【作者】 张松；

【导师】 周智广；

【作者基本信息】 中南大学 ， 内科学， 2007， 博士

【摘要】 1型糖尿病（type 1 diabetes mellitus，T1DM）是一种以分泌胰岛素的胰岛β细胞特异性损伤为特征的自身免疫性疾病。研究已经表明，CD4⁺和CD8⁺T淋巴细胞、巨噬细胞等均参与了对胰岛β细胞的破坏，而且凋亡是胰岛β细胞主要的死亡形式。T1DM的发病机制十分复杂，虽然目前仍不十分清楚，但越来越多的证据显示，Th1细胞及其细胞因子是导致胰岛β细胞的破坏的重要原因，而使免疫平衡向Th2方向偏移能够延缓甚至预防T1DM的发病。此外，CD4⁺CD25⁺调节性T细胞在维持机体外周免疫耐受中发挥着十分重要的作用，诱导CD4⁺CD25⁺调节性T细胞的产生可以有效地预防和治疗T1DM。研究发现，在T1DM患者发展成为显性糖尿病之前存在一个相对较长的临床前期，在此期进行有效的免疫干预有可能延缓甚至预防糖尿病的发生发展。因此，T1DM早期有效的干预方式已越来越受到人们的关注。机体重建对胰岛素自身抗原的免疫耐受是预防和治疗T1DM的重要手段之一，在目前报道的胰岛自身抗原中，谷氨酸脱羧酶（glutamic acid decarboxylase，GAD）65被认为是T1DM发生发展的关键抗原，而且研究显示GAD65能够通过诱导免疫耐受或者其他的机制，减轻胰岛炎，预防T1DM。DNA疫苗由于其具有安全性高、制备简单经济、易于储存运输等特点，近年来已经在感染性疾病和肿瘤的预防中取得了较好的疗效。目前已有运用编码自身抗原GAD65的DNA疫苗对T1DM预防研究的报道，但是结果很不一致，甚至出现相反的结论。我们以前的研究结果也证明，编码人全长GAD65基因的DNA疫苗来说仅使人类T1DM理想的动物模型——非肥胖糖尿病（non-obese diabetic，NOD）鼠糖尿病的发病率降低30％左右，预防效果尚不理想，还有许多可以改进之处。综上所述，本研究的目的在于：优化我们以前构建的编码全长GAD65基因的DNA疫苗，并对NOD鼠进行预防，进一步探讨其可能的机制，旨在为人类T1DM的预防进一步奠定实验基础。本研究共分为三个部分：1．通过改良抗原基因序列、与细胞因子基因或其他胰岛自身抗原基因共表达的方法，构建和鉴定8种新的GAD65片段DNA疫苗，并在检测其表达。2．将构建好的8种GAD65片段DNA疫苗对NOD鼠进行预防实验，观察不同GAD65片段DNA疫苗对NOD鼠糖尿病发病和胰岛炎的作用。3．检测几种有效的GAD65片段DNA疫苗免疫对NOD鼠胰岛p细胞凋亡、免疫耐受、Th1／Th1免疫平衡、以及CD4⁺CD25⁺调节性T细胞的作用，探讨不同GAD65片段DNA疫苗预防NOD鼠糖尿病可能的作用机制。第一部分GAD65片段DNA疫苗的构建、鉴定与优化目的构建、鉴定和优化以预防1型糖尿病为目的的编码不同谷氨酸脱羧酶（GAD）65基因片段的DNA疫苗。方法从GAD65质粒中扩增出GAD_190-315（GAD1）片段和GAD_490-570（GAD2）片段的cDNA，以ovedap PCR法将之分别与IL-2信号肽cDNA拼接，得到带信号肽的SGAD1、SGAD2融合基因。并分别以IL-4、IL-10及胰岛原质粒为模板，用PCR方法扩增出IL-4、IL-10和胰岛素B链（InsB）基因。SGAD1、SGAD2、IL-4、IL-10和InsB基因先克隆入pGEM-T载体后，先将SGAD1、SGAD2融合基因分别克隆入双启动子真核表达载体pBudCE4.1中，构建出2种单基因重组真核表达载体pBud-SGAD1和pBud-SGAD2，再分别将IL-4、IL-10、InsB基因克隆入真核表达载体pBud-SGAD1和pBud-SGAD2中，构建出pBud-SGAD1／IL-4、pBud-SGAD1／IL-10、pBud-SGAD1／InsB、pBud-SGAD2／IL-4、pBud-SGAD2／IL-10、pBud-SGAD2／InsB6种双基因重组真核表达载体。重组真核表达载体经测序鉴定正确后，用脂质体介导的方法体外转染COS-7细胞，转染后以蛋白质印迹法检测SGAD1、SGAD2及InsB的表达，ELISA方法检测细胞因子IL-4和IL-10的表达。结果（1）核酸序列测定表明克隆的SGAD1、SGAD2融合基因、IL-4、IL-10和InsB基因序列与报告序列一致，开放读码框正确。（2）蛋白质印迹法检测到SGAD1、SGAD2和InsB在COS-7细胞中的表达，其中融合蛋白SGAD1和SGAD2可以分泌表达。（3） ELISA方法检测到细胞因子IL-4和IL-10在COS-7细胞中的表达。结论首次成功构建了pBud-SGAD1、pBud-SGAD2、pBud-SGAD1／IL-4、pBud-SGAD1／IL-10、pBud-SGAD1／InsB、pBud-SGAD2／IL-4、pBud-SGAD2／IL-10、pBud-SGAD2／InsB8种新的GAD65片段DNA疫苗，为1型糖尿病的DNA疫苗预防研究提供了实验基础。第二部分GAD65片段DNA疫苗对NOD鼠糖尿病和胰岛炎的作用目的探讨优化后的GAD65片段DNA疫苗对NOD鼠糖尿病发病和胰岛炎的作用。方法3-4周龄NOD雌鼠随机分为PBS组（n=20）、pBudCE组（n=18）、GAD65组（n=18）、SGAD1组（n=17）、SGAD2组（n=18）、SGAD1／IL-4组（n=19）、SGAD1／IL-10组（n=17）、SGAD1／InsB组（n=20）、SGAD2／IL-4组（n=18）、SGAD2／IL-10组（n=17）和SGAD2／InsB组（n=19）11个组，分别肌肉注射PBS、pBudCE空质粒、pcDNA3.1（+）／GAD65 DNA疫苗和经过优化后的pBud-SGAD1、pBud-SGAD1／IL-4、pBud-SGAD1／IL-10、pBud-SGAD1／InsB、pBud-SGAD1、pBud-SGAD2／IL-4、pBud-SGAD2／IL-10、pBud-SGAD2／InsB DNA疫苗，注射量为75μg，一周后重复一次，两次共150μg。