【作者】 陈璐；

【导师】 谢青；

【作者基本信息】 上海交通大学 ， 内科学（感染病学）， 2015， 博士

【摘要】 背景慢性乙型肝炎、药物性肝损害等其他多种因素可造成我国患者肝脏损伤,在肝损伤发生过程中患者个体之间差异较大,同种病因往往可导致完全不同的转归,因此其他潜在的造成这种个体差异的因素需要进一步探究。角蛋白是三大细胞骨架蛋白中间丝蛋白的最大亚群,它在肝细胞中与肝细胞结构、功能密切相关,特别是研究发现角蛋白基因突变可导致宿主容易造成肝脏损伤,因此它是否作为一种潜在因素影响着患者不同的转归,有待进一步研究。肝脏特异性角蛋白K18 R90C突变造成其结构改变,使表达该类角蛋白的肝细胞变得脆弱且使宿主容易发生肝脏损伤。寻找能够逆转突变角蛋白结构的方法,从而恢复其结构功能,是保护及治疗肝脏损伤的一种新方法。目的本研究希望能够找到逆转突变角蛋白结构的小分子物质,稳定其突变结构,恢复角蛋白功能并为肝脏损伤提供保护,并研究相关小分子物质的保护机制,进一步阐述角蛋白结构改变的原因。本研究为肝脏损伤提供了新的治疗前景,同时也为以肝损伤为代表的“中间丝蛋白相关疾病”的治疗提供了新的方向。并且本研究为将来进一步探究角蛋白是否与肝损伤个体差异有关,以及我国患者中角蛋白突变情况的研究提供了依据。方法本研究将通过以免疫荧光图像为基础的小分子药物筛选方法,寻找能够稳定突变角蛋白结构的小分子物质,在突变细胞及转基因小鼠中分别验证该小分子的结构稳定作用,并将之作为预防性药物检测其在突变细胞和小鼠中的肝损伤保护作用,并进一步通过免疫组化、免疫共沉淀以及重建实验探究小分子物质的作用机制。结果PKC412是一种多蛋白激酶抑制剂,本研究发现它能够改变K18 R90C突变结构,在荧光显微镜下观察发现它能使突变结构恢复到正常的细胞骨架蛋白结构。并且PKC412能够为突变细胞及转基因小鼠提供保护,与未经PKC412处理的细胞相比,其能够减少细胞凋亡相关信号的表达,而与对照组小鼠相比,它能够大大减少急性肝损伤的发生(PKC412处理小鼠平均血清ALT水平为266 U/L,而对照组小鼠平均ALT水平为6546 U/L,P<0.05,且H&E染色表明PKC412能够减少小鼠急性肝损伤后肝脏出血)。本研究中还进一步探究了PKC412产生保护作用的机制,发现PKC412是通过改变非肌肉型肌球蛋白重链(NMHC)IIA的磷酸化程度,从而调节其与角蛋白之间的结合程度来改变突变角蛋白结构的,而研究中的重建实验进一步表明,PKC412作为蛋白激酶抑制剂对NMHC IIA的磷酸化改变而非对角蛋白的磷酸化改变是其发挥作用的关键。结论本文发现了多蛋白激酶抑制剂PKC412能够逆转突变角蛋白结构,并且能够为突变细胞提供凋亡保护,同时也能减少带有突变的转基因小鼠的急性肝损伤。PKC412的保护作用与其促进K8/K18和NMHC IIA的结合相关,并且受到NMHC IIA的磷酸化调节。更多还原

【Abstract】 Background There are a lot of different etiologies could result in the human liver injury, and among those the Hepatitis B is the major reason in China. Regardless of Hepatitis B or other etiologies, like DILI, NASH or biliary diseases, there could be huge individual differences of the outcomes from the patients with the same etiology, which suggests that other potential reasons may contribute to it. Keratin is the biggest family of the intermediate filaments, and is found play an important role in the liver injury. We hypothesis that keratin maybe one of the reason to trigger individual differences of the liver injury, and it also will provide a new treatment area of the liver injury through keratin. Liver specific keratin 8 and 18 mutation could result in the keratin structure disruption in the hepatic cells, and make the cells easy to be apoptotic and the transgenic mice predispose to the liver injury. Exploring the treatment for the keratin 8/18 mutation-related predisposition to liver injury may could provide a new way for the treatment for the liver injury.Aim In this study, we will focus on the liver specific keratin 8/18 mutation mediated structure disruption, using the drug screening to find small molecular that could stable the mutated keratin structure, thereby rescue the keratin function and provide the protect for the cells and mice. This study will provide a new treatment idea to the Intermediate filament-related diseases and more importantly, it will give the different outcomes of the liver injury a potential reason and show a new treatment area for the liver injury.Methods We will use the imaged-based drug screening, which relied on the structure changes seen by immunoflorensence after the drug use. The hits will be confirmed in the mutated cells and transgenic mice, and we will also detect the mechanism of the small molecular function.Results In this paper, we demonstrate that PKC412, which is the multi-kinase inhibitor, could stable the mutated K18 R90 C structure and provide the protection in the mutated cells and transgenic mice form apoptosis and acute liver injury. And we also show that the mechanism of PKC412 is to induce the hypophosphorylation of non-muscle myosin IIA and trigger its binding to the keratins.Conclusions We defined PKC412 as the compound that could alleviate mutated keratin structure, and provide protection for the mutated cells and mice from apoptosis and acute liver injury. The mechanism of under that is because that PKC412 could increase keratin and NMHC-IIA binding, which also related to NMHC-IIA’s phosphorylation.更多还原