【作者】 祁赞梅；

【导师】 姜奕；

【作者基本信息】 中国医科大学 ， 免疫学， 2004， 博士

【摘要】 实验目的 系统性红斑狼疮(systemic lupus erythematosus，SLE)是一种病因不清的全身性自身免疫性疾病，以抗核抗体的产生为主要免疫学特征。SLE的发病涉及遗传因素和环境因素的复杂相互作用，这些因素可能通过促进免疫耐受丧失、免疫调节紊乱以及器官损伤等方式导致SLE发生。 近年来，T细胞在SLE发病机制中的作用逐渐受到重视，一方面由于主要的致病性自身抗体—IgG类自身抗体的产生需要T细胞的辅助，另一方面由于T细胞具有重要的免疫调节作用。很久以前就已发现SLE患者和小鼠模型中存在多种T细胞数量与功能异常。80年代后期以来，在SLE T细胞内发现了越来越多的与数量和功能异常有关的信号转导异常和生化改变，有些信号转导路径异常与疾病的活动性和治疗无关，更像是T细胞本身的改变。因此近几年来，探索SLE T细胞内在异常的根本原因已经成为自身免疫病发病机制研究的热点之一。虽然已经发现部分SLE易感位点与T细胞活化和表型异常有关，但并未阐明其遗传基础。因此有必要从更深的水平了解SLE T细胞异常的原因。 自发产生与人类SLE相似症状的小鼠模型一直是研究SLE的极好工具。新西兰黑色品系小鼠(New Zealand Black，NZB)产生自身免疫性溶血性贫血，老年时发生轻微的SLE症状。白色品系小鼠(New Zealand White，NZW)为非自身免疫鼠系。两者杂交的子一代小鼠(NZB×NZW)F1小鼠，从生后5、6个月开始至12个月，几乎全部自发典型的SLE症状，免疫异常与人类SLE极为相似。Steinberg等通过实验证实了来自NZB和NZW的基因是子一代小鼠狼疮表型加速出现的主要原因。因此，研究子一代小鼠中遗传相关的免疫学异常并确定其遗传基础，对于揭示SLE的发病机制具有重要意义。 已有实验表明(NZB×NZW)F1小鼠中存在T细胞活化异常和T细胞亚群异常，为了了解(NZB x NZW)F1小鼠的T细胞异常是否与来自NZB和NZW的遗传因素有关，并确定其遗传基础，本实验比较了NZB、NZW和(NZB x NZW)F1小鼠的T细胞亚群数量，并在此基础上，通过建立回交小鼠模型，确定了CDS‘T细胞数量异常的易感基因位点，并对候选易感基因进行了序列分析。试验方法 一、采用流式细胞分析技术检测6一12月龄NZB、NZW和(NZBxNZW)Fl小鼠外周血淋巴细胞中CD4‘T细胞、CDS十T细胞百分比: 眼球后静脉丛采血looul，加人标记好的流式细胞仪用染色管中。每个试管预先加人0.5此FITC标记大鼠抗小鼠CDS抗体、1林gPE标记大鼠抗小鼠CD4抗体。另有一试管加人FITC标记大鼠IgGZa和PE标记大鼠lgGZa作为阴性对照。室温下避光反应30分钟，每管加入2时红细胞裂解液溶血，PBS洗一次。每管加人500闪2%多聚甲醛固定液，充分混匀，静置巧分钟，待检。流式细胞仪激发波长488run，利用Cell quest软件获取细胞，每个样品分析1 0000个细胞。以二维点阵图显示，记录阳性细胞百分率。 二、采用基因组范围内的数量性状位点(Quantitative trait loeus，QTL)分析法确定(NZB x NZW)Fl小鼠外周血淋巴细胞中CDS‘T细胞数量异常的易感位点: 1.建立(NzB x NZW)Fl x NZW回交小鼠模型。实验中共使用211只回交小鼠，全部为雌性。 2.采用流式细胞分析技术检测8月龄回交小鼠外周血淋巴细胞中CDS‘T细胞百分比。 3.筛选在NZB、NZW小鼠间具有长度多态性的微卫星DNA遗传标记，在小鼠全部19条常染色体上平均每隔5一10cM选择一个多态性微卫星DNA遗传标记，共选择约190个遗传标记。 4 .PCR一SSLP(POI卿erase Chain Reaction一Simple Se，enee此ngthPolym呷hism)分析法确定微卫星遗传标记基因型。取小鼠尾部组织1 cm左右，酚、氯仿抽提DNA。用特异性微卫星引物扩增微卫星DNA片段。PCR反应在%孔板中进行，总反应体积7.5闪，包括基因组DNA 20ng，Taq酶0.5 unit。反应条件:94℃预变性3 min，三温度循环(94℃30sec、58℃40seC、72℃1 min)共45个循环，末次延伸72℃3而n。用10%一18%聚丙烯酞胺凝胶电泳分离PCR产物，根据分离后的微卫星DNA扩增片段的大小，决定回交小鼠基因型。 5.将每只回交小鼠的全部微卫星DNA遗传标记的基因型数据及8月龄小鼠外周血淋巴细胞中CDS十T细胞百分比输入MAPMAKE份Q，rL和MAPMANAGE印QTL分析软件数据库中，进行基因组范围内的QTL分析，确定与CDS辛T细胞数量异常易感基因紧密连锁的微卫星DNA遗传标记。在此基础上，查阅基因组数据库，筛选候选基因。 三、CD48 cDNA编码区和CD48基因启动子区域的序列分析: 1 .RT一PCR扩增CD48 cDNA编码区 制备6周龄NZB和NZW小鼠的胸腺细胞，ISOGEN法提取总RNA。用01190(dT)引物合成单链cDNA。PCR扩增CD48 cDNA编码区，引物为F:5’一ATGTGCTI’CATAAAACAGGGAT一3‘(从转录起始点开始41一62)，R:5‘一AGAGTTCTrGTCAGGTTAACAG一3‘(773一752)。反应体系为100林l，含1卜1 eDNA、lu Taq酶。反应条件为:94℃预变性10min，pCR三温循环(95℃40see、55℃60see、72℃90see)共30个循环，末次延伸7而n。 2.PCR扩增CD48基因启动子区域 酚氯仿法抽提更多还原

