【作者】 钱芳；

【导师】 曾抗； 孙乐栋；

【作者基本信息】 南方医科大学 ， 皮肤病与性病学， 2008， 硕士

【摘要】 系统性红斑狼疮(Systemic lupus erythematosus,SLE)是一种累及多器官、多系统的自身免疫性疾病,其患病率高达70-100/10万。如果以全国16亿人口计算,我国的SLE患者已达112-160万之多,且多为年轻女性。其发病机制复杂,临床变化多端,诊治困难,死亡率高。目前治疗SLE的方法很多,首选的治疗方法是糖皮质激素制剂。开始时剂量需较大,以期在1-2周内控制病情,必要时可应用皮质类固醇激素冲击疗法或加用免疫抑制剂治疗。但在皮质类固醇激素和(或)免疫抑制剂治疗过程中,由于用药量大,时间长,容易出现副作用和并发症,如感染(病毒、细菌、真菌等)、诱发糖尿病、消化道溃疡、穿孔或消化道出血、骨折及骨缺血性坏死、精神异常等,很多患者最后死于激素的副作用和/或并发症,严重影响患者的生存期和生活质量。同时,由于上述药物的选择性不理想,部分患者经正规治疗仍不能有效控制病情。大剂量免疫球蛋白冲击疗法、血浆置换疗法以及周身淋巴结照射治疗等,由于未能针对病因治疗,且疗效短暂、费用较高,不能广泛应用。造血干细胞移植治疗虽有令人鼓舞的报告,然而技术条件要求高,价格也甚昂贵,很难普遍推广。因此,SLE的治疗仍是目前国内外公认的医学难题,尚无有效根治方法。1981年BEU-NUN等人在研究由髓磷脂碱性蛋白(MBP)主动免疫诱导的Lewis大鼠实验性自身免疫性脑脊髓炎(EAE)时,第一次提出了T细胞疫苗(T cell Vaccine,TCV)的概念。目前国内外学者对一些与T细胞介导有关的自身免疫性疾病(Autoimmune deseases,AID),器官、组织移植术后进行了T细胞疫苗治疗的实验研究,如类风湿性关节炎(Rheumatoid arthritis,RA)、自身免疫性甲状腺炎、免疫性心肌炎等,都取得令人鼓舞的结果。并且临床上将之应用于治疗RA,也取得了初步疗效。SLE是以T和B淋巴细胞功能异常为特点的自身免疫性疾病,自身反应性T淋巴细胞的活化及由此介导的自身免疫反应是本病发生的中心环节。早在上世纪90年代国外已有学者研究显示,抑制自身反应性T细胞的活性,可降低SLE病情的严重性及延长狼疮鼠的寿命。因此清除自身反应性T细胞可能是SLE又一新的治疗途径。识别自身抗原的自身反应性T细胞,是正常T细胞库的一部分,存在于外周循环中。独特型—抗独特型网络理论认为,在正常机体中,自身反应性T细胞受抗独特型的调节性T细胞的制约,处在数量较少和活性较低的状态,不引起AID,一旦这一免疫自稳状态被破坏,自身反应性T细胞便可活化、增殖并引起病理损害。针对这一理论,用灭活的自身反应性T细胞,诱导机体产生大量的调节性T细胞来抵抗自身反应性T细胞的致病作用,即为TCV的治疗原理,这项治疗措施是目前研究AID治疗的新方向。CD4~+CD25~+T细胞是一种重要的调节性T细胞,最近越来越多的研究显示,CD4~+CD25~+T细胞数量及功能异常与许多AID有密切关系,免疫学上目前将CD4~+CD25~+T细胞视为调节性T细胞的代名词。CD4~+CD25~+调节性T细胞在SLE中的作用也是目前SLE发病机制的研究热点,多项研究发现,CD4~+CD25~+调节性T细胞在SLE患者中存在数量减少,或功能异常,与SLE患者病情密切相关。故本实验制备T细胞疫苗,免疫BXSB鼠后,检测CD4~+CD25~+ T细胞,研究TCV对该细胞水平的影响,从而评价T细胞疫苗在SLE治疗上的应用前景。另外,转录因子Foxp3与CD4~+CD25~+ T细胞的关系也是近年研究的热点,国内外多项研究已经证实,Foxp3是CD4~+CD25~+调节性T细胞的功能相关基因,它作为CD4~+CD25~+调节性T细胞一个相对特异的标志,它的表达是该细胞发育及发挥功能的前提。有学者研究显示,SLE患者外周血Foxp3的表达下降,同时其表达水平与CD4~+CD25~+调节性T细胞数量呈依赖关系。所以Foxp3基因的检测及相关分析,也是SLE治疗免疫学上的一个新指标,并有可能揭示TCV的治疗机制。T细胞是在双信号刺激下发生活化和增殖反应的。TCR/CD3复合物与抗体递呈细胞上的MHC结合形成第一信号,进而CD28与相应配体CD80或CD86结合产生共刺激信号,致T淋巴细胞活化从而引发细胞免疫。Salomon B通过给狼疮鼠注射CD28的拮抗剂,发现ds-DNA抗体的产生与CD28-CD86共刺激分子的作用有关,且注射CD28拮抗剂能降低狼疮肾炎的发病率,延长小鼠的寿命。由此可见CD28-CD86共刺激通路在SLE的发病中具有重要的作用。国内有学者研究提示,CD8~+CD28~+T细胞在SLE患者外周血中表达下降,提示该细胞也可能是反映和监测SLE病情的一个指标。但有关CD8~+CD28~+T细胞在SLE疾病发生发展中的作用的研究报道甚少。因此本文另一目的是通过检测经TCV免疫的BXSB鼠前后CD8~+CD28~+T细胞的变化,来分析该细胞亚群在SLE发病中的变化及与SLE病情的关系,以从另一方面揭示TCV的治疗作用。T细胞疫苗在系统性红斑狼疮中的实验研究国内做的不多,本文旨在结合上述SLE发病机制中免疫学方面新的进展,研究T细胞疫苗免疫BXSB鼠后,CD4~+CD25~+T细胞及相关基因Foxp3,和CD8~+CD28~+T细胞的变化,来探讨TCV治疗的可能机制和应用前景。目的1.研究T细胞疫苗免疫对BXSB鼠外周血CD4~+CD25~+调节性T细胞、CD8~+CD28~+T细胞表达水平的影响,探讨T细胞疫苗治疗SLE的可能机制。2.研究BXSB鼠经T细胞疫苗免疫后基因Foxp3表达的变化,及其与CD4~+CD25~+调节性T细胞的关系,从基因水平分析T细胞疫苗的可能机制。材料与方法1实验动物2.5月龄BXSB雄性小鼠12只,体重22-27g,北京大学医学院免疫系提供,饲养在南方医院SPF实验室。2方法(1)狼疮鼠T细胞的分离运用目前T细胞疫苗实验研究中常用的提取方法,即从BXSB鼠脾脏提取细胞。一切操作均严格遵守无菌操作及试剂说明书规则进行。(2) T细胞疫苗的制备将提取的脾脏细胞悬液经伴刀豆球蛋白Con A(2.5ug/ml)有丝分裂、增殖,后用丝裂霉素C(25ug/ml)处理细胞,调整细胞浓度备用。(3) T细胞疫苗皮下免疫取1×10~7细胞皮下免疫,并于首次免疫后第1,2周分别重复免疫。(4)检测:于免疫前,首次免疫后1周、2周和4周分别从尾静脉取血1ml,采用流式细胞术检测CD4~+CD25~+T细胞、CD8~+CD28~+T细胞的细胞比例及基因Foxp3表达比率。(5)统计学处理组间CD4~+CD25~+T细胞、CD8~+CD28~+T细胞水平及Foxp3比率的比较应用SPSS11.5统计软件处理数据,进行重复测量数据的方差分析,结果中数据以“(?)±s”表示。