蛋白酪氨酸磷酸酶SHP-1在非酒精性脂肪性肝炎中的作用及其机制研究

Inflammation is an important factor for the occurrence and development of non-alcoholic steatohepatitis (NASH). Src-homology domain 2-containing protein tyrosine phosphatase-1 (SHP-1) is a negative regulator of multiple inflammatory related signal pathways, but the role of SHP-1 in NASH has not been reported. Our previous studies revealed that the expressions of inflammatory cytokines in the liver were remarkably upregulated in the hepatocyte-specific SHP-1 knock-out mice. These results suggest that SHP-1 is involved in the regulation of inflammation in the liver. In this study, the hepatocytes from the hepatocyte-specific SHP-1 knock-out mice and the control mice will be isolated and stimulated with oleic acid or fructose firstly. The effects of SHP-1 knock-out on hepatocytes steatosis and the inflammatory related signal pathways will be evaluated. Secondly, the isolated hepatocytes will be infected with the adenovirus expressing SHP-1 (AdSHP-1) while stimulated with oleic acid or fructose. The effect of SHP-1 upregulation on hepatocytes steatosis and inflammation will be further studied. Thirdly, the role of SHP-1 knockout on the occurrence and development of NASH would be studied in vivo with the SHP-1 conditional knockout mice. And the therapeutic effects of AdSHP-1 on NASH will be further investigated with the rat NASH model. Our study will ascertain the role of SHP-1 in NASH and provide a new method and the basic evidence for the treatment of NASH.

炎症是影响非酒精性脂肪性肝炎（NASH）发生发展的一个重要因素。含SH2结构域蛋白酪氨酸磷酸酶-1 (SHP-1)是多种炎症相关信号通路负调控因子,但SHP-1在NASH中的作用迄今尚未报道。本课题组前期研究发现，肝细胞特异SHP-1敲除小鼠肝组织中炎症细胞因子IL-6、TNF-α等表达显著上调，提示SHP-1参与调控肝脏炎症反应。本课题将分离肝细胞特异SHP-1敲除小鼠肝细胞及对照细胞，观察油酸或果糖刺激下SHP-1缺失对肝细胞脂肪变性、炎症相关信号通路等影响，并利用腺病毒载体上调SHP-1表达（AdSHP-1），探讨SHP-1对肝细胞脂肪变性的抑制作用；利用SHP-1条件缺失小鼠，体内观察SHP-1缺失对NASH发生发展的影响及AdSHP-1对NASH的治疗作用和机制。该研究将明确SHP-1在NASH中的作用，为临床NASH治疗提供新的思路及理论依据。

炎症是影响非酒精性脂肪性肝炎（NASH）发生发展的一个重要因素。含SH2结构域蛋白酪氨酸磷酸酶-1（SHP-1）是多种炎症相关信号通路负调控因子。本研究首先分离培养小鼠原代肝细胞，利用油酸诱导肝细胞发生脂肪变性，结果发现AdSHP-1感染后能抑制肝细胞脂肪变性，抑制肝内炎症相关基因TGFβ、TNFα、IL-6、IL-10、TLR2、TLR4表达，同时能下调脂肪合成相关基因ACC、FAS、SREBP、PPARγ表达，而上调脂肪分解指标PPARα和CPT表达。体内利用肝细胞特异性SHP-1敲除小鼠（Ptpn6H-KO），给予MCD饮食喂养4 W构建NASH模型，结果发现SHP-1敲除后肝脏脂肪沉积较对照组（Ptpn6f/f）明显加重，胶原沉积增多；real-time RT-PCR及免疫组化结果显示SHP-1敲除后肝内炎症加重，炎症相关指标IL-6、TGFβ、TNFα、TLR2及TLR4表达均明显上调，脂肪代谢相关基因ACC、SCD1、PPARγ表达明显上调，而CPT、PPARα、SREBP表达则下调，提示肝脏脂肪代谢更加紊乱。最后，我们经尾静脉注射AdSHP-1或对照病毒AdGFP，观察其对MCD诱导小鼠NASH治疗作用，结果发现与对照病毒组相比，AdSHP-1治疗能明显改善肝内脂肪沉积，肝组织内炎性细胞浸润明显减轻，胶原沉积减少；real-time RT-PCR及免疫组化结果显示上调SHP-1表达能抑制肝内炎症相关基因IL-6、IL-10、TGFβ、TNFα、TLR2及TLR4表达，同时改善肝脏脂肪代谢紊乱。本研究进一步明确SHP-1在NASH发生发展中的作用，为临床NASH治疗提供了新的思路及理论依据。