【Abstract】 The exact patho - aetiology of systemic lupus erythematosus ( SLE ) remains elusive. An extremely complicated and multifactorial interaction among various genetic and environmental factors is probably involved. These factors contribute to SLE through three pathways, which are the loss of immune tolerance, dysfunction of immune regulation, and the enhancement of tissue damage.Since T cells play important role in the immune regulation and the production of autoreactive antibodies of IgG class, more and more effort has been made to study the role T cell plays in the pathogenesis of SLE. In fact, studies with SLE patients and SLE murine models have showed various T cell abnormalities, such as the aberrant T cell number, the impairment of inhibitory T cell function, the shifting of cytokine spectrum to Th2 pattern and the loss of self tolerance. An increasing evidence showed abnormal signal transduction and biochemical events in SLE T cells, which were believed to be associated with T cell dysfunction, and phenotype changes. Some signal transduction abnormalities seem to be intrinsic for they exist previous to the emergence of symptoms and are not modified by treatment. Thus, the investigation of the mechanisms leading to these intrinsic T cell defects becomes one of the hot - spots in the field of the stuHy of SLE pathogenesis. Some progresses have been made in searching for the susceptibility loci for abnormal T cell activation and aberrant T cell phenotypes. But the genetic defects in these susceptibility loci remain unknown. So it is necessary to clarify the genetic basis of T cell abnormalities.Lupus -prone mice are useful tools for the studies of SLE. The New Zealand Black ( NZB) strain spontaneously develops autoimmune hemolytic anemia. In addition, a mild form of lupus nephritis can occur in later life of this strain.New Zealand White ( NZW) mice are principally a non - autoimmune strain. In the Fl hybrid progeny of the NZB and NZW strain, florid SUE with a much earlier onset and a higher incidence of nephritis spontaneously develops from a young age onward. It is evident that in (NZB xNZW) Fl mice, NZB -derived genes determine specific aspects of autoimmune phenotypes and that genes from the NZW parent modify the diseases. To investigate the inherited immune abnormalities in Fl mice and determine their susceptibility gene is undoubtedly significant to the study of the pathogenesis of SLE.Studies showed polyclonal T cell activation and aberrant T cell subsets in (NZB xNZW) F1 mice. In order to elucidate whether these T cell abnormalities are related to the genetic background of NZB and NZW and to determine the causing genetic defect, we compared the percentage of T cell subset in the peripheral blood among NZB, NZW and (NZB x NZW) Fl mice and mapped the susceptibility gene of aberrant CD8 + T cell number in ( NZB x NZW) Fl mice by setting up backcross mice model. Based on these results, we selected candidate genes and detected potential defects on these genes by sequencing.Method1. Determination by flow cytometry of the percentage of the CD4+ and CD8 + T cells in the peripheral lymphocytes of mice.Peripheral blood of mice was collected with glass capillary tube from post-bulbar venous plexus and transferred into labeled 12 x 75 mm test tubes in which 0. 5ug FITC - labeled rat - anti - mouse CD8 antibody and 1 ug PE - labeled rat anti - mouse CD4 antibody was added previously. One tube in which FITC - labeled rat IgG2a and PE - labeled rat IgG2a was added was used as negative control. Incubated for 30 minutes at room temperature in the dark cabinet. Then added 2 ml of red cell lysis buffer to each tube. After washing, 500ul 2% formaldehyde was added to each tube to fix the sample. Fluorescence intensity was measured using a FACScan flow cytometer and CellQuest software. Flow cyto-metric analysis utilized linear forward light scatter (FLS) , linear side light scatter (SS) and log fluorescence parameters.2. Gene mapping using genome -wide Quantitative Trait Locus analysis.2. 1 (N更多还原