and the EV71 infection HBMEC can lead to HBM by using rhodamine phiric cyclic peptide staining. The rearrangement of EC cytoskeleton, and JC-1 kit confirmed that EV71 could induce apoptosis in early HBMEC by 8h, and annexin-V FITC/PI apoptosis detection kit was used to detect the apoptosis of HBMEC, but with the prolongation of the infection time, it was mainly cell necrosis, and the time of infection had a significant effect on the apoptosis and necrosis induced by EV71 infection. Ringing (P0.01).
3, two dimensional gel electrophoresis and mass spectrometry were used to identify 28 differentially expressed proteins of HBMEC infected with EV71 in 0,1,16,24h.
4, the differential expression protein of HBMEC and HBMEC infected by EV71 was further verified by immunoblotting. It was confirmed that EV71 infection HBMEC could induce the downregulation of Vimentin expression, which was consistent with the proteomic results.
Five. Conclusion
1, we isolated and identified EV71 wild strains from severe HFMD patients, and simulated the EV71 infection HBMEC model in vitro.
2, EV71 can infect HBMEC by morphological observation, immunofluorescence and transmission electron microscopy, and Real-time PCR method is used to further confirm that EV71 can be replicated in HBMEC, which indicates that HBMEC is a susceptible cell of EV71, but the replication efficiency is lower than that of EV71 receptor on RD cell.HBMEC. EV71 and specific receptors on endothelial cells are specific. In combination with endocytosis, it enters the cell to replicate, resulting in its morphological and functional changes and wearing BBB into the brain.
3, the JC-1 mitochondrial apoptosis detection kit confirmed that EV71 infection HBMEC could induce the decrease of mitochondrial membrane potential, and the Annex-V FITC/PI staining method proved that EV71 could induce apoptosis of HBMEC. With the prolongation of the infection time, the cells were mainly necrotic. It shows that EV71 can induce the apoptosis of HBMEC mitochondria, and the mitochondrial structure and functional barrier can be induced by EV71. It is the central event of inducing apoptosis by various stimulant factors, and thus leads to the release of apoptosis inducing factors in mitochondria, and participates in the regulation of apoptosis, and causes the apoptosis of HBMEC.
4, EV71 infection with HBMEC can lead to rearrangement of HBMEC cell microfilament structure, and the change of microfilament structure may play a supporting role in virus replication and activation.
5, a total of 28 differentially expressed proteins in the process of EV71 infection were found in the proteomics study, and they were divided into 9 categories according to their function. The function of the protein expressed in the differential spectrum and the changes in the expression of EV71 and uninfected HBMEC protein were analyzed, and some important molecular targets in the infection were found.
6, Vimentin differentially expressed proteins may play a role in EV71 infection of HBMEC.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R373

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 羅文英;吳顯勁;彭亮;何肖龍;張立科;張文炳;曹虹;;腸道病毒71型誘導(dǎo)人腦微血管內(nèi)皮細(xì)胞的凋亡[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2014年13期

本文編號：1870396