CD4~+CD25~+T细胞与基因Foxp3表达水平进行双变量相关分析。结果1 TCV对BXSB鼠外周血CD4~+CD25~+T细胞的影响1.1 TCV免疫BXSB鼠前后不同时间段内CD4~+CD25~+T细胞的表达Mauchly球形检验统计量W=0.429,P=0.261本研究显示,不拒绝球形假设,应用单变量检验方法时无需ε(epsilon)校正。TCV免疫前后不同时间段内CD4~+CD25~+T细胞表达水平差异有显著性意义(F=16.441,P=0.000)。1.2 TCV免疫BXSB鼠后CD4~+CD25~+T细胞水平与免疫前比较CD4~+CD25~+T细胞在经TCV免疫后不同时间段均有不同程度的表达,且免疫后该细胞表达水平均值均高于免疫前。其中,免疫后1周与免疫前相比差异无统计学意义(P=0.075);免疫后2周、4周与免疫前相比差异有统计学意义(P值均为0.000)。1.3 TCV免疫BXSB鼠后各时间段之间的CD4~+CD25~+T细胞水平的比较TCV免疫后2周、4周CD4~+CD25~+T细胞水平与免疫后1周相比差异均有统计学意义(P值分别为0.002,0.003);免疫后4周与免疫后2周相比差异无统计学意义(P=0.559)。2 TCV对BXSB鼠基因Foxp3的影响2.1 TCV免疫BXSB鼠前后不同时间段内基因Foxp3的表达水平Mauchly球形检验统计量W=0.409,P=0.231本研究显示,不拒绝球形假设,应用单变量检验方法时无需ε(epsilon)校正。TCV免疫前后不同时间段内基因Foxp3的表达水平差异有显著性意义(F=15.388,P=0.000)。2.2 TCV免疫BXSB鼠后Foxp3基因的表达水平与免疫前比较基因Foxp3在经TCV免疫后不同时间段的表达均值均高于免疫前。其中,免疫后1周和免疫前比较,二者无统计学意义(P=0.251);免疫后2周、4周与免疫前相比差异均有统计学意义(P值分别为0.003,0.002)。2.3 TCV免疫BXSB鼠后各时间段之间基因Foxp3表达水平的比较免疫后2周、4周基因Foxp3的表达水平与免疫后1周相比差异均有统计学意义(P值分别为0.004,0.002);免疫后4周与免疫后2周相比差异无统计学意义(P=0.143)。3基因Foxp3的表达与CD4~+CD25~+T细胞水平的相关分析经相关分析,Spearmon相关系数r_s=0.631,P=0.000(双侧),即认为基因Foxp3的表达与CD4~+CD25~+T细胞的水平存在正相关关系。4 TCV对BXSB鼠外周血CD8~+CD28~+T细胞的影响4.1 TCV免疫BXSB鼠前后不同时间段内CD8~+CD28~+T细胞表达Mauchly球形检验统计量W=0.498,P=0.375本研究显示,不拒绝球形假设,应用单变量检验方法时无需ε(epsilon)校正。T细胞免疫前后不同时间段内CD8~+CD28~+T细胞表达水平差异有显著性意义(F=16.172,P=0.000)。4.2 TCV免疫BXSB鼠后CD8~+CD28~+T细胞水平与免疫前比较CD8~+CD28~+T细胞在T细胞疫苗免疫后不同时间段均有不同程度的表达,且免疫后该细胞表达水平均值均高于免疫前。且免疫后1周、2周及4周与免疫前相比差异均有统计学意义(P值分别为0.010,0.001,0.001)。4.3 TCV免疫BXSB鼠后各时间段之间的CD8~+CD28~+T细胞水平的比较免疫后2周、4周CD8~+CD28~+T细胞水平与免疫后1周相比差异均有统计学意义(P值分别为0.025,0.007);免疫后4周与免疫后2周相比差异无统计学意义(P=0.074)。结论1.BXSB鼠经T细胞免疫后,可促进CD4~+CD25~+T细胞、CD8~+CD28~+T细胞水平的升高,提示T细胞疫苗可影响BXSB鼠体内的免疫状态。这可能是T细胞疫苗发挥治疗作用的主要机制。2.CD4~+CD25~+T细胞的水平与基因Foxp3的表达呈正相关,提示T细胞疫苗可能从基因水平发挥治疗作用。更多还原

【Abstract】 SLE(Systemic lupus erythematosus) is an autoimmune disease that involves multi-organ and multi-system.Its prevalence rate is as high as 70-100/100000 people. and if the population of our country is 1.6 billion,there are 112-116 ten thousand people suffering from SLE,and the desease is particularly prevalent in young women.SLE has many features,such as complex pathogenesis,high prevalence rate, the variety of clinical manifestion,difficulty of treatment and high mortality.Currently there are many ways of treatment for SLE.It is the preferred method to combine glucocorticoids with immunosuppressant drugs,generally larger doses are used at the beginning to control the illness in 1-2 weeks.And if necessary, we can use the pulse therapy of glucocorticoid or combined immunosuppressive agents.But in this course of treatment,because of large drug consumption and long time,it is prone to emerge side effects or complications,such as infections(viruses, bacteria,fungi,etc.),the risk of diabetes,gastrointestinal ulcers,perforation or gastrointestinal bleeding,and bone fractures or ischemic necrosis,mental disorders, many patients died last on account of these side effects.It has affected the survival and quality of patients’ life seriously.At the same time,because the selectivity of the drug is not satisfactory,part of patients is still unable to effectively control condition. The pulse therapy of large dose immunoglobulin,plasma replacement therapy or the irradiation treatment to lymph node all over the body,all don’t aim at etiological treatment directly,and its effect is temporal,so they also can not be widely applied. Although there are encouraging reports of hematopoietic stem cell transplantation, but it needs high technology and prices are also very expensive,so it is difficult to promote also.Therefore,the treatment of SLE is still recognized as a difficult medical problem at home and abroad,and there is no effective cure method.At 1981, TCV(T-cell Vaccine) concept was first proposed by BEU-NUN etc,when they researched Lewis rats experimental autoimmune encephalomyelitis(EAE) of active immunizating by myelin basic protein(MBP).At present some scholars at home and abroad have researched the TCV treatment on autoimmune disease related with T cell-mediated,such as rheumatoid arthritis,organs and tissues after transplantation, autoimmune thyroiditis,autoimmune myocarditis etc,and have some encouraging results.A few also had a pilot clinical trial,such as rheumatoid arthritis,has also made a preliminary efficacy.At present,it’s regarded that T and B lymphocyte dysfunction are characteristics of SLE.Self-reactive T lymphocyte activation and the mediated autoimmune reaction is a central link in this disease.As long ago as 1990s,abroad scholars researched and shown that inhibitting self-reactive T-cell activity can reduce the severity of SLE disease,and extend the life span of mice.Therefore removing self-reactive T cells may be another new therapeutic approach for SLE.Autoreactive T lymphocyte which can recognize their own autoantigen is a normal part of the T-cell,existing in peripheral circulation,but does not cause autoimmune disease(AID).Idiotype-anti-idiotypic network theory confirms,the autoreactive T cells are constrainted by the anti-idiotypic regulatory T cells in the normal body,and are in a relatively small number and lower state.Once the steady state of immune was damaged,atuoreactive T cells can activate,proliferate and cause pathological damage.In response to this theory,the treatment theory of TCV is using inactivated autoreactive T cells to make the body produce large amounts of regulatory T cells to resist the pathogenic role of autoreactive T cells,the treatment is the new direction in the current treatment of autoimmune diseases.The CD4~+CD25~+T cells is an important regulatory T cells,and more and more studies have shown that recently,the abnormity of amount and function of CD4+ CD25~+ T cells related to many autoimmune diseases closely.Immunology has regard CD4~+CD25~+T cells as a pronoun for regulatory T cells.And CD4~+CD25~+ regulatory T cells play the role in the pathogenesis of SLE