IGFBP3参与酒精性肝细胞损伤的分子机制研究

Alcoholic consuming is one of the main factors causing liver injure, and there is no effective and specific agent for patients. IGFBP3 is closely related to liver metabolism, but its role and regulation mechanism in alcoholic liver disease (ALD) are still unclear. Our previous studies showed that IGFBP3 was significantly down-regulated after alcoholic treatment both in vitro and in vivo, and the overexpression of IGFBP3 in cells would enhance alcohol-induced hepatocyte injury, suggesting that hepatocyte may resist the influences of alcohol by inhibiting IGFBP3. Numerous studies have shown that IGFBP3 can regulate Akt and Caspase signals, respectively, via an IGF1-dependent pathway and an IGF1-independent pathway that directly binding membrane receptor TMEM219. This study aims to carry out point mutation on IGF1 binding site of IGFBP3, and then overexpress wild-type and mutant IGFBP3 in cells and mice by the methods of transfection or adenovirus tail intravenous injection, meanwhile, the TMEM219 transgenic mice also be used to investigate both pathways. This project may shed light on the identification on novel specific therapeutic targets of ALD.

酒精是造成肝脏损伤的重要因素之一，目前尚缺乏有效的特异性治疗药物。IGFBP3与肝脏代谢密切相关，但在酒精肝（ALD）中的作用和调控机制还不清楚。申请人前期研究发现，IGFBP3在ALD的细胞和动物模型中都有明显的下调，而在细胞中过表达IGFBP3会促进酒精引起的肝细胞损伤，提示在酒精肝中机体可能通过下调IGFBP3来缓冲酒精对肝细胞的影响。IGFBP3在细胞中能通过IGF1依赖或直接结合膜受体TMEM219的IGF1非依赖途径，分别调控下游的Akt和Caspase通路信号。本项目拟通过对IGFBP3的IGF1结合位点进行点突变，再用转染和腺病毒尾静脉注射的方法在体外和体内ALD模型中对野生型和突变型的IGFBP3进行过表达，以及结合TMEM219转基因小鼠对两条途径展开研究。以期能明确IGFBP3在ALD中的具体作用和调控机制，寻找到新的治疗靶点和为ALD的发生发展提供一定的理论支撑。

酒精性肝病（alcoholic liver disease, ALD）是由长期大量饮酒导致的一类广谱性疾病，包括酒精性脂肪肝、肝炎、肝纤维化、肝硬化、肝癌等，严重损害人民身体健康并给社会带来巨大的经济负担。随着我国经济的发展， ALD的发病率呈现逐年上升，已成为病毒之后导致肝损伤的第二个大病因。目前，除了戒酒及加强营养支持外，临床上尚缺乏有效和特异性的酒精性肝损伤治疗药物。IGF系统在肝脏生理及病理活动中起着重要的作用，通过对细胞和动物ALD模型的IGF系统家族各成员的表达情况进行分析，发现酒精能导致IGFBP3的表达发生明显下调。通过质粒转染和慢病毒尾静脉注射以及构建转基因小鼠的方法，分别在细胞和小鼠中过表达或敲除IGFBP3，检测细胞的增殖、凋亡及脂代谢等指标，发现过表达IGFBP3能促进酒精对肝细胞增殖的抑制，诱导细胞的凋亡，提升肝细胞内ROS的水平，促使脂滴的堆积，而敲除IGFBP3能缓解酒精对肝细胞的影响。对IGFBP3的IGF1特异性结合位点进行点突变，发现IGFBP3可以通过IGF1依赖和非依赖两条途径，调控下游Akt和caspase通路信号。此外，RNA-seq结果显示，IGFBP3主要通过影响脂代谢相关通路应该ALD的发生发展。综上，IGFBP3以期能够通过IGF1依赖和非依赖两种途径介导ALD的发生，深入理解IGFBP3及其相关通路蛋白的作用，可以为ALD治疗提供新的靶点，并对进一步认识ALD发病机制和防治ALD提供坚实的理论基础