【作者】 张杰；

【导师】 辛永宁；

【作者基本信息】 青岛大学 ， 内科学（消化系病）， 2021， 硕士

【摘要】 研究目的:非酒精性脂肪性肝病(Non-alcoholic fatty liver disease,NAFLD)是以肝脏脂质过度蓄积为主要特征的临床病理综合征,已成为目前患病率最高的慢性肝脏疾病之一。肝组织活检虽为NAFLD诊断金标准,但其为有创性检查,患者接受度低,因此目前急需开发灵敏度高、特异性好的无创性检查方法。外泌体的数量及内容物在不同疾病状态下改变,使其逐渐成为疾病诊断的生物标志物。目前已有研究表明外泌体在非酒精性脂肪性肝炎患者、高脂饮食喂养小鼠和脂肪酸处理的肝细胞中均分泌增多,但是目前在NAFLD中外泌体的生物发生和诊断价值仍不明确。本研究的目的为探究外泌体在NAFLD发生发展过程中的生物发生机制及诊断价值。研究方法:(1)本研究使用三种方法,包括磷脂酰丝氨酸酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)、超速离心抽提外泌体后的纳米颗粒跟踪分析(nanoparticle tracking analysis,NTA)和蛋白质定量(bicinchoninic acid,BCA),检测NAFLD患者血浆外泌体水平,并比较三种方法的检测效能。(2)通过GEO数据库和基因富集分析,筛选到在NAFLD发生发展中与外泌体分泌相关的基因。(3)利用CRISPR/Cas9技术构建DRAM全身敲除的小鼠模型,使用BCA、NTA、ELISA、Western Blot四种方法检测小鼠血浆外泌体水平。(4)在Hep G2细胞中敲低或者过表达DRAM基因,提取外泌体后采用Western Blot、BCA的方法及直接采用ELISA法三种方法检测外泌体水平。(5)使用溶酶体LGALS3/半乳糖凝集素聚集实验和提取溶酶体后检测胞浆和溶酶体的组织蛋白酶B实验两种方法检测溶酶体膜通透性,给予小鼠注射巴佛洛霉素A1,一种溶酶体抑制剂,探究是否抑制溶酶体功能可逆转敲除DRAM基因对外泌体分泌的抑制效应。(6)通过免疫共沉淀和免疫荧光实验验证DRAM基因与STOM基因之间的相互作用。结果:(1)本研究发现ELISA、NTA和BCA三种方法在检测血浆外泌体水平方面具有类似的检测效能,NAFLD患者的血浆外泌体水平高于健康受试者。(2)通过GEO数据库和基因富集分析,发现DRAM基因在单纯性非酒精性脂肪肝人群中表达水平高,表达水平与肝损伤、脂肪变性相关指标呈正相关,与外泌体分泌相关基因在DRAM基因高表达组富集,DRAM可能是NAFLD患者中与外泌体分泌相关的基因之一。(3)结果显示,在敲除DRAM基因的小鼠模型和敲低DRAM基因的细胞模型中,DRAM基因的敲除和敲低均可下调肝细胞分泌的外泌体水平。(4)高脂饮食和脂肪酸刺激可导致肝细胞DRAM基因表达升高,增加肝细胞外泌体分泌。敲低DRAM基因可抑制脂肪酸诱导的外泌体分泌。(5)过表达DRAM可促进肝细胞溶酶体膜透化,沉默DRAM表达可逆转脂肪酸诱导的肝细胞溶酶体膜透化,使用巴佛洛霉素A1抑制溶酶体功能可回复小鼠敲除DRAM引起的外泌体分泌降低。(6)DRAM可与STOM相互作用,促进STOM溶酶体定位,敲低STOM可以抑制DRAM诱导的外泌体分泌。结论及意义:本研究表明,脂质可诱导肝细胞DRAM表达升高,表达升高的DRAM与STOM相互作用,促进STOM溶酶体定位,并诱导溶酶体膜透化,进一步促进肝细胞外泌体的分泌。本研究揭示了在NAFLD发生发展过程中肝细胞外泌体分泌的机制,为外泌体作为NAFLD患者新的生物标志物提供了理论基础。更多还原

【Abstract】 Objective: Non-alcoholic fatty liver disease(NAFLD)characterized by excessive fat deposition in liver cells refers to a clinical pathological syndrome,which becomes currently one of the most common chronic liver diseases.Although liver biopsy is the gold standard for the diagnosis of NAFLD,it is an invasive examination with low acceptance by patients.Therefore,there is an urgent need to develop a non-invasive examination method with high sensitivity and specificity.The amount and contents of exosomes change in different disease states,making them gradually become biomarkers for disease diagnosis.Previous studies have shown that exosomes are all significantly increased in patients with non-alcoholic steatohepatitis,mice treated with high-fat diet(HFD),and hepatocytes treated with fatty acids(FAs).However,the biogenetic and diagnostic value of exosomes in NAFLD is still unclear.The purpose of this study is to explore the mechanism of exosome biogenesis and its diagnostic value during the occurrence and development of NAFLD.Methods:(1)We used three methods,including nanoparticle tracking analysis(NTA),bicinchoninic acid(BCA)assays after exosome isolation using ultracentrifugation and phosphatidylserine Enzyme-linked immunosorbent assay(ELISA)to evaluate the level of plasma exosomes in patients with NAFLD,and the efficacy of the three methods was compared.(2)Genes related to exosome secretion in NAFLD were screened by GEO database and Gene Set Enrichment Analysis.(3)DRAM knockout mice were constructed using CRISPR/Cas9 technology,and the level of plasma exosomes in mice was determined by BCA,NTA,ELISA and Western Blot.(4)After knockdown or overexpression of DRAM in Hep G2 cells,three methods including Western Blot,BCA after exosome isolation and ELISA,which detected exosomes’ level directly,were used to detect the level of exosomes.(5)Lysosomal LGALS3/galectin puncta assay and detection of cathepsin B level in the cytoplasm and lysosome after lysosome extraction were used to determine lysosomal membrane permeability,and mice were injected with bafilomycin A1(a lysosomal inhibitor)to explore whether inhibition of lysosomal function can reverse the inhibitory effect of DRAM knockdown on exosome secretion.(6)Co-Immunoprecipitation experiment and Immunofluorescence assay were conducted to explore the interaction between DRAM and STOM.Results:(1)We found that NTA,BCA and ELISA have similar detection capability in detecting the number of exosomes,and the exosome level in NAFLD patients was significantly higher than that in healthy people.(2)By analyzing the GEO database and gene enrichment analysis,it is confirmed that DRAM was highly expressed in nonalcoholic fatty liver population,and that the expression was positively correlated with indicators related to liver injury and steatosis.And genes related to exosome secretion were enriched in the DRAM high expression group,which confirmed that DRAM might be one of the genes related to exosome secretion in NAFLD.(3)The results showed loss or knockdown of DRAM could down-regulate exosome secretion from hepatic cells using a knock-out mouse model and a knock-down cell model.(4)HFD and FAs can induce the expression of DRAM and increase the exosome secretion of hepatic cells.Knockdown of DRAM could reverse the increase of exosomes induced by FAs.(5)Overexpression of DRAM could promote the lysosomal membrane permeability of hepatocytes,and silencing DRAM could reverse FAs-induced lysosomal membrane permeabilization(LMP)and exosome secretion in hepatic cells.Inhibition of lysosomal function by baflomycin A1 could reverse the decrease of exosome secretion in DRAM knockout