【作者】 林鹏；

【导师】 陈丽；

【作者基本信息】 山东大学 ， 内科学， 2010， 博士

【摘要】 第一部分Pdx1抗原免疫治疗延缓了NOD小鼠糖尿病的发病我们的免疫系统针对为我们提供着有效的保护,防止致病因子或外源性物质对机体的损害,然而,我们的免疫系统并不完美。胸腺对自身反应性T细胞阴性选择的失败,加上体内调节性T细胞(Regulatory T cell, Treg)的缺乏(可调控自身反应性T细胞),会导致自身反应性T细胞攻击自身组织,促进自身免疫性疾病的发生。近年来,采用抗原特异性的免疫治疗以特异性敲除自身反应性T细胞或诱导Treg,被视为自身免疫性疾病很有希望的治疗方法。1型糖尿病特点为自身反应性T细胞识别一系列胰岛特异性抗原,导致胰岛p细胞的选择性破坏。多种自身抗原可用于指导免疫治疗,包括胰岛素抗原、谷氨酸脱羧酶抗原、胰岛细胞抗原等。自身反应性T细胞针对这些抗原的反应存一定的等级,自身反应性T细胞对胰岛素抗原的识别反应被认为是位于对其他抗原反应的上游。胰岛素特异性的免疫治疗在1型糖尿病啮齿类动物模型身上显示出一些保护作用,然而,胰岛素抗原特异性的免疫治疗在糖尿病临床治疗方面疗效不佳。这暗示着抗原特异性的免疫治疗原理十分复杂,或许,糖尿病患者体内存在其他一些未知的自身抗原,利用这些未知的自身抗原免疫可能会产生更好的治疗效果。胰腺十二指肠同源盒1(Pancreatic and duodenal home box factor-1, Pdx1)是胰腺发育中关键的转录因子,在p细胞的分化和功能维持方面也起着十分重要的作用。在成熟的β细胞,Pdx1参与胰岛素基因表达的调控。在我们以前发表的文章中,我们报道了基因重组Pdx1蛋白可改善脲链佐菌素所致的糖尿病小鼠的血糖水平,通过促进胰腺的再生和促进肝细胞分化为胰岛素分泌细胞而起作用。进而,我们发现Pdxl是非肥胖糖尿病小鼠(Nonobese diabetic mice, NOD)小鼠体内新的胰岛p细胞特异性的自身抗原,在NOD小鼠糖尿病的初期和糖尿病期,以及一部分糖尿病患者体内都可检测到针对Pdxl的自身抗体。NOD小鼠是模拟1型糖尿病较好的动物模型,雌性的NOD小鼠一般在12-14周龄左右自发进展为糖尿病,其特点为T细胞介导的胰岛免疫浸润和胰岛β细胞免疫破坏。在这个试验中,我们尝试不同的抗原输注方法,观察Pdxl蛋白(或M-Pdx1蛋白,M-Pdx1蛋白丧失Pdxl的生物学活性,但仍保留其免疫原性)免疫治疗是否可延缓NOD小鼠糖尿病的发病,并进行了脾细胞过继移植实验、体内T细胞增殖实验、胰腺免疫组化、细胞流式分析、实时定量PCR及体外T细胞增殖实验等相关研究,以分析其免疫治疗机制。目的研究Pdxl(或M-Pdx1蛋白)免疫治疗是否会延缓雌性NOD小鼠糖尿病的发病及可能的免疫治疗机制。方法在这个试验中,七或十周龄的NOD小鼠接受Pdxl(或M-Pdx1蛋白、对照蛋白)的免疫治疗。因为治疗效果可以受很多因素的影响,我们试验中尝试了不同的抗原输注方法,以及不同的抗原输注剂量和时间。不同的抗原输注方法包括腹腔注射、皮下注射或口服；不同的输注剂量和时间是指用于实验的NOD小鼠分别选自7周或10周,接受Pdxl(或M-Pdx1蛋白、对照蛋白)的免疫治疗维持的周数也有所不同。每周检测NOD小鼠血糖状况,血糖水平大于11.1mmol/1并持续两天被认为是糖尿病发病。为明确其可能的机制,进行了以下分析：(1)过继移植实验：将接受Pdxl (M-Pdx1或对照蛋白)免疫的NOD小鼠的脾细胞分离出,移植入非肥胖糖尿病、重症联合免疫缺陷(Nonobese diabetic-severe combined immunodeficient mice, NOD-SCID)小鼠体内,观察NOD-SCID小鼠糖尿病的发病情况；(2)体内T细胞增殖实验：将CD4+T细胞从NOD.BDC2.5小鼠的脾脏中分离出,体外经荧光染料羧基荧光素二醋酸盐琥珀酰亚胺酯(Carboxyfluorescein diacetate succinimidyl ester, CFSE)标记后,经尾静脉注射入Pdxl蛋白(M-Pdx1或对照蛋白)处理过的NOD小鼠体内；5天以后,将NOD小鼠的胰腺淋巴结细胞和腹股沟淋巴结细胞分离出,细胞流式评估CFSE标记的CD4+T细胞在NOD小鼠体内增殖的情况：(3)胰腺免疫组化研究：M-Pdx1蛋白(或对照蛋白)处理的NOD小鼠接受胰腺免疫组化研究。胰岛组织切片行HE染色和胰岛素染色,并对胰岛炎进行评分；(4)细胞流式：细胞流式分析M-Pdx1蛋白(或对照蛋白)免疫NOD小鼠是否可改变脾脏、胰腺淋巴结中CD4+/CD8+T细胞的比例,分析CD4+FoxP3+Treg、IL-10分泌细胞和Th-17细胞等的变化情况,细胞流式分析IL-4、IL-10、IFN-γ等细胞因子表达情况；(5)实时荧光定量PCR分析M-Pdx1蛋白(或对照蛋白)免疫NOD小鼠对各种细胞因子基因表达的影响,包括IL-2、IL-4、IL-10、IL-17、IFN-γ、TGF-α、TGF-β及FoxP3等；(6) T细胞体外增殖实验：观察Pdx1蛋白(或对照)免疫的NOD小鼠,脾脏淋巴细胞对不同抗原刺激的体外增殖情况；结果与对照组相比,将Pdxl蛋白(或M-Pdx1蛋白)腹腔或皮下输注免疫NOD小鼠可较好地延缓NOD小鼠糖尿病的发病,但口服Pdx1蛋白(或M-Pdx1蛋白)没有显示出明显的免疫预防效果。对照组PBS处理的NOD小鼠的脾细胞过继转移给NOD-SCID小鼠时,转移后大多3周内发生糖尿病。将Pdx1(或M-Pdx1蛋白)处理的NOD小鼠的脾细胞过继转移给NOD-SCID小鼠时,NOD-SCID小鼠发生糖尿病的时间明显推迟。这提示Pdxl(或M-Pdx1蛋白)免疫NOD小鼠可能激活免疫调节细胞,且这种保护作用是可转移的。CFSE标记的CD4+T细胞过继转移实验：在腹股沟淋巴结,M-Pdx1蛋白(或对照蛋白)处理的NOD小鼠,CFSE标记的NOD.BDC2.5 CD4+T细胞都无明显增殖,无明显的统计学差异(p>0.05)；而在胰腺淋巴结,对照组NOD小鼠胰腺淋巴结内CFSE标记的NOD.BDC2.5 CD4+T细胞增殖明显,与M-Pdx1蛋白处理的NOD小鼠形成明显的反差,M-Pdx1蛋白处理的NOD小鼠NOD.BDC2.5 CD4+T细胞增殖受到抑制(p<0.05)。此结果表明M-Pdx1蛋白处理可能诱导NOD小鼠体内Treg的产生,从而对移植入体内的CFSE标记的NOD.BDC2.5 CD4+T细胞的增殖产生抑制调节作用。胰腺免疫组化结果显示：对照组NOD小鼠胰岛淋巴细胞浸润较严重,胰岛素染色阳性的β细胞数量降低,胰高血糖素细胞代偿性增多。