自10周龄开始，每周测尿糖一次，尿糖阳性后监测血糖，连续两次血糖≥16.7mmol／L即诊断为糖尿病，所有动物观察至发生糖尿病或者至30周龄。各组再取6-8只12周龄未发病小鼠胰腺HE染色观察胰岛炎情况。结果（1）与PBS组相比，SGAD1、SGAD1／IL-4和SGAD1／IL-10组NOD鼠糖尿病的发病明显降低（P＜0.05或P＜0.01）。（2）无论与GAD65组相比，还是与SGAD1组相比，SGAD1／IL-4和SGAD1／IL-10组NOD鼠糖尿病的发病明显降低（P＜0.05或P＜0.01）。（3）与PBS组相比，pBudCE、SGAD1／InsB、SGAD2、SGAD2／IL-4、SGAD2／IL-10和SGAD2／InsB组NOD鼠糖尿病的发病差异无显著性。（5） 12周龄时，无论与PBS组或与GAD65组相比，pBud-SGAD1、pBud-SGAD1／IL-4、pBud-SGAD1／IL-10 DNA疫苗能够降低NOD鼠胰岛炎的严重程度（P＜0.05或P＜0.01）。结论pBud-SGAD1、pBud-SGAD1／IL-4、pBud-SGAD1／IL-10DNA疫苗减轻胰岛炎和预防NOD鼠糖尿病的疗效优于pcDNA3.1（+）／GAD65 DNA疫苗，其中pBud-SGAD1／IL-4和pBud-SGAD1／IL-10 DNA疫苗的疗效更好。第三部分GAD65片段DNA疫苗预防NOD鼠糖尿病的机制探讨目的探讨优选的GAD65片段DNA疫苗预防NOD鼠胰岛炎和糖尿病的机制。方法PBS组、pBudCE组、GAD65组、SGAD1组、SGAD1／IL-4组、SGAD1／IL-10组各取6-8只12周龄未发病NOD雌鼠并处死，TUNEL加SP免疫组织化学方法检测胰岛β细胞的凋亡情况；制备脾细胞悬液后，[³H]掺入法检测脾细胞对特异性抗原GAD65的刺激增殖反应；流式细胞仪检测脾细胞中CD4⁺CD25⁺调节性T细胞的数量；ELISA方法测定抗原刺激后脾细胞培养上清中Th1细胞因子IFN-γ，Th2细胞因子IL-4、IL-10水平；RT-PCR方法检测脾脏内细胞因子IFN-γ、IL-4、IL-10和转录因子foxp3 mRNA的表达。结果（1） SGAD1、SGAD1／IL-4和SGAD1／IL-10组NOD鼠胰岛β细胞凋亡率均低于PBS组（均P＜0.05）。（2） GAD1、SGAD1／IL-4和SGAD1／IL-10组NOD鼠脾细胞特异性抗原刺激增殖反应低于PBS组（均P＜0.01）。（3） SGAD1、SGAD1／IL-4和SGAD1／IL-10组脾细胞培养上清中IFN-γ水平均低于PBS组（均P＜0.01）和GAD65组（均P＜0.01）；SGAD1、SGAD1／IL-4和SGAD1／IL-10组脾脏中IFN-γmRNA表达水平也均低于PBS组（均P＜0.01）和GAD65组（均P＜0.01）。（4） SGAD1、SGAD1／IL-4和SGAD1／IL-10组脾细胞培养上清中IL-4水平均高于PBS组（P＜0.05或P＜0.01），SGAD1／IL-4组的IL4水平高于GAD65组（P＜0.05）GAD1、SGAD1／IL-4和SGAD1／IL-10组脾脏中IL-4 mRNA表达水平高于PBS组（P＜0.05或P＜0.01）和GAD65组（P＜0.05或P＜0.01）。（5） SGAD1／IL-10组脾细胞培养上清中IL-10水平高于PBS组（P＜0.05）和GAD65组（P＜0.05）；SGAD1／IL-4和SGAD1／IL-10组脾脏中IL-10 mRNA表达水平高于PBS组（P＜0.05或P＜0.01）和GAD65组（P＜0.05）。（6）各组间NOD鼠脾细胞中CD4⁺CD25⁺调节性T细胞的数量，以及脾脏中Foxp3 mRNA表达水平差异无显著性。结论pBud-SGAD1、pBud-SGAD1／IL-4、pBud-SGAD1／IL-10DNA疫苗免疫NOD鼠后，下调Th1细胞因子IFN-γ，上调Th2细胞因子IL-4和IL-10，导致免疫平衡向Th2方向偏移，使NOD鼠胰岛炎减轻，胰岛β细胞凋亡减少，并诱导NOD鼠对自身抗原GAD65的免疫耐受，从而预防NOD鼠发生糖尿病。更多还原

【Abstract】 Type 1 diabetes mellitus （T1DM） is an autoimmune diseasecharacterized by selective destruction of the insulin-producingβcells inthe islets of Langerhans. A variety of immune effector cells, includingCD4⁺ T cells, CD8⁺ T cells, and macrophages have been implicated asmediators of pancreatic isletsβcells destruction in T1DM. Previousresearches indicated that programmed cell death, or apoptosis is the mainform ofβcells death responsible for the development of T1DM in animalmodels. Although the etiology of autoimmune diabetes is complex andremain largely unknown, accumulating experimental evidences suggestedthat the insulin-secreting pancreaticβ-cells were damaged by cytotoxicThl cells and their cytokines. Hence, a fine tuning of the immune systemtoward Th2-like immunity may represent an attractive and reasonablepreventive