CD40siRNA慢病毒载体的构建及其对EAM大鼠Treg细胞的影响

分类号：R7254密级：单位代码：10422学 号：201013252菇办孚硕士学位论文论文题目：CD40siRNA慢病毒载体的构建及其对EAM大鼠Treg细胞的影响Construction of CD40siRNA lentiviral vector and effectof CD40siRNA on Treg cells in rats with EAM作者 姓 名学院 名 称专业 名 称指导 教 师合作 导 师王介忠医学院儿科学韩 波教授2013年4月20日原创性声明本人郑重声明：所呈交的学位论文，是本人在导师的指导下，独立进行研究所取得的成果。除文中已经注明引用的内容外，本论文不包含任何其他个人或集体已经发表或撰写过的科研成果。对本文的研究作出重要贡献的个人和集体，均已在文中以明确方式标明。本声明的法律责任由本人承担。论文作者签名：互葺L 日 期：矽l罗f关于学位论文使用授权的声明本人完全了解山东大学有关保留、使用学位论文的规定，同意学校保留或向国家有关部门或机构送交论文的复印件和电子版，允许论文被查阅和借阅；本人授权山东大学可以将本学位论文的全部或部分内容编入有关数据库进行检索，可以采用影印、缩印或其他复制手段保存论文和汇编本学位论文。(保密论文在解密后应遵守此规定)论文作者签名：秀牡导师签名：目 录中文摘要1英文摘要3符号说明6前 言7日U 舌7第一部分大鼠CD40基因RNAi慢病毒载体的构建与鉴定材料与方法8结 果18；口7K16讨 论1 9小 结21参考文献22第二部分CD40siRNA对自身免疫性心肌炎大鼠Treg细胞的影响材料与方法23结 果26讨 论28参考文献3 1附 图32综 述39致 谢45CONTENTSAbstract in Chinese1Abstract in English3Symbol description6Intl70duction7Part One：Construction and Identification of RNA Interference Lentiviral VectorTargeting CD40 Gene in RatsMaterials andmethods8Results1 8Discussion。19Conclusion21References。22Part Two：Effect of CD40siRNA on regulatory T cells in rats with experimentalautoimmune myocarditisMaterials and methods23Results26Discussion：18Conclusion30References31Figures32Reviews39Acknowledgment45山东大学硕士学位论文CD40siRNA慢病毒载体的构建及其对EAM大鼠Treg细胞的影响专 业：硕士研究生：导 师：儿科学王介忠韩波教授中文摘要研究背景、目的：病毒性心肌炎(VMC)是儿科心血管系统常见疾病，严重危害儿童身体健康，其发病机制尚未完全阐明，亦无特效治疗。目前研究认为，主要是病毒感染及其诱导的免疫应答导致的心肌损伤，其中T淋巴细胞介导的细胞免疫起主导作用，研究病毒性心肌炎的免疫机制己成为当前国内外热点内容。实验性自身免疫性心肌炎(EAM)大鼠模型因其病情进展与人心肌炎极为相似，为研究心肌炎的免疫机制提供了最佳的动物模型。T淋巴细胞介导的细胞免疫需双信号的活化，其中CD40与CD40配体(CD40L)是重要的共刺激信号通路之一。研究表明应用CD40Ig阻断CD40CD40L通路，能显著减轻病毒性心肌炎小鼠心肌炎症。能否应用RNA干扰技术从免疫反应的源头即通过沉默CD40的表达来减轻心肌炎症反应，国内外尚未见文献报道。本实验首先针对大鼠CD40基因构建了CD40siRNA慢病毒载体，然后应用于EAM大鼠，探讨CD40siRNA对心肌免疫损伤的治疗作用及对CD4+CD25+调节性T细胞(Tregs)的影响。研究方法：1针对大鼠CD40基因RNAi设计有效靶序列，合成靶序列的寡核苷酸序列。退火形成双链DNA，与经HpaI和XhoI双酶切后的GVl 18载体连接产生短发卡RNA慢病毒载体，PCR筛选阳性克隆，测序鉴定。随后进行Westernblot外源筛选最佳干扰靶点、慢病毒包装及病毒滴度测定。1山东大学硕士学位论文240只雄性Lewis大鼠随机分成4组，即正常对照组、EAM模型组、CD40siRNA治疗组及siRNA对照组，每组10只大鼠。于实验第1天、第8天，正常对照组大鼠双后肢足垫区皮下注射PBS缓冲液02ml只；另外三组大鼠双后足足垫区皮下注射充分混合的猪心肌球蛋白02ml只。并且于实验第8天，CD40siRNA治疗组尾静脉注射25p,1 CD40siRNA慢病毒表达载体：siRNA对照组尾静脉注射25Itl siRNA慢病毒表达载体。于实验第21天处死所有大鼠，光镜下观察心肌病理变化并计算病理积分；流式细胞仪检测大鼠脾脏中CD4+CD25+Treg的表达。研究结果：1CD40 siRNA成功插入慢病毒载体。慢病毒载体经293T细胞包装成功，测定病毒的滴度为20109 TUmL。2各组大鼠均无死亡，正常对照组大鼠饮食、精神状态良好，其他3组于实验第4天渐出现精神不振、耸毛、进食少、懒动，部分大鼠双足垫出现溃疡、踝关节肿胀，以EAM模型组明显。3EAM模型组心肌病理组织可见炎症细胞浸润，伴有心肌细胞肥大变性，相邻细胞间隙增大，核大深染；CD40siRNA治疗组心肌组织病理积分明显低于EAM模型组(234士060 vs 340士035，p005)。4CD40siRNA治疗组大鼠脾脏CD4+CD25+Treg的表达较EAM模型组明显上调(40701400 W 1220士110，p005)。研究结论：1成功构建大鼠CD40基因的RNAi慢病毒载体，为后期进一步研究病毒性心肌炎发病机制及新的治疗方法提供工作基础。2CD40siRNA可减轻EAM大鼠的心肌炎症，其机制可能与上调CD4+CD25+Treg的表达有关。