与此对比,经M-Pdx1处理的NOD小鼠淋巴细胞浸润减少,胰岛素染色阳性的β细胞占胰岛细胞的绝大部分,胰岛炎评分优于对照组。细胞流式结果表明：M-Pdx1免疫NOD小鼠可上调脾细胞内CD4+Foxp3+ Treg的比例和IL-10分泌细胞的比例。M-Pdx1免疫NOD小鼠可下调Th-17细胞的比例；而CD4+/CD8+的比值、IFN-γ分泌细胞的比例等无明显变化。Real-time PCR结果显示：利用M-Pdx1蛋白免疫处理NOD小鼠可上调Th2相关基因的表达,如IL-4,IL-10,Foxp3,TGF-α及TGF-p；下调Thl相关基因的表达,如IL-2,IFN-γ等。同时,利用M-Pdx1蛋白免疫NOD小鼠可提高FoxP3基因的表达。体外T细胞增殖实验：对照组NOD小鼠分离出的T细胞对Pdx1蛋白抗原刺激表现出明显的增殖,与Pdx1蛋白免疫处理的NOD小鼠相比,p<0.05；这提示利用Pdxl蛋白免疫NOD小鼠可导致体内Pdx1抗原特异性的自身反应性T细胞功能降低。通过Pdxl蛋白免疫NOD小鼠,T细胞对胰岛素的增殖反应也降低。利用Pdxl蛋白免疫的NOD小鼠T细胞分泌IFN-γ的水平降低。结论利用Pdxl蛋白(或M-Pdx1蛋白)免疫NOD小鼠可较好地预防NOD小鼠糖尿病的发病,其机制可能为诱导自身反应性T细胞无反应性或克隆清除,诱导CD4+Foxp3+ Treg细胞的产生,上调IL-10分泌细胞比例,下调Th-17细胞的数量,以及影响Thl细胞与Th2细胞的比例。利用Pdxl抗原免疫治疗1型糖尿病是一个很有希望的治疗方法。第二部分利用共培养微流控细胞芯片观察骨髓间充质干细胞是否可改善IL-1β/IFN-γ所导致的INS-1细胞凋亡及功能不全目的利用微流控细胞芯片技术制作共培养细胞芯片,将骨髓间充质干细胞(Bone marrow-derived mesenchymal stem cells, BM-MSCs)和INS-1细胞进行共培养,我们观察BM-MSCs所分泌的一些细胞因子是否可改善IL-1β/IFN-γ所导致的INS-1细胞凋亡及功能不全。方法设计制备共培养微流控细胞芯片。BM-MSCs从糖尿病患者中获取,细胞流式分析细胞表面分子的表达。将BM-MSCs和INS-1细胞分别接种于微流控芯片不同的培养小室,保持培养液从BM-MSCs向INS-1细胞的单向流动。INS-1细胞培养基中添加细胞因子IL-1β及IFN-γ,同时INS-1细胞也接受BM-MSCs培养液的影响。Annexin V/PI双染分析BM-MSCs对IL-1β/IFN-γ所致的细胞凋亡的抑制作用：应用RIA分析BM-MSCs是否可改善IL-1β/IFN-γ所致的INS-1细胞胰岛素分泌功能不全。免疫荧光和实时荧光定量PCR分析IL-1β/IFN-γ及BM-MSCs对INS-1细胞胰岛素含量及胰岛素基因表达的影响。结果微流控细胞芯片可以确保培养液从BM-MSCs向INS-1细胞的单向持续的流动,是体外观察两种细胞间单向作用的好工具。在此试验中,在不同的时间可观察到IL-1β/IFN-γ可导致INS-1细胞凋亡和胰岛素分泌功能受损,BM-MSCs所分泌的一些细胞因子可改善IL-1β/IFN-γ所导致的INS-1细胞凋亡和胰岛素分泌功能受损,增加细胞内胰岛素的含量,促进胰岛素基因的表达。结论共培养微流控细胞芯片是体外观察干细胞与其他细胞作用的好工具。BM-MSCs可能通过分泌一些抗炎性、抗凋亡和营养因子促进INS-1细胞的存活,促进INS-1细胞胰岛素分泌及胰岛素基因的表达。更多还原

【Abstract】 Our immune system provides effective protection from infectious agents and foreign substances, however, this immune system is not perfect. Failure in the negative deletion of self-reactive T cells in the thymus, combined with the deficiency of regulatory T cell (Regulatory T cell, Treg) cells which can control the activity of self-reactive T cells, can result in autoimmune disease attacking the individuals own tissues. Recently, antigen-specific immunotherapy for the specific blockade of self-reactive T cells or induction of Treg cells has been viewed as a promising means of curing autoimmune diseases.Type 1 diabetes is characterized by the selective destruction of pancreatic isletβcells by self-reactive T lymphocytes recognizing a list of autoantigens. Many self-antigens have guided in designing therapeutic strategies, including insulin, glutamic acid decarboxylase antigen and islet cell antigen. There exists a sequential hierarchy in self-reactive T-cellular responses directed against these islet autoantigens, and the T-cellular response against insulin is viewed to be upstream of the responses to other self-antigen. Insulin-specific immunotherapy has shown some protective results in rodent models of Type 1 diabetes, however, insulin antigen-driven immunotherapy