and therapeutic strategy for T1DM. Additionally, CD4⁺CD25⁺regulatory T cells have described recently as an important powerfulmediator of peripheral tolerance in autoimmune diabetes, and couldefficiently protect and cure of animal model from diabetes development.Many previous studies suggested that individuals could have a pre-clinical period for a long time before it finally progressed to overtT1DM, and overt T1DM could been delayed or prevented by efficientimmune intervention at the early stage. Thus, an increasing number ofresearchers put their interests in finding efficient therapies to cure T1DM.Re-establish the tolerance to autoantigen is one of the important mannersto prevent T1DM. Glutamic acid decarbocylase （GAD） 65 has beenrecognized as a key antigen among islets autoantigens in the progressionof T1DM, and has been shown to lessen insulitis and prevent T1DM byinduction of immune tolerance or other mechanisms in previous studies.DNA vaccine has been applied to treat infectious disease and cancer,and promising effects have been observed in recent years. The establishedand potential advantages of DNA vaccination over other methods ofimmunization have been recognized. The fact that DNA vaccine is safety,and can be constructed easily and produced with low cost and also onlyrequires convenient condition for storage and transportation. Moreover,GAD65 DNA vaccines have been reported recently to prevent T1DM inanimal models. However, there are inconsistent results in those previousstudies. More importantly, our previous study showed that incidence ofspontaneous autoimmune diabetes only decreased by thirty percent byDNA vaccination encoding full-length GAD65 gene in that study in anexcellent animal model of human T1DM, the non-obese diabetic （NOD）mice. Therefore, preventive efficacy of GAD65 DNA vaccine should to be improved.In conclusions, the aims of present study are to optimize the DNAvaccine containing full-length GAD65 gene for prevention of T1DM, andto investigate the roles of different optimized GAD65 DNA vaccines indiabetes onset and insulitis development in NOD mice, and also toexplore the possible mechnisms of optimized GAD65 DNA vaccines inpreventing T1DM in NOD mice, which provide an experimentalfoundation for prevention of human T1DM.In this study, we firstly optimize DNA vaccine encoding full-lengthGAD65 gene by means of autoantigen geng sequence modification, andaddition of signal peptide sequence, and co-expression with protectivecytokine or other autoantigen gene, to construct and identify eight newGAD65 fragment DNA vaccines. Moreover, we