SLC7A2致病变异通过影响L型精氨酸转运抑制巨噬细胞功能导致麻风发病的分子机制研究

SLC7A2, encodes protein which essential for the activation of macrophage via transferring L-arginine into cell, has been identified as one of the susceptibility genes of leprosy. High-throughput re-sequencing has determined the rare variant rs13259978（D28H）was significantly associated with leprosy. Functional analysis showed that rs13259978（D28H）may impair membrane localization of this protein. Previous study of our group also confirmed SLC7A2 was upregulated by infection of Mycobacterium leprae. These results implied that rs13259978（D28H）may be involved in pathogenesis of leprosy via impairing the function of macrophage, but the exact mechanism remains unclear. In this study, the differential expression of SLC7A2 will be analyzed between leprosy cases and health controls by qPCR and immunostaining. Furthermore, other molecular experiments, such as immunofluorescent staining, L-arginine uptake study and intracellular bacterial survival assays will be conducted to clarify how the candidate causal variant affects the function of SLC7A2. The in vivo mechanism of candidate causal variant regarding to the phenotype analysis of the granuloma formation, macrophage function and relative genes expression will be explored in Zebrafish. This study will greatly advance the biological understanding of leprosy as well as other mycobacterial infectious diseases.

SLC7A2是课题组最新发现的麻风易感基因，其通过转运L型精氨酸在巨噬细胞活化及杀伤胞内菌的过程中起重要作用。深度重测序显示SLC7A2编码区的rs13259978位点与麻风紧密相关，分析显示其可能会影响SLC7A2蛋白在细胞膜上的定位，课题组前期免疫组化及qPCR结果也证实了SLC7A2由麻风分枝杆菌感染诱导高表达，提示rs13259978可通过抑制巨噬细胞功能进而导致麻风发生，但具体机制尚不明确。课题组拟在此基础上，通过免疫组化，qPCR技术探索麻风与正常对照中SLC7A2基因表达情况，并利用细菌活力测定实验对rs13259978位点影响SLC7A2转运L型精氨酸进而抑制胞内菌杀伤的作用进行分析；同时获取SLC7A2基因敲除斑马鱼模型，通过接种海鱼分枝杆菌，深入研究SLC7A2基因在分枝杆菌感染时，对机体免疫应答的影响，以明确麻风发病分子机理，为疾病风险预测与干预等提供理论依据。

SLC7A2是课题组最新发现的麻风易感基因。前期的研究显示此基因可通过转运L型精氨酸促进巨噬细胞活化，进而在胞内菌杀伤的过程中起到重要作用。课题组通过免疫组化技术研究发现，麻风及海分枝杆菌感染形成的肉芽肿部位，SCL7A2显著高表达，提示其在分枝杆菌感染过程中的重要作用；进一步构建体外细胞感染模型，海分枝杆菌感染后可显著上调人单核细胞来源的巨噬细胞系THP-1以及小鼠巨噬细胞系RAW264.7中的SLC7A2基因的表达，且随着感染菌量及感染时间的增加，其表达量也随之增加，呈现出明显的剂量和时间依赖现象。然后课题组构建SLC7A2稳定敲降及过表达THP-1细胞株，流式细胞术研究显示SLC7A2基因的敲降和过表达均不影响THP-1细胞的增殖；利用荧光标记的海分枝杆菌感染上述细胞系，流式细胞实验结果显示，SLC7A2的敲降可显著增加THP-1的吞噬能力，菌落形成实验显示SLC7A2的敲降和过表达也不影响巨噬细胞的杀菌能力。由以上结果可知，SLC7A2基因的敲降可促使巨噬细胞的吞噬能力增加，但无法有效的对分枝杆菌进行杀伤，进而影响疾病的进程。