that could counter diabetes in the clinic has yet to be achieved successfully. These suggest that principles guiding the antigen-based immunotherapy are very complicated and theβcells-specific T cell reactivity in patients is largely ill defined. Perhaps, there exist some other unknown antigens in diabetic patients, and administration of these unknown autoantigens may be more effective for human application.Pancreatic and duodenal home box factor-1 (Pdx1) is a key transcription factor that plays an important role in pancreas development,βcells differentiation and functional maintenance ofβcells. In matureβcells, Pdxl is also involved in insulin gene expression. In our previously published paper, we reported that treatment of streptozotocin-induced diabetes with recombinant pancreatic duodenal homeobox 1 protein reverses diabetes by stimulatingβ-cell regeneration and liver cell reprogramming into insulin-producing cells. More existed, we found that Pdx1 is a novelβ-cell-specific autoantigen existing in the nonobese diabetic (NOD) mice. Autoantibodies against Pdx1 were observed in serum samples from both prediabetic and diabetic NOD mice and a subset of Type 1 diabetic patients. The NOD mouse is an ideal experimental model of autoimmune type 1 diabetes. In female mice, diabetes onset typically occurs at 12 to 14 weeks of ages, characterized by T cell-mediated inflammation of the pancreatic islets and the destruction of theβcells. In this study, we investigated whether Pdx1 protein (or M-Pdx1, with no biological function, but intact immunologically active component) could delay the onset of diabetes in NOD mice and the underlying mechanisms. Adoptive transfer assay of splenocytes, in vivo T cells proliferative assay, histology of pancreas, cell flow, real-time PCR assays and in vitro T cells proliferative assay were performed to investigate the underlying mechanism.OBJECTIVE- In this study, we investigated whether immunization with Pdx1 (or M-Pdx1) protein could delay the onset of diabetes in female NOD mice and the underlying mechanism.RESEARCH DESIGN AND METHODS- In this experiment, NOD mice at the age of seven or ten weeks were immunized with Pdx1 protein (or M-Pdx1 protein, control proteins). Because the therapeutic effects could be influenced by many factors, in our experiments we tried different route and form, as well as different dosage and timing of administration. For different route and form of administrations, NOD mice were treated with protein intraperitoneally, subcutaneously or orally; For different dosage and timing of administrations, NOD mice were chosen for experiment at the age of seven week or ten week, and treated with Pdx1 protein (or M-Pdx1 protein, control proteins) for different weeks. Blood glucose was measured weekly and mice with a blood