关键词：CD40siRNA；自身免疫性心肌炎；大鼠；调节性T细胞山东大学硕士学位论文Construction of CD40siRNA lentiviral vector and effect ofCD40siRNA on Treg cells in rats with EAMSpecialty：Post Graduate：Supervisor：Background and Objective：PediatricsWang JiezhongProfHanBoEnglish AbstractViral myocarditis is a common disease of the pediatric cardiovascular system，which seriously influence the childrenS healthTo date，the pathomechanism is notyet fully elucidated and there is no specific or effective treatement for the diseaseIt is reported that viruses infection induced immune responses cause myocardialinjuryIn this process，T lymphocyte-mediated cellular immunity plays a dominantroleTherefore，study on the mechanism of viralmyocarditis has become a hot point inthe worldS current researchExperimental autoimmune myocarditis rat model wassupported as the best model for the study of myocarditisT lymphocyte-mediated cellular immunity needs activation of dual signalCD40and CD40 ligand(CD40 L)is one of the important costimulatory signal pathwaysStudies revealed that block the CD40CD40L signal by CD40一Ig could alleviateviralmyocarditisIt was unknown whether RNA interference could reduce theexpression of CD40 silence from the source of the immune response inflammatoryreactionIn this studN we first construct the CD40 siRNA lentiviral vector anddetermined the therapeutic effect of CD40 siRNA in viralmyocarditis and the effect ofCD4+CD25+regulatory T cellsMethods：1Effective target sequences that target at CD40 gene were designed，thensynthetized the oligonucleotide sequences After annealing of the complementarystrands，the DNA fragments were connected to the GV l l 8 vectors by double digestionwith HpaI and XhoI，to construct the lentiviral vectors which expressed short hairpin3山东大学硕士学位论文RNAThe lentiviral vectors were identified by PCR and DNA sequencingWesternblot was employed to sort the best interfering targets exogenously,and lentiviralpackaging and assaying viral titer were also accomplished240 male Lewis rats were randomly divided into four groups，The normalcontrol group，the EAM model group，CD40 siRNA treatment group and siRNAtreatment group，there are 1 0 rats in each groupAt 1 st day，8th day,subcutaneousinjection PBS buffer 02ml in the two hind footpad of the rats of the control group；while the other three groups of rats subcutaneous injection of mixed sufficientlyporcine cardiac myosin 02ml in the two 11ind footpadAnd at the 8th day,251ACD40siRNA lentiviral expression vector was inj ected through tail vein in theCD40siRNA treatment group；Lentiviral siRNA group Was injected with 259l siRNAexpression vectorAll rats were sacrificed in 2 1 th day,myocardial pathologicalchanges and calculate the pathological integral under light microscope；CD4+CD25+Treg expression in rat spleen was determined by flow cytometerResults：1CD40siRNA successfully 1nserted Into the 1entiviral vectors and the Ientiviralvector was packaged in 293T cellsThe determination of virus titer was 201 09 TUML2There was no death in each groupThe normal control rats eat well and werein good spiritsIn the 4m day the other three groups gradually appears lack of energytowering hair，eating less，lazy movingPart of the rats ulcer in feet pad，ankleswelling，which was obvious in EAM model group3Inflammatory cell infiltration was observed in pathologic tissue of EAMmodel，accompanied with myocardial the cell hypertrophy degeneration，the adj acentcell gap increases，and a large deeply staine