has gradually become the hot spot of research.Many studies have found that,the number of the CD4~+CD25~+ regulatory T cells in SLE patients is decrease,and the function is abnormal.Following the improvement of SLE disease,CD4~+CD25~+ T cells will also be resumed.So the CD4~+CD25~+ T cells can be used as an indication to monitor the development of SLE disease.Therefore,we prepare the TCV and use it to immune the BXSB rat,then detect the leves of the CD4~+CD25~+T cells in the peripheral blood at arranged time, thereby speculate the potential therapeutic effect of the TCV in SLE desease.In addition,the relationship between transcription factor Foxp3 and CD4~+CD25~+ T cells are also researchful hotspot.Both home and abroad a number of researches indicate that Foxp3 is the function-related gene of CD4~+CD25~+ regulatory T cell.As a relatively specific signs of CD4~+CD25~+ regulatory T cells,the Foxp3 expression is the premise of cell development and function.Furthermore research shows that expression of Foxp3 decline in peripheral blood of SLE patients,and its expression level has dependent relationship with CD4~+CD25~+ regulatory T cells.As a result,Foxp3 gene detection and correlation analys is a new research area in SLE immunology.In the situation of stimulated by dual signal,T cells is in response to activation and proliferation.The MHC combines and creates first signal on TCR/CD3 complex and antigen presenting cell,then while CD28 and the corresponding ligands CD80 or CD86 are combined costimulatory signal is create.This situation results in the activation of T lymphocytes to trigger immune cells.When Salomon B injected lupus mice with the CD28 antagonists,he found that the generation of dsDNA antibody is related to CD28-CD86 costimulatory molecules.Furthermore the injection of CD28 antagonist can reduce the incidence of disease and extend the life span of lupus mice. It can be seen that CD28-CD86 costimulatory pathway is very important at SLE pathogenesis.Another purpose of this paper is by detecting the CD8~+CD28~+T cells change of T cells vaccine BXSB mouse,to analyze the relationship between SLE illness and changes of cell subsets in SLE pathogenesis.Some domestic scholars have suggested that,the level of CD8~+CD28~+T cell is decreased in peripheral blood of SLE patients.This indicates that CD8~+CD28~+T cell is possibly an index to reflect or monitor the state of SLE.But the mechanism about CD8~+CD28~+ T cell in SLE desease has reported very few.At home few scholars do experimental study of T cell vaccination in systemic lupus erythematosus.With the progress in the fields of immunology of SLE pathogenesis,this paper research the changement of CD4~+CD25~+ T cells and related gene Foxp3,CD8~+CD28~+ T cells T-cell about vaccine BXSB rats,and dicuss the application prospects of T-cell vaccine therapy.Objective1.To research the diversify of the CD4~+CD25~+T cells and the CD8~+CD28~+T cells in peripheral blood of BXSB mice after vaccined by TCV,and then explore the mechanism of this treatment. 