glucose level>11.1 mmol/l for 2 consecutive days were considered diabetic.In order to investigate the underlying mechanism, we tried to do the following assays:(1) Spleen adoptive transfer experiment:Splenocytes from Pdx1 protein (or M-Pdx1 protein, control proteins) treated mice were isolated and subsequently transferred into nonobese diabetic-severe combined immunodeficient mice (NOD-SCID) mice, and the diabetic incidence of NOD-SCID mice was observed. (2) In vivo T cell proliferative assay:CD4+ T cells isolated from the spleen of NOD.BDC2.5 mice were marked with carboxyfluorescein diacetate succinimidyl ester (CFSE) and subsequently injected intravenously into the Pdx1-treated (or M-Pdx1 protein, control proteins) NOD mice. Five days later, pancreatic lymph node cells and inguinal lymph node cells were harvested from NOD mice and analyzed by cell flow cytometry aiming to evaluate CFSE labeled CD4+ T cell proliferation.(3) Histology of pancreas:Female NOD mice immunized by either M-Pdx1 protein or control proteins underwent pancreatic histological studies. Tissue section of the islet stained with hematoxylin and eosin or with anti-insulin antibodies. The slides were coded and an insulitis score was determined.(4) Cell flow:Female NOD mice were immunized by either M-Pdx1 protein or control proteins. Cell flow assays were performed to evaluate the ratio of CD4+/CD8+ T cells in spleen or pancreatic lymph nodes, including the changes of CD4+ FoxP3+ regular cells, IL-10 secreting cells and Th-17 cells. The expression of cytokines such as IL-4、IL-10、IFN-γwere determined.(5) Real-time PCR assays:The expression of cytokines such as IL-2、IL-4、IL-10、Th-17、IFN-γ、GF-α、TGF-βand FoxP3 were determined by Real time PCR to evaluate the effects of M-Pdxl protein (or control proteins) on NOD mice.(6) In vitro T cell proliferative assay:To observe the proliferation of spleen lymphocytes from NOD mice immunized by either M-Pdx1 protein or control proteins in presence of different antigens.RESULTS-Compared with control group, Pdxl protein (or M-Pdx1 protein) specific immunotherapy (intraperitoneal or subcutaneous administration) delayed the onset of diabetes in NOD mice, however, oral administrations of Pdx1 protein (or M-Pdx1 protein) did not prevent diabetes obviously.Spleen adoptive transfer experiment:NOD-SCID mice injected with splenocytes from PBS-treated NOD mice in control group showed no significant therapeutic effect, and most of mice become diabetic within 3 weeks of cell transfer. However, non-diabetic incidence analysis revealed a significant delay in the onset of diabetes among the NOD-SCID mice that received infusions of splenocytes from Pdxl or M-Pdxl immunized donors. This suggested the activation of immunoregulatory cells in NOD mice treated with Pdx1 protein (or