基于MMP-9/CXCR4靶向的多模态活体分子成像在系统性红斑狼疮肾损害的早期精准识别及作用机制研究

In lupus nephritis(LN), glomerular lesions often have different degrees of proliferation, such as mesangial cells and matrix. Degradation and reorganization of ECM can be regulate by MMPs, through hydrolysis the matrix components. Simultaneously, the expression of MMP-9 is regulated by SDF-1 / CXCR4 axis. Result of preliminary test showed that, the expression of MMP-9 is relate with the degree/time of the renal damage in Faslpr lupus mice. We also found the expression of MMP-9/CXCR4 in serum and kidney tissue can reveal the degree of the renal damage. Why it happened? The specific mechanism is unclear. As we all know, SDF-1 can be bind by CXCR4, so we consider that, perhaps through the PI3K/AKT signal pathway, SDF-1/CXCR4 axis regulate the expression of MMP-9, and thereby further affecting the degree/stage of the renal damage in LN mice. In our project, we will use multimodality molecular imaging, gene interference and gene transfection, to research the relationship between the expression of MMP-9/CXCR4 and the renal damage, to find out the target of renal damage in early stage, and the mechanism about the regulation of MMP-9 through by SDF-1/CXCR4 axis. To provide the theory basis for multiple target combination therapy in LN, and improve the prognosis of the LN patient.

目前已证实LN的肾脏损伤程度与肾小球系膜细胞和基质增生相关，MMPs可通过对基质成分的水解来调控ECM降解与重组，且MMP-9的表达受SDF-1/CXCR-4轴的调控。我们前期研究发现狼疮小鼠肾组织MMP-9信号强度表达与肾脏损害程度及肾损伤出现时间相关，且预试验亦发现狼疮小鼠肾组织及血清中MMP-9及CXCR4蛋白表达水平可反映肾脏损害程度，但具体机制尚不清楚。我们推测LN小鼠不同程度、不同时期的肾脏损伤可能与LN肾组织高表达的CXCR4通过与其配体SDF-1结合，并可能通过PI3K/AKT信号通路干预了MMP-9的表达所致。因此本课题利用多模态分子成像等技术，并通过基因干扰及基因转染等方法以期了解MMP-9/CXCR-4表达与肾损伤相关性、早期识别靶点以及SDF-1/CXCR-4轴调控MMP-9表达的可能机制，为将来多靶点联合疗法治疗LN、改善LN患者预后提供依据。

课题以系统性红斑狼疮模型小鼠为研究对象，借助分子成像技术活体水平研究疾病不同阶段肾组织损伤情况，并在体外实验中通过慢病毒、CXCR4拮抗剂等作用于肾小球内皮细胞、系膜细胞、足细胞，干扰MMP/CXCR4信号通路，观察其相关基因及蛋白的表达及调控机制，并进一步在体内实验中加以验证，从而深入探讨系统性红斑狼疮肾损害过程中SDF-1/CXCR4 轴对肾组织 MMP的调控机制及时间节点，进一步揭示系统性红斑狼疮肾脏损害机制，并为精准治疗提供新的思路及依据。实验结果：①活体成像结果显示， MMP的信号表达主要来自于Faslpr小鼠的肾脏组织，且信号强度显著高于Fas+小鼠，提示MMP在Faslpr小鼠的肾脏组织存在高表达。离体器官成像结果进一步证实，MMP在Faslpr小鼠肾脏的表达水平较Fas+小鼠显著升高，且仅定位于肾脏，在其他器官中未检测到明显的信号表达。②原代分离培养的肾小球内皮细胞和足细胞纯度较高，系膜细胞的纯度较低。干扰CXCR4表达的慢病毒显著降低了肾小球内皮细胞中CXCR4的mRNA的表达。在肾小球内皮细胞、系膜细胞和足细胞中CXCR4干扰表达慢病毒转染组、T140处理组、AMD 3100处理组和LY294002处理组中CXCR4、Collagen IV、MMP-9、PI3K、Rac1和VCAM-1的蛋白质表达水平均低于对照组。对照组中蛋白质表达和空载慢病毒转染组差别不显著。故抑制CXCR4活性能够降低原代分离培养的肾小球细胞的肾纤维化相关基因表达，即使在非炎症条件下此效果依然有效。③CXCR4干扰或者使用CXCR4抑制剂和PI3K抑制剂显著改善了系统性红斑狼疮肾组织的病理损伤，减少了肾小管上皮细胞坏死、脱落，免疫组化和Western blot都显示这些抑制剂都显著下调了红斑狼疮模型大鼠中CXCR4、MMP-9的表达，且Western blot结果显示这些抑制剂显著下调PI3K、VCAM-1和Collagen IV的蛋白表达水平，上调了 Rac1的蛋白表达水平，增加了血清中补体蛋白C3的含量。MMP-9/CXCR4的表达与红斑狼疮肾损害程度正相关，且CXCR4可能通过PI3K信号通路干预MMP-9的表达，且调控了PI3K、VCAM-1和Collagen IV的表达水平，进而参与红斑狼疮肾损害过程。