2.To research the expression of Foxp3 gene in BXSB mice after immunization with TCV,and analyse the relationship between Foxp3 gene and CD4~+CD25~+ regulative T cells.Accordingly we analyse the possible mechanism of T-cell vaccine in the level of gene.Methods1.AnimalsTwelve 2.5-month-old BXSB male mice,weight 22-27g,provided by medical college of Beijing university,and breeded in SPF laboratory of NanFang hospital.2.Methods(1) The extraction of T cellsApply the generally method in many studies recently to prepare the TCV,that is to extract T cells from spleen of BXSB mice.Every procedure abide by the aseptic technique,and follow the instruction of agentia.(2) The preparation of TCVWe use Con A(2.5ug/ml) to promote the caryocinesis of T cells extracted from spleen,then use mitomycin C to deal with cells,and adjust the concentration of cells to finish the reserve of TCV.(3) Immune BXSB mice under skin with prepared TCV.Get 1×10~7 cells from prepared TCV to immune BXSB mice first,and then respectively repeat immune after 1 and 2 weeks.(4) Detection:Flow cytometry was used to measured the level of CD4~+CD25~+T cells,CD8~+CD28~+T cells and gene Foxp3 before immunization and at 1,2,4 weeks after first immunization.(5) Statistical analyseWe use SPSS 11.5 to analyse the results of this study.Results1.The alteration of CD4~+CD25~+T cell after immunization with TCV 1.1 The level of CD4~+CD25~+T cell in different periods before and after immunization with TCV.Mauchly globular test statistics W=0.429,P=0.261.This study show that globular hypothesis isn’t rejected,so applicating univariate analysis needn’t correction ofε(epsilon).So the diversity of CD4~+CD25~+T cell between before and after immunization with T cell vaccination has statistical significance.(F=16.441, p=0.000).1.2 The comparison of the level of CD4~+CD25~+T cells between preimmune and after immunization with TCV.CD4~+CD25~+T cells appear different levels in respective period after immunizaition with TCV,and the levels are higher than preimmune.And the comparison between preimmune and first week after immunization has no statistical significance(P=0.075).The diversity between preimmune and second/forth week after immunization have statistical significance(both P is 0.000).1.3 The comparison of the level of CD4~+CD25~+T cells among different periods after immunization with TCV.In both second and forth week the level of CD4~+CD25~+T cells are higher than first week after immunization(P is respectively 0.002,0.003).And the diversity between the forth week and the second week has no statistical significance (P=0.559).2 The alteration of the gene Foxp3 after immunization with TCV2.1 The level of the gene Foxp3 in different periods before and after immunization with TCV.Mauchly globular test statistics W=0.409,P=0.231.This study show that globular hypothesis isn’t rejected,so applicating univariate analysis needn’t correction ofε(epsilon).So the diversity of the level of the gene Foxp between before and after immunization with TCV have statistical significance.