M-Pdx1 protein), and protection from diabetes could be adoptively transferred to NOD-SCID recipients.In vivo T cell proliferative assay:In the inguinal lymph node of M-Pdx1 treated or control protein treated NOD mice, CFSE labeled NOD.BDC2.5 CD4+T cells barely proliferated (p>0.05). CFSE labeled NOD.BDC2.5 CD4+T cells proliferated obviously in the pancreatic lymph node of control mice, however, cell flow cytometry revealed a depressed T cells proliferation in the pancreatic lymph node of M-Pdx1 treated mice (p<0.05). These results suggested that M-Pdx1 maybe could increase the number of Treg cells in NOD mice, which displayed a depressing effect of autoreactive NOD.BDC2.5 CD4+T cells proliferation.Histology of pancreas:In control group, histological examination of pancreas revealed islets heavily infiltrated by leukocytes. The few islets with remaining insulin containingβ-cells were infiltrated by abundant leukocytes, and the number of glucagon-positive cells was increased compensatorily. In contrast, in M-Pdx1 protein treated mice, the extent of lymphocyte infiltration of the islets was reduced, and immunohistochemistry of pancreas revealed abundant insulin containingβ-cells. Insulitis severity scores in NOD mice treated with M-Pdx1 were much better than that in control group.Cell flow:The number of Foxp3+ regulatory T cells among the total CD4+ T cells in the spleen cells of M-Pdxl-treated NOD mice increased, as compared with controls. Similarly, the number of IL-10 secreting cells were also increased. Moreover, the result showed that CD4+ cells expressing Th17 were decreased in NOD mice treated with M-Pdx1. Clow cytometric analyses revealed no significant differences in the the ratio of CD4+/CD8+ in spleen cells. Besides, cells expressing IFN-γremained unchanged.Real-time PCR assays showed an increased expression of Th2 gene, such as IL-4, IL-10, Foxp3, TGF-αand TGF-β,in the spleen and pancreatic lymph node from NOD mice treated with M-Pdxl protein. Down regulation of Thl gene, including IL-2, IFN-γ, was noted in the spleen and pancreatic lymph node of the M-Pdx1-treated mice, Moreover, the expression of FoxP3 gene was up regulated in NOD mice immunized with M-Pdx1 protein.T cells isolated from NOD mice in control group exhibited a more efficient proliferation towards Pdx1 protein than cells from NOD mice treated with Pdx1 (p<0.05) These data indicated that the function of Pdxl-specific T cells were decreased by immunizing NOD mice with Pdxl protein. Moreover, T cells proliferation against insulin were also decreased by immunizing NOD mice with Pdx1 protein. In Pdx1 immunized NOD mice, IFN-γwas decreased and Th2 cytokine IL-10 were increased.CONCLUSIONS-Our results demonstrated that immunization with Pdx1 antigen (or M-Pdxl protein) could delay the onset of diabetes