investigate the effects ofdifferent optimized GAD65 fragment DNA vaccines on development ofdiabetes and insulitis in NOD mice. Finally, we examine the roles ofdifferent optimized GAD65 fragment DNA vaccines in apoptosis ofβcells, immune tolerance to GAD65, Th1/Th2 immune balance and statusof CD4⁺CD25⁺ regulatory T cells in this model. Part I Construction, Identification and Optimization of GlutamicAcid Decarbocylase （GAD） 65 DNA VaccineObjective To construct and optimize DNA vaccines containingdifferent GAD65 gene fragments for prevention of type 1 diabetes.Methods The cDNAs of GAD_190-315 （GAD1） and GAD_490-570（GAD2） fragments were amplified from GAD65 plasmid, and linked withIL-2 signal peptide cDNA respectively through overlap PCR. And thefusion genes, SGAD1 and SGAD2, were inserted into pGEM-T vectors.IL-4, IL-10, insulin B chain （InsB） genes were amplified from IL-4,IL-10, proinsulin plasmids and cloned into pGEM-T vectors, respectively.Then, pBudCE4.1-SGAD1 and pBudCE4.1-SGAD1 were constructedwhen SGAD1 and SGAD2 were cloned into an eukaryotic expressionvector pBudCE4.1, which contains two multiple cloning sites under thecontrol of different promoters. Moreover, IL-4, IL-10, InsB genes werecloned respectively after the other promoter into pBudCE4.1-SGAD 1 andpBudCE4.1-SGAD2 plasmid to establish six recombinant eukaryoticexpression vectors, pBud-SGAD 1/IL-4, pBud-SGAD 1/IL-10,pBud-SGAD1/InsB, pBud-SGAD2/IL-4, pBud-SGAD2/IL-10 andpBud-SGAD2/InsB. After being identified by DNA sequencing, all of therecombinant eukaryotic expression vectors were transfected into COS-7cells respectively by liposome in vitro. The expression products in theCOS-7 cells transfected with recombinants were detected using Westernblot or ELISA. Results（1） DNA sequencing of the target genes cloned into recombinanteukaryotic expression vectors were in accordance with the reportedsequence, with correct open reading frame.（2） Western blot showed that the products of those recombinanteukaryotic expression vectors containing SGAD1 or SGAD2 gene wereexpressed and secreted. InsB gene was also expressed in COS-7 cells invitro.（3） The expression products of IL-4, IL-10 gene in COS-7 cellstransfected with those recombinant eukaryotic expression vectors containcytokines gene were determined by ELISA.Conclusions The pBudCE4.1-SGAD1, pBudCE4.1-SGAD1,pBud-SGAD1/IL-4, pBud-SGAD1/IL-10, pBud-SGAD1/InsB,pBud-SGAD2/IL-4, pBud-SGAD2/IL-10 and pBud-SGAD2/InsB DNAvaccines were constructed, being a foundation for further development ofDNA vaccine against type 1 diabetes. PartⅡEffects of GAD65 DNA Vaccines on Development