紫外线照射诱导的CRT寡聚化在系统性红斑狼疮发展中的作用及机制研究

As the commonest autoimmune disease, systemic lupus erythematosus (SLE) exert a strong impact on the life of patients especially the women of childbearing age. SLE is characterized with large amounts of auto antigens and auto antibodies. Anti calreticulin(CRT) antibodies are found in the sera of 40% of SLE patients. At the same time, ultraviolet B (UVB) irradiation can traditionally worsen the condition of SLE. ..Our previous work had shown that CRT could oligomerize under UVB irradiation, so we postulate that oligomerization of CRT might expose cryptic epitopes or generate new ones, thus activate the immune system, initiating or exacerbating SLE disease. This project contains three parts. Firstly, to compare the levels of CRT antibodies in the sera between photosensitive SLE patients and the health controls; to purify CRT proteins from sera, analyze whether the CRT exists in the oligomer form in SLE patients; and to examine the pathological change of the tissue sections from skins. Secondly, to test the tissue damage of skin and kidney of UVB irradiated BXSB mice; to measure CRT antibodies in the sera, and analyze the change of CRT conformation; Thirdly, to extract CRT proteins from mouse livers through ammonium sulfate precipitation combining with anion exchange, to induce CRT oligomerization by UVB irradiation, then to test the antigenicity and immunogenicity of CRT monomers and oligomers, including the potential of activating of DC cells, the ability of generating autoantibodies...The data from above experiments would tell us why CRT becames the target of immune system after UVB irradiation and how it impacts on SLE development, this work might provide new insights into the pathogenic mechanisms of how UVB deteriorating SLE disease.

系统性红斑狼疮（SLE）严重影响患者的生活质量。紫外线B（UVB）照射会促进病情恶化，同时抗钙网蛋白(CRT)抗体在SLE患者血清中的水平升高，其原因尚不清楚。我们前期工作表明CRT在UVB照射下会由单体变为寡聚体，由此推测UVB促使CRT寡聚化，导致隐蔽抗原的暴露或者新的抗原表位生成，激活免疫系，促进了SLE的发展。本研究拟比较光敏感性SLE患者和健康人CRT抗体的水平，分析CRT是否寡聚化，进行皮肤病理检测；用UVB照射BXSB小鼠，检测组织的损伤，CRT抗体的水平，皮肤组织中CRT表达水平和寡聚化情况；采用离子交换层析法从小鼠组织提取CRT，检测CRT经UVB照射形成寡聚体后对DC等免疫细胞的激活作用和诱导抗体的产生。本研究以患者，动物，细胞为载体，从病理，免疫反应，蛋白质的变化等方面揭示UVB诱导的CRT寡聚化在SLE中的作用及机制，为UVB促进SLE进展提供新的解释。

系统性红斑狼疮（SLE）严重影响患者的生活质量。紫外线B（UVB）照射会促进病情恶化，同时抗钙网蛋白 (CRT)抗体在SLE患者血清中的水平升高，其原因尚不清楚。我们的前期工作表明CRT在UVB照射下会由单体变为寡聚体，由此推测UVB促使CRT寡聚化，导致隐蔽抗原的暴露或者新的抗原表位生成，激活免疫系统，促进了SLE的发展。本研究首先比较了SLE患者和健康人CRT抗体的水平，表明了SLE患者CRT抗体水平明显高于正常人。在动物实验用UVB照射SLE易感的MRL/gld小鼠，观测到UVB照射组小鼠被照射的背部皮肤毛发生长受到抑制，肾脏组织的胶原沉积明显增多；CRT抗原在两组间没有明显差异，但CRT抗体的水平在UVB照射组明显上升；SLE特异性自身抗体 anti-dsDNA随着小鼠年龄的增长会升高，但在照射组升高的程度更大。采用硫酸铵沉淀结合离子交换层析法从小鼠组织中提取CRT，经UVB照射后CRT形成寡聚体；寡聚化和单体CRT与单克隆抗体的识别结合能力不一致，表明CRT寡聚化后生成了新的抗原表位或者暴露了隐蔽的抗原表位；寡聚化的CRT比单体CRT对DC细胞具有更强的激活作用，刺激其分泌更高水平的IL-6和TNF-α，与脾细胞共培养时可以刺激其中的T细胞有更明显的增殖，但对巨噬细胞和T及B细胞的直接刺激作用两组间没有差异；CRT寡聚体免疫正常小鼠能够诱导抗CRT特异性抗体的产生。此外用双分子荧光互补实验验证了细胞内的CRT在经过紫外线照射后也会发生寡聚化，表明了机体经紫外线照射后CRT也有寡聚化的倾向。本研究以患者，动物，细胞为载体，从病理，免疫反应，蛋白质的变化等方面揭示UVB诱导的CRT寡聚化导致了新的自身抗原的生成，激活了免疫细胞和免疫系统，加重了炎症反应，加速了肾脏的病变，促进了SLE的发展，为紫外线照射恶化SLE提供了新的解释。这将为临床上SLE患者病情的控制抑或治疗扩展了新的思路。