Scara1受体在小胶质细胞吞噬和清除PrPSc中作用机制的研究

As we known ，the pathologic features that characterize prion diseases are the protease-resisitant pathogenic form of prion protein-PrPSc and the accumulation of fibrils or amyloid plaques . As macrophages of the central nervous system, the phagocytic capacity of microglia is well recognized, and it is possible that the activated microglia express several receptors involving in the adhension of cells and phagocytosis. However, the mechanism of microglial cells in gulfing and clearing PrPSc and fibrils or amyloid plaques is unclear. We hypothesis that the scavenger receptor Scara1 expressed on myeloid cells may as a key receptor in clearance of PrPSc and fibrils or amyloid plaques. To determine the role of Scara1 in this process, we use shRNA technique to construct a Scara1-deficient microglial cell line and gene recombination technology to establish a Scara1-deficient mice model in vitro and in vivo, respectively .In contrast, we construct the overexpression model of Scara1 by pharmacological upregulation as well as construction of Scara1 eukaryotic expression plasmid. The systematic study of the deficiency or overexpression of Scara1 on the influence for the phagocytic and scavenging capacity of microglia to PrPSc and fibrils or amyloid plaques illustrates the phagocytic mechanism of microglia in removing PrPSc and fibrils or amyloid plaques, which may be important for the basic regulatory mechanism of prion diseases and associated neurodegenerative diseases and provides a novel therapeutic target for prion diseases in vivo .

众所周知，病原因子PrPSc和淀粉样蛋白的蓄积是朊蛋白疾病的重要病理特征。已有研究证实，激活的小胶质细胞表面表达大量的受体,可以介导细胞粘着和胞饮作用。然而，参与小胶质细胞吞噬和清除PrPSc的受体及其作用机制尚不清楚。本课题以清道夫受体A族（Scara1）成员可能作为小胶质细胞吞噬和清除PrPSc和淀粉样蛋白的关键受体为切入点，通过shRNA干扰技术建立Scara1基因缺失的小胶质细胞模型,利用基因重组技术建立Scara1基因缺失小鼠，用于体内实验研究模型。通过药理方式和构建真核表达质粒，建立Scara1基因的过表达模型。系统的研究Scara1基因的缺失或过表达对小胶质细胞吞噬和清除PrPSc和淀粉样蛋白能力的影响，进而阐明小胶质细胞对PrPSc的吞噬和清除机制，为朊蛋白疾病的治疗靶位点筛选提供理论依据和技术支持。

Prion疾病是神经退行性疾病的一种，其主要特点是引起中枢神经系统的错误折叠蛋白的蓄积，海绵状病变以及小胶质细胞激活导致炎症反应。之前研究表明，Prion疾病中，小胶质细胞的激活可能是通过Scara1受体来实现的，且小胶质细胞激活后，就会启动固有免疫系统。本实验室前期研究发现，Prion疾病感染的模型中，炎症复合体NALP3可被激活，且可引起Caspase1的活化及IL-1b的分泌。而众所周知，自噬是机体分解代谢的过程，可以清除损伤的细胞器以及蛋白质的蓄积等，自噬也可清除Prion疾病中的部分PrPSc，减少其蓄积。已有研究报道，在多种神经退行性疾病中，自噬参与了炎症复合体的调控，但在Prion疾病中，NALP3和自噬之间的调控关系还不清楚。 经过本研究，我们发现抑制自噬可明显促进IL-1β的释放，而用基因沉默的技术抑制NALP3炎症复合体，则可明显的激活自噬。通过siRNA干扰TRIF和TLR4可明显减弱Prion疾病引起的自噬，而抑制Caspase1则可阻止其对TRIF的剪切，进而增强自噬。结果表明，TLR4-TRIF信号通路参与了Prion疾病调控的自噬。我们的研究结果显示，Prion疾病中，NALP3炎症复合体负调控自噬可能是通过激活Caspase1诱导的TRIF剪切来实现的。同时暗示，抑制Caspase-1的过度激活，可激活自噬，促进错误折叠蛋白的降解，可能为Prion疾病的治疗提供新的思路。