(F=15.388, P=0.000).2.2 The comparison of the level of the gene Foxp3 between preimmune and after immunization with TCV.The mean of the gene Foxp3 in respective period after immunizaition with TCV is higher than preimmune,and the comparison between preimmune and first week after immunization has no statistical significance(P=0.251).The diversity between preimmune and second/forth week after immunization have statistical significance(P is respectively 0.003,0.002).2.3 The comparison of the level of the gene Foxp3 among different periods after immunization with TCV.In both second and forth week the level of the gene Foxp3 are higher than first week after immunization(P is respectively 0.004,0.002).And the diversity between the forth week and the second week has no statistical significance(P=0.143).3 The correlation analysis between levels of the gene Foxp3 and CD4~+CD25~+T cells.The correlation analysis show that,the Spearmon coefficient correlation is 0.631(r_s=0.631),P=0.000(two-tailed).So positive correlation exists between gene Foxp3 and CD4~+CD25~+T cells.4 The altration of CD8~+CD28~+T cell after immunization with TCV4.1 The level of CD8~+CD28~+T cell in different periods before and after immunization with TCV.Mauchly globular test statistics W=0.498,P=0.0.375.This study show that globular hypothesis isn’t rejected,so applicating univariate analysis needn’t correction ofε(epsilon).So the diversity of CD8~+CD28~+T cell between before and after immunization with T cell vaccination has statistical significance.(F=16.172, P=0.000).4.2 The comparison of the level of CD8~+CD28~+T cells between preimmune and after immunization with TCV.CD8~+CD28~+T cells appear different levels in respective period after immunizaition with TCV,and the means of the level of post-immune all are higher than preimmune.And the diversity between preimmune and post-immune has statistical significance(P is respectively 0.010,0.001,and 0.001).4.3 The comparison of the level of CD8~+CD28~+T cells among different periods after immunization with TCV.In both second and forth week the level of CD8~+CD28+T cells are higher than first week after immunization(P is respectively 0.025,0.007).And the diversity between the forth week and the second week has no statistical significance (P=0.074)Conclusion1.Immunization of TCV can promote the level of CD4~+CD25~+T cells and CD8~+CD28~+T cells in peripheral blood of BXSB mice,so we can presume that,the TCV can intervene with the status of immunity in body of BXSB mice,and this may be the main mechanism of TCV.2.Immunization of TCV can enhance the expression of Foxp3 gene,this indicated that TCV may possibly produce a marked effect in level of gene.更多还原