in NOD mice. The underlying mechanism includes induction of self-reactive T cell clearance or nonfunction, up-regulation of CD4+Foxp3+ regulatory T cells and IL-10 secreting cells, and down-regulation of Th-17 cells. Besides, immunization with Pdxl antigen can also change the ratio of Th1 cells to Th2 cells. This novel Pdx1-based protein therapy maybe can offer a promising way for the treatment of type 1 diabetes. OBJECTIVE- The coculture microfluidic chip was fabricated based on microfluidic technology to facilitate the coculture of bone marrow mesenchymal stem cells (BM-MSCs) together with INS-1 cells, and we tried to observe whether some cytokines secreted by BM-MSCs can ameliorate IL-1β/IFN-γ-induced apoptosis or dysfunction of INS-1 cells.RESEARCH DESIGN AND METHODS-A coculture microfluidic chip were designed and fabricated. BM-MSCs were obtained from diabetes mellitus patients and the representative cell surface antigen expression profiles were analyzed by flow cytometric analysis. BM-MSCs and INS-1 cells were seeded into two champers on the chip separately, permitting the medium to flow from the BM-MSCs culture area to the INS-1 cells culture area unidirectionally. INS-1 cells were cultured in medium supplemented with IL-1βand IFN-y on the chip, and INS-1 cells were cocultured with BM-MSCs on a microfluidic chip with persistent perfusion of medium. Annexin V/PI double staining analysis were performed to evaluate the depressive effects of bone marrow mesenchymal stem cells on IL-1β/IFN-γinduced INS-1 cell apoptosis. RIA analysis was performed to investigate whether bone marrow mesenchymal stem cells could ameliorate IL-1β/IFN-γ-induced dysfunction of INS-1 cells. Immunofluorescence and real time PCR assay were performed to study whether bone marrow mesenchymal stem cells could enhance the insulin content and gene expression.RESULTS- This microfluidic chip assures continuous and single-line perfusion from BM-MSCs to INS-1 cells without returning, which is a better platform for us to investigate the protective effect of BM-MSCs on INS-1 cells in vitro. In this experiment, exposure to the cytokine combination IL-1β/IFN-γresulted in a marked increase in the number of apoptotic INS-1 cells at different observing times. Some cytokines secreted by BM-MSCs partially rescued INS-1 cells from cytokine-induced apoptosis and dysfunction. Simultaneously, insulin content and the mRNA expression of insulin and were also markedly ameliorated. CONCLUSIONS- This microfluidic device is a better platform for us to investigate the relationship between stem cells and other cells in vitro. BM-MSCs may enhance the survival of INS-1 cells、insulin content and the expression of insulin gene through excreted cytokines such as anti-inflammatory, anti-apoptotic and nutrient factors.更多还原