ofAutoimmune Diabetes and Insulitis in NOD MiceObjective To investigate the roles of different GAD65 fragmentDNA vaccines in diabetes onset and insulitis severity in NOD mice.Methods Female NOD mice at 3-4 weeks of age were randomlydivided into eleven groups and received intramuscular injection of eitherpBudCE4.1 blank vector alone （n=18） or pBudCE4.1 carrying theSGAD1 （n=17）, SGAD2 （n=18）, SGAD1/IL-4 （n=19）, SGAD2/IL-4（n=17）, SGAD1/IL-10 （n=20）, SGAD2/IL-10 （n=18）, SGAD1/InsB（n=17）, SGAD2/InsB （n=19） construct, or pcDNA3.1（+） vector encodingfull-length GAD65 gene （n=18）, and mice received injection of PBS（n=20） as control. All mice were injected with 75μg DNA vaccine or PBSinto the tibialis anterior muscle and an identical set of injection wereperformed 1 week later, for a total of 150μg of DNA vaccine per mouse.The incidence of spontaneous diabetes was monitored up to 30 weeks ofage. From 10 weeks of age, mice were determined once a week forglycosuria, and diabetes was diagnosed when the blood glucose levelsexceeded 16.7mmol/L on two consecutive readings. Six to eight ofnon-diabetic animals from each group were killed at 12 weeks of age andpancreas were removed to score insulitis by routine H&E staining.Results（1） Compared with PBS group, there was a significant reduction ofdiabetes onset in NOD mice treated with either pBud-SGAD1,pBud-SGAD1/IL-4, or pBud-SGAD1/IL-10 DNA vaccine （P＜0.05 or P＜0.01）.（2） pBud-SGAD1/IL-4 and pBud-SGAD1/IL-10 DNA vaccinationdecreased the incidence of diabetes in NOD mice, compared with eitherGAD65 group or SGAD1 group （P＜0.05 or P＜0.01）.（3） No significant difference of diabetes onset was observed amongthe PBS, pBudCE, GAD65, SGAD2, SGAD1/InsB, SGAD2/IL-4,SGAD2/IL-10 groups （all P＞0.05）.（4） Insulitis scoring showed that mice received either pBud-SGAD1,pBud-SGAD1/IL-4, or pBud-SGAD1/IL-10 DNA vaccine significantlysuppressed islet inflammation as compared to mice received PBS andpcDNA3.1（+）/GAD65 DNA vaccine （all P＜0.01）.Conclusion Immunization with optimized GAD65 fragment DNAvaccines, pBud-SGAD1, pBud-SGAD1/IL-4, and pBud-SGAD1/IL-10,prevents diabetes onset and lessens insulitis in NOD mice, which is moreeffective than former DNA vaccine containing full-length GAD65 gene. PartⅢThe Preventive Mechanisms of GAD65 DNA Vaccines onAutoimmune Diabetes in NOD MiceObjective To explore the mechanisms of optimized GAD65fragment DNA vaccines in preventing autoimmune diabetes and insulitisin NOD mice.Methods Six to eight of non-diabetic mice from either PBS,pBudCE, GAD65, SGAD1, SGAD1/IL-4, or SGAD1/IL-10 group, weresacrificed at 12 weeks of