新西兰小鼠遗传易感基因功能分析和肾小球蛋白谱表达的研究

We have set up New Zealand black(NZB)xNew Zealand white(NZW)F1xNZB backcross mice and New Zealand White(NZW)xNew Zealand Black(NZB)F1xNZW backcross mice model and used polymorphic microsatlite marker quantitive trait locous(QTL)analysis and amplified fragment length polymorphism to map and screen the susceptibility genes for Lupus Nephritis. Susceptibility genes of LN were linked to microsatlite marker of D11Mit263 on chromosome 11 and the D17 Mit61 on chromosome 17 in NZB and D4Mit71 on chromosome 4 and Tnfa on chromosome 17 in NZW according to QTL analysis before. There are also other candidate genes in these areas such as histone acetyl (Hdac1,), interleukin -14 (IL-14), Lck , N-multi-ligand proteoglycans proteoglycans 3 (Sdc3), high mobilityfamily 2 (Hmgn2) and platelet-activating factor receptor (Ptafr) gene. These molecules are associated with the onset of systemic lupus erythematosus or lupus nephritis, its gene is likely to be susceptibility genes in SLE or LN .In this subject we intend to screen, clone and functional analysis the lupus nephritis (LN) positioning results of susceptibility genes further,it is called Positional candidate gene cloning method. Using DNA sequencing, PCR-SSCP technical means for QTL analysis of candidate genes. Select the higer increased LOD score genes as candidate genes to study their functions. The aim of this study was also to attain proteomic profile in glomeruli of LN mice by using two-dimensional differential gel electrophoresis and mass spectrometric analysis, and to investigate the effect of accordingly blockers and agonists treatment.By combining it with the function of susceptibility genes ,we will easier to find aberrant expression of proteins and their metabolic pathways in the body in order to further identify candidate genes and proteins. Then by transfected glomerular epithelial cells, mesangial cells and kidney fibroblasts and constructing a recombinant DNA plasmid to study of these genes on regulation of downstream molecules. Study their cellular localization and function of candidate proteins to investigate the role in the development in LN.If funding permit ,we can also study human SLE / LN specimens, in order to obtain protein markers associated with early diagnosis , relapse ,clinical phenotypes and pathological type of SLE/LN.

本课题拟在狼疮性肾炎（LN）易感基因染色体定位结果的基础上，进一步开展LN易感基因的筛选、克隆及功能分析。利用定位候选基因克隆法在小鼠4号染色体D4Mit72及11号染色体D11Mit263区域筛选候选基因，采用DNA测序、PCR-SSCP等技术对候选基因进行QTL分析。选取LOD值升高的基因进行功能研究。通过构建重组DNA质粒，肾小球上皮细胞、系膜细胞及肾脏间质成纤维细胞转染等技术研究这些基因对下游分子的调控。同时应用双向差异凝胶电泳及质谱法观察LN发病过程肾小球蛋白表达谱的变化及和激素及免疫抑制剂治疗过程中对LN肾小球蛋白表达谱的影响。旨在寻找与LN发病过程密切相关的蛋白质，与前面基因功能测定相结合，更容易找到LN的致病基因及异常表达蛋白和它们在体内的代谢通路，以进一步确定候选基因和蛋白质，如有相应的抑制剂和阻断剂可以进一步进行研究，以期找到诊断和治疗SLE/LN的新方法。

我们研究小组在既往的研究课题中分别以 (NZB x NZW)F1 x NZB回交小鼠及(NZB x NZW)F1 x NZW回交小鼠为研究对象，进行 QTL分析，最终确定了二个来源于NZB小鼠狼疮性肾炎候选易感基因的染色体定位。三个来源于NZW小鼠的狼疮性肾炎易感基因的染色体精确定位。. 此实验中我们利用定位候选基因克隆法，在包含有狼疮性肾炎易感基因的NZB小鼠第 4染色体D4Mit71及NZW第11染色体 D11Mit263附近区筛选与免疫学相关的候选基因，对候选基因进行全程测序。. 我们根据Mouse Genome Informatics (MGI)数据库确定候选基因41个，针对基因结构区设计引物69对。测序结果利用Chromas软件判读，对存在碱基变异处进一步确定有无氨基酸序列变化。对比测序结果并经双向确认，共发现在狼疮易感区存在变异的基因11个，突变点41个。查询GeneBank数据库，发现41个突变点中有14个可导致氨基酸序列的改变，影响了6个基因的最终产物。我们把重点放在了Traf6（Tnf receptor-associated factor 6）。近年来有人发现Traf6在一些自身免疫病如系统性红斑狼疮，干燥综合征，硬皮病等有基因或者蛋白表达异常。. 我们同时选取了NZB/NZWF1新西兰雌性小鼠作为研究对象，利用磁珠灌注法提取大量高纯度的小鼠肾小球，应用差异凝胶电泳和质谱分析法对肾小球蛋白谱进行分析，主要研究应用激素后的蛋白组学变化。蛋白组学的研究结果发现，应用激素后小鼠肾小球的蛋白质潜伏膜蛋白1(LMP1的恶化也会导致自身免疫性疾病,如系统性红斑狼疮)，IRAK，NF-κBp等与Traf6相关蛋白的表达有所变化（数据尚在统计中）。. 我们可以简单看出，这些基因和蛋白与B和T细胞的功能密切相关。因此下一步我们将以它们作为突破口，观察其在狼疮性肾炎中作用机制。