基于瘦素基因敲除大鼠模型的非酒精性脂肪肝的致病机制研究和实验性治疗

Nonalcoholic fatty liver disease (NAFLD) has become one of the most common liver diseases. Leptin (Lep) is an important factor during the pathogenesis of NAFLD due to its close correlation with obesity, type 2 diabetes and insulin resistance, etc. However, the detailed mechanism by which Lep involves in NAFLD is not clear yet. In our previous work, we have already used the CRISPR genome editing technology to generate Lep-knockout rat models which showed NAFLD phenotypes. In this proposed research, we will examine a variety of physiological and biochemical parameters of the animals during NAFLD pathogenesis. And we will verify whether Lep mediates NAFLD through the central nervous system or the peripheral system by ELISA and immunohistochemistry methods. Meanwhile, we will perform RNA-Seq and transcriptome analysis on liver tissues of the rats, in which we will examine the differential expression of enzymes and proteins which are related to hepatic lipid metabolism and possibly other metabolic pathways, and will identify new biological targets for NAFLD. Then, we will directly modulate the expression of the known and potential targets at transcriptional level by the CRISPR transcriptional regulation technology. Finally we will validate the function of the potential biological targets, and evaluate their efficacy on the treatment of NAFLD. This research will elucidate the pathogenic mechanism of Lep mediated NAFLD, discover and verify new biological targets, and eventually provide a strong theoretical and technical support for the treatment of NAFLD.

非酒精性脂肪肝（NAFLD）已经成为最普遍的肝脏疾病之一。瘦素（Lep）与肥胖症、2型糖尿病以及胰岛素抵抗等紧密关联，在NAFLD的发生发展过程中起着重要作用，然而具体致病机制尚不明确。申请人已经成功制作具有NAFLD表型的Lep基因敲除大鼠模型。本研究将在此基础上鉴定大鼠模型NAFLD的发病进程，从而明确Lep通过中枢或者外周系统介导NAFLD的分子机制。同时，将对大鼠模型的肝组织进行转录组分析，进一步解析脂肪代谢以及其他代谢途径相关的酶和蛋白的表达差异，寻找新的生物靶标。最后，本研究将通过CRISPR基因转录调控技术直接在肝脏中对已知和新发现的候选生物靶标进行表达调控，验证这些生物靶标在NAFLD发病中的分子机制，并分析和评价这些NAFLD治疗方案的效果。本研究将阐明Lep在NAFLD中的致病机制，为NAFLD的临床治疗提供新的理论和技术基础。

非酒精性脂肪性肝炎（NASH）是一种严重威胁公众健康的肝病，但其发病进程尚不明确。已知瘦素（LEPTIN，LEP）是主要由白色脂肪细胞分泌的一种激素，对调节人和动物的饱腹感和能量稳态至关重要。我们首先利用CRISPR/Cas9技术构建了一株Lep突变大鼠模型，该大鼠的LEP蛋白缺失了第14位异亮氨酸（LEP∆I14）。由于反馈回路的作用，成熟的LEP∆I14不断以更高的水平释放到血液中。通过计算模拟突变型LEP∆I14与LEP受体（LEPR）的结合构象，发现LEP∆I14的构象变化导致其与LEPR的结合减弱。因此，在荧光素酶报告实验中，LEP∆I14无法激活下游的STAT3信号通路。表型研究表明，与Lep缺失突变大鼠的表型一致，Lep∆I14/∆I14大鼠具有肥胖、高胰岛素血症、脂肪肝、糖尿病肾病以及不孕不育等一系列症状。..与ob/ob小鼠不同，Lep∆I14/∆I14大鼠在出生后第16周出现独特的NASH表型，包括脂肪变性、淋巴细胞浸润和气泡样等。我们以Lep∆I14/∆I14大鼠为NASH模型，综合分析了出生后4-48周肝脏转录组的动态变化，从而研究NASH的发病进展。我们发现脂代谢中编码ACC和FASN等9种限速酶的基因表达显著上调。然而，炎症正调控相关基因的表达在16周达到高峰，然后一直维持稳定表达，直到第48周。CCL2、TNFα、IL6和IL1β等细胞因子和趋化因子高表达，并与JNK和NF-κB途径中关键分子的磷酸化相关。Lep∆I14/∆I14大鼠肝脏中MPO+中性粒细胞、CD8+ T细胞、CD68+巨噬细胞和CCR2+炎性单核细胞源性巨噬细胞的浸润增加。同时，浸润的巨噬细胞呈现从M2到M1的极化。与众多已建立的小鼠模型和食蟹猴自发性NAFLD模型相比，Lep∆I14/∆I14大鼠与NASH患者共享更多上调或下调的同源基因。转录组学分析表明，许多临床试验中的药物靶点可以在Lep∆I14/∆I14大鼠模型上进行评价。..综上所述，通过长时程（出生第4至48周）的整合转录组分析，本研究构建和鉴定了新型Lep∆I14/∆I14大鼠NASH模型。同时，本研究揭示了NASH进程中肝脏脂代谢和炎症相关基因的动态表达，对理解人类NASH疾病进程和新药研发具有重要的理论意义和应用价值。