age. Firstly, the apoptotic isletsβcells wereevaluated by TUNEL and immunohistochemistry. Moreover, splenocyteswere isolated and cultured in vitro with the rGAD65, the proliferationwas quantified by determining [3H]-thymidine incorporation. Thepopulation of CD4⁺CD25⁺ regulatory T cells was analyzed by flowcytometry. Furthermore, cytokine IFN-γ, IL-4 and IL-10 releasestimulated by rGAD65 were measured by ELISA. Finally, total RNA wasisolated from fresh spleen tissue of NOD mice with or without treatmentof GAD65 DNA fragment vaccines. The levels of cytokine IFN-γ, IL-4,IL-10 and transcription factor Foxp3 mRNA expression in spleen weredetected by RT-PCR.Results（1） Isletβcells apoptosis rates decreased in NOD mice receivedeither SGAD1, SGAD1/IL-4, or SGAD1/IL-10 DNA vaccine treatmentcompared with mice received PBS injection （all P＜0.05）.（2） Splenocyte proliferation response to autoantigen GAD65 inSGAD1, SGAD1/IL-4, or SGAD1/IL-10 DNA vaccine treated mice were suppressed as compared to control mice （all P＜0.01）.（3） The release of IFN-γin supematants of splenocytes in eitherSGAD1, SGAD1/IL-4, or SGAD1/IL-10 DNA vaccine treated micegroup, were lower than those in PBS （all P＜0.01） and GAD65 group （allP＜0.01）. The similar difference was observed in level of IFN-γ, mRNAexpression.（4） The secretion of IL-4 in supematants of splenocytes in eitherSGAD1, SGAD1/IL-4, or SGAD1/IL-10 DNA vaccine treated micegroup, were higher than those in PBS group （P＜0.05 or P＜0.01）. Theonly SGAD1/IL-4 group showed significant difference compared toGAD65 group （all P＜0.05）. And IL-4 mRNA expression were increasedin either SGAD1, SGAD1/IL-4, or SGAD1/IL-10 DNA vaccine treatedmice compared with PBS and DNA vaccine containing full-lengthGAD65 gene treated mice （P＜0.05 or P＜0.01）.（5） SGAD1/IL-10 DNA vaccine treatment increased IL-10 levels insupematants of splenocytes in mice as compared to those in PBS group（P＜0.05） or GAD65 group （P＜0.05）, and IL-10 mRNA expressionincreased in mice received either SGAD1/IL-4 or SGAD1/IL-10 DNAvaccine treatment compared with mice in PBS （P＜0.05 or P＜0.01） orDNA vaccine containing full-length GAD65 gene injection （all P＜0.05）.（6） No significant differences in both CD4⁺CD25⁺ regulatory T cellpopulation and Foxp3 mRNA expression were found between the DNAvaccine treated mice and the untreated mice group. （all P＞0.05）.Conclusion A shift to Th2 immune response, inhibition of the apoptosis of isletβcells, and induction of tolerance of autoreactive Tcells against autoantigen GAD65, are found as the preventivemechanisms in immunization with the optimized GAD65 DNA fragmentvaccines.更多还原