ECSIT调控mtROS在假体松动中的作用机制研究

Prosthesis loosening is the main complication of joint arthroplasty. Its pathogenesis and mechanism have not been identified. Recently, prosthetic wear particles has been shown to stimulate macrophages leading to chronic inflammation. In addition , excessive production of ROS can intensify the inflammatory response.These are closely related to prosthesis loosening. Our previous study has indicated that enzymes promoting the generation of ROS are highly expressed in membrane tissues at the interface of aseptic loosening , which may results in the overproduction of ROS and aggravates prosthesis loosening. Lastest research shows that the ubiquitination of ECSIT is an important way of mitochondrial ROS (mtROS) produced , which results in an increase of the total amount of ROS and promotes inflammation.Our further experiment shows that wear particles can increase the expression of ECSIT and production of mtROS. We hepothesize that ECSIT can enhance the inflammatory response induced by wear particles and participate in the occorrence of prosthesis loosening. Based on our previous study and latest researches, we planned to: (1) collect tissues of interface membranes around implants to analyze ECSIT expression by immunofluorescence method; (2)shRNA macrophage model is constructed to test whether MAPK pathway or NF-κB pathway is involved in the regulation of mtROS by ECSIT; (3) animal model of ECSIT-/- mice is required to explore the effection of ECSIT gene defect on the prosthesis loosening situation . After the researches above, we hope to find out the correlation between ECSIT and mtROS in local periprosthetic inflammation and verify the mechanism of regulation of ECSIT on mtROS in the development of loosening .Therefore it may provide a scientific basis for ECSIT and mtROS as a target for prevention and cure of loosening.

假体松动是关节置换术后最主要的并发症，尚缺乏有效防治。已证实假体磨损颗粒刺激巨噬细胞导致慢性炎症，ROS过量产生可加重炎症反应，均与假体松动密切相关。申请者前期发现,松动假体的界膜中促进ROS生成的酶类表达增高，导致ROS产生过量从而加重假体松动。最新研究表明，ECSIT泛素化是mtROS产生的重要途径，可导致ROS总量增加并促进炎症反应。申请者预实验表明：磨损颗粒可上调ECSIT的表达及mtROS的产生。由此假设，ECSIT介导了磨损颗粒诱发的炎症反应并参与假体松动的发生。本研究拟利用免疫荧光技术检测假体界膜中ECSIT的表达情况；同时，通过建立shRNA巨噬细胞模型，观察ECSIT基因沉默对mtROS水平、MAPK与NF-κB通路活性的影响；并进一步探究ECSIT基因缺陷小鼠的假体松动情况，综合分析ECSIT调控mtROS在假体松动中的作用，为防治假体松动提供新靶点。

项目背景：假体松动是关节置换术后最主要的并发症。已证实假体磨损颗粒刺激巨噬细胞导致慢性炎症，ROS过量产生可加重炎症反应，均与假体松动密切相关。申请者前期发现松动假体的界膜中促进ROS生成的酶类表达增高，导致ROS产生过量从而加重假体松动。研究表明，ECSIT泛素化是mtROS产生的重要途径，可导致ROS增加并促进炎症反应。我们预实验证实：磨损颗粒可上调ECSIT的表达及mtROS的产生。由此假设，ECSIT介导了磨损颗粒诱发的炎症反应并参与假体松动的发生。另外，免疫耐受的调控因子（SOD、SPHKs、PDI等）在假体松动中的作用及机制，以及巨噬细胞自噬／凋亡对于磨损颗粒诱导的ROS、慢性炎症的影响和具体机制仍不明确，需进一步研究。.研究内容：探究了ECSIT调控相关氧化途径导致的炎症、免疫改变在假体松动发生发展过程中的相关作用和机制；发现假体松动中对ROS、慢性炎症、免疫耐受的调控因子（SOD、SPHKs、PDI等）的作用及机制，进一步研究发现磨损颗粒诱导的慢性炎症、凋亡异常可能是受巨噬细胞自噬的增强所调控；巨噬细胞的炎症活性改变损害假体周围组织的局部先天免疫力，导致炎症性骨溶解吸收，进一步导致假体松动。.重要结果：磨损颗粒可通过ESCIT、SPHK2、PDI等在假体周围组织诱导ROS、慢性炎症的长期存在，释放多种炎性因子；通过对自噬的调节可进一步导致过多病理性的凋亡引起炎症反应，所致的免疫耐受、炎性骨溶解／吸收可能是假体松动发生的关键环节。相关自噬抑制剂可能是抑制假体无菌性松动的有效药物；通过干预ESCIT、SPHK2、PDI对假体周围组织的ROS、慢性炎症、自噬/凋亡及免疫耐受进行相对应的调控，可能抑制假体松动的发生。.关键数据及其科学意义：在前期证实ROS对假体松动的作用的基础上，我们通过本研究进一步探讨了假体周围组织ECSTI调控mtROS作用机制；我们的研究进一步发现了假体松动中ROS、慢性炎症／免疫耐受、自噬／凋亡的调控因子（SOD、SPHKs、PDI）的作用及机制，以上成果为ECSIT、SPHK2、PDI及mtROS、自噬调控作为防治关节假体松动的靶点提供新的科学依据。