NAFLD特异性功能核酸适体的靶标确定及功能研究

Non alcoholic fatty liver disease(NAFLD) is the first cause of chronic liver disease in the world, and the prevalence is still rising rapidly. There has neither specific nor effective treatment for NAFLD. We used Cell-SELEX to obtain a specific functional nucleic acid aptamer NAFLD01 for NAFLD cells, which can improve the degree of steatosis after combined with NAFLD cells.Confocal microscopy showed that NAFLD01 was localized to the cell membrane after binding to the target cells, and this specific binding could be digested by proteases.Thus, we propose that NAFLD01 may regulate the signaling pathways associated with lipid metabolism to improve hepatic steatosis in NAFLD cells. Our project using NAFLD01 as a molecular probe, explore its binding targets, and then identified by mass spectrometry; improve the function and mechanism of NAFLD01 and target protein binding in the further study. This study is to explore the pathogenesis of NAFLD from a new perspective, and is expected to provide an effective molecular tool for the treatment of NAFLD.

非酒精性脂肪性肝病(NAFLD)是全球慢性肝病的首位病因，且患病率迅速攀升。目前尚无特异性治疗该病的有效方法。我们采用Cell-SELEX技术获得了一条特异性针对NAFLD细胞的功能核酸适体NAFLD01，它在与NAFLD细胞结合的同时能改善其脂肪变性的程度。通过激光共聚焦成像发现NAFLD01与靶细胞结合后定位于细胞膜上，而且这种特异性结合能被蛋白酶所消化。由此，我们提出核酸适体NAFLD01可能通过结合NAFLD细胞膜上某种与脂代谢相关的膜蛋白，调控相关信号通路，改善肝细胞脂肪变性的科学假说。本项目以NAFLD01为分子探针，探寻其结合的靶标，并利用质谱技术进行鉴定；进一步研究NAFLD01与靶蛋白结合后改善肝细胞脂肪变性的作用及机制；本研究从一个新的角度探索NAFLD相关发病机制，并有望为治疗NAFLD提供有效的分子工具。

非酒精性脂肪性肝病(NAFLD)是全球慢性肝病的首位病因，且患病率迅速攀升。目前尚无特异性治疗该病的有效方法。我们采用Cell-SELEX技术获得了一条特异性针对NAFLD细胞的功能核酸适体NAFLD01，它在与NAFLD细胞结合的同时能改善其脂肪变性的程度。通过激光共聚焦成像发现NAFLD01与靶细胞结合后定位于细胞膜上，而且这种特异性结合能被蛋白酶所消化。由此，我们提出核酸适体NAFLD01可能通过结合NAFLD细胞膜上某种与脂代谢相关的膜蛋白，调控相关信号通路，改善肝细胞脂肪变性的科学假说。我们前期实验中证实了核酸适体NAFLD01的靶标类型为细胞膜蛋白，然后通过提取膜蛋白的方法提取出蛋白质，用核酸适配体介导的亲和富集实验即aptamer-pull down实验富集出NAFLD01所结合的靶标蛋白，经SDS-PAGE分离后，发现潜在靶标蛋白所在的差异条带，用质谱法分析NAFLD01作用下NAFLD细胞的不同膜蛋白，确定候选靶点，并检测NAFLD01与靶点蛋白沉默的NAFLD细胞的亲和力，验证其有效性。CD36敲除NAFLD01的结合，其结合亲和力与膜结合CD36相关。NAFLD01与NAFLD细胞的亲和力与CD36表达水平成正比。此外，与随机序列相比，NAFLD01对小鼠和人的NAFLD组织切片都有更好的识别能力。重要的是，在体外，NAFLD01可以通过与CD36相互作用改善肝脏脂肪沉积。因此，适配体NAFLD01可以作为一种有效、安全的NAFLD靶向药物。NAFLD01是第一个报道的CD36特异性适配体。该适配体可通过特异性结合CD36改善肝细胞脂肪变性。本研究为我们研究CD36在NAFLD中的作用机制提供了分子工具。