Cd180在SLE B细胞的异常表达及其抑制IFN-α通路的机制研究

发布时间：2018-05-02 03:40

本文选题：系统性红斑狼疮 + B细胞　；参考：《南京大学》2016年硕士论文

【摘要】：系统性红斑狼疮(Systemic lupus erythematosus, SLE)是一种慢性的、累及多器官多系统的自身免疫性疾病,其主要特征是患者血清中存在大量的IFN-α和自身抗体。B细胞作为抗体产生细胞,其异常活化在SLE的发病过程中发挥关键作用,然而其异常活化的分子机制仍不完全清楚。许多研究报道,SLE的B细胞中IFN-α通路是高度活化的,且IFN-α可通过直接或间接的方式参与B细胞的生存与功能,进而参与SLE的病程发展；在SLE患者体内,CD180-B细胞数量明显增加,并且参与自身抗体的产生,提示CD180可能参与SLE中B细胞的异常活化。那么,CD180的表达变化是否影响IFN-α通路活化?导致CD180表达变化的原因又是什么?因此,本研究针对上述问题,分析了CD180在SLE来源B细胞的表达变化,鉴定了CD180-B细胞的表型,探讨了CD180调控IFN-α通路影响B细胞的分子机制。1.发现SLE来源B细胞低表达CD180,且CD180-B细胞可能由生发中心B细胞和浆细胞组成；我们首先各取5例正常人与SLE患者外周血进行流式分析,验证SLE患者外周血CD180-B细胞异常增多；从分选出B细胞后检测CD180 mRNA水平,发现CD180表达水平在SLE患者中是显著降低的。表明CD180-B细胞的增多可能是由于某些原因导致的B细胞CD180表达降低有关。接下来我们在狼疮模型小鼠中进行了验证,发现在狼疮小鼠脾脏、外周血和骨髓中,CD180-B细胞数量都是明显增加,且分选出的脾脏B细胞的CD180表达降低。我们进一步研究了CD180-B细胞的表型,发现CD180-B细胞可能由生发中心B细胞和浆细胞组成。2.发现激活CD180能够显著抑制B细胞的IFN-α通路活化,提示anti-CD180可能是治疗SLE的潜在策略：我们探讨了CD180与IFN-α通路的关系及影响其表达变化的原因,发现激活CD180在体外和体内均能显著抑制B细胞IFN-α通路的活化并降低IFN-α下游基因的表达。其具体机制是CD180通过lyn-PI3K-BTK这一级联通路引起IFN-α下游STAT-2磷酸化水平下降,进而影响IFN-α通路的活化。进一步发现,用TLR7配体R848和TLR9配体CpG刺激小鼠脾脏B细胞能显著抑制CD180的表达,并调节CD180对IFN-α通路的抑制作用。综上所述,我们证实了MRL/lpr小鼠中CD180-B细胞显著增多；发现激活CD180能够显著抑制IFN-α通路的活化；还发现SLE来源B细胞CD180的表达变化可能归因于TLR7和TLR9通路活化。由于靶向IFN-α治疗SLE具有很大的前景,我们的发现提示,anti-CD 180可能是治疗SLE的潜在策略。
[Abstract]:Systemic lupus erythematosus (lupus erythematosus, SLE) is a chronic, multi-organ and multi-system autoimmune disease characterized by the presence of a large number of IFN- 伪 and autoantibody. B cells as antibody producing cells. Its abnormal activation plays a key role in the pathogenesis of SLE, but the molecular mechanism of its abnormal activation is still unclear. Many studies have reported that IFN- 伪 pathway is highly activated in B cells of SLE, and IFN- 伪 can participate in the survival and function of B cells directly or indirectly, and then participate in the course of SLE, and the number of CD180-B cells in patients with SLE increases obviously. It is suggested that CD180 may be involved in the abnormal activation of B cells in SLE. Does the expression of CD180 affect the activation of IFN- 伪 pathway? What is the cause of the change in CD180 expression? Therefore, in this study, we analyzed the expression of CD180 in SLE derived B cells, identified the phenotype of CD180-B cells, and explored the molecular mechanism of CD180 regulating IFN- 伪 pathway affecting B cells. It was found that SLE derived B cells were low expressed CD180, and CD180-B cells might be composed of germinal center B cells and plasma cells. Firstly, we took 5 normal persons and 5 SLE patients for flow analysis to verify the abnormal increase of CD180-B cells in peripheral blood of SLE patients. The level of CD180 mRNA was detected from B cells, and the expression of CD180 was significantly decreased in SLE patients. The results suggest that the increase of CD180-B cells may be related to the decrease of CD180 expression in B cells. Then we demonstrated that the number of CD180-B cells in spleen, peripheral blood and bone marrow of lupus mice was significantly increased, and the CD180 expression of selected B cells in spleen was decreased. We further studied the phenotype of CD180-B cells and found that CD180-B cells may consist of germinal center B cells and plasma cells. It was found that activation of CD180 could significantly inhibit the activation of IFN- 伪 pathway in B cells, suggesting that anti-CD180 might be a potential strategy for the treatment of SLE. We investigated the relationship between CD180 and IFN- 伪 pathway and the causes of its expression changes. It was found that activation of CD180 could significantly inhibit the activation of IFN- 伪 pathway and decrease the expression of IFN- 伪 downstream gene in B cells in vitro and in vivo. The mechanism is that CD180 leads to the decrease of phosphorylation level of STAT-2 downstream of IFN- 伪 through lyn-PI3K-BTK, which affects the activation of IFN- 伪 pathway. It was further found that TLR7 ligand R848 and TLR9 ligand CpG could significantly inhibit the expression of CD180 and regulate the inhibitory effect of CD180 on IFN- 伪 pathway. In conclusion, we confirmed that the number of CD180-B cells in MRL/lpr mice was significantly increased, the activation of CD180 could significantly inhibit the activation of IFN- 伪 pathway, and the change of CD180 expression in SLE derived B cells might be attributed to the activation of TLR7 and TLR9 pathway. Our findings suggest that anti-CD180 may be a potential strategy for the treatment of SLE.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R593.241

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