突变基因Nasp在狼疮性模型小鼠自身免疫病发病中的作用及机制研究

Systemic lupus erythematosus (SLE) is a prototype autoimmune disease with many organs affected. The evidences from clinic and basic study suggest that genetic factor plays a dominant role in SLE development. The lupus-susceptibility sublocus Sle2c1rec1c was identified in lupus murine NZM2410 model. Our preliminary study demonstrated that Sle2c1rec1c sublocus interacted with apoptosis-deficient lpr gene in epistatic manner to result in total T cell increase and T subset abnormality as well as to promote nephritis. Our evidences of murine exome sequencing confirmed that only nuclear autoantigenic sperm protein (Nasp) gene in Sle2c1rec1c sublocus shows mutation in an exon with change of two sequential amino acids. This project proposes to explore the function, pathogenicity and mechanisms of the mutated Nasp gene in autoimmune disease. For this purpose, we design several strategies in order to identify in vitro if the ability of the mutated Nasp binding histones and regulating cell cycles is different from its wild type, as well as to investigate in vivo whether or how this mutated Nasp gene is involved in lupus pathogenesis through establishing four new congenic strains. To sum up, this project will provide insights into the genetic mechanisms of SLE and contribute discovery of therapeutic drug.

系统性红斑狼疮（SLE）是多器官受累的典型自身免疫性疾病，遗传因素在SLE的发病过程中发挥主导作用。小鼠狼疮性易感基因亚座Sle2c1rec1c来自SLE小鼠模型NZM2410品系。我们的初步研究显示，小鼠Sle2c1rec1c基因亚座能够与缺陷的凋亡基因lpr发挥上位乘积效应，促进T细胞增多、亚群异常以及肾脏炎症；并且，我们对该基因亚座内所有基因的外显子进行了测序，发现只有细胞核自身抗原性精子蛋白（Nasp）基因发生突变，导致两个连续氨基酸异常。因此，确定这一新发现的Nasp基因突变与SLE的关系成为本研究的核心内容。我们将在体外分析突变的Nasp蛋白质结合组蛋白的能力和对细胞增殖的影响；通过新建四个同类系小鼠模型，体内研究突变基因Nasp对SLE发病的促进作用和效应机制。本课题的最终目的是鉴定突变基因Nasp的功能变化、致病性以及作用机理，这将有助于阐明SLE发病的遗传缺陷原因和机制。

本课题的目的是研究小鼠核内自身抗原性精子蛋白质（nuclear autoantigenic sperm protein， NASP）突变基因对于狼疮性疾病的影响。首先，我们采用原核细胞表达系统和合适的纯化方法，获得了高度纯化的重组小鼠NASP蛋白质。 然后， 我们采用表面等离子共振技术（surface plasmon resonance technology, SPR）（Octet K2 System）测定了小鼠NASP蛋白质与组蛋白的亲和力。我们的结果发现小鼠变异的NASP蛋白质与组蛋白H3和H3/H4多聚体的亲和力不同于正常小鼠的NASP蛋白质，表明前者的分子功能发生了改变。其次，我们采用小鼠基因工程技术成功的将小鼠NASP突变基因的两个突变碱基敲入（Knock in） B6小鼠的基因组内，构建了B6.NaspM新品系。通过对小鼠的杂交和回交，我们获得了纯合子的B6.NaspM小鼠，以及纯合子的B6.NaspM.lpr小鼠，B6.NaspM.Sle1.Yaa小鼠，和B6.NaspM Fcgr2b-/-小鼠。最后，我们系统的检测了这些小鼠品系的免疫病理变化。我们的结果发现，与对照B6.lpr小鼠相比，B6.NaspM.lpr小鼠血清中IgG类型的抗染色质抗体和抗双链DNA抗体水平明显增多； B6.NaspM.lpr小鼠的脾脏和淋巴结显著增大；B6.NaspM.lpr小鼠的尿蛋白水平也异常升高。这些结果显示，B6.NaspM.lpr小鼠出现比较严重的自身免疫反应和肾脏炎症，表明小鼠NASP突变基因能够促进自身免疫病的致病基因。本项研究的结果发现了新的促进系统性红斑狼疮发病的突变基因，提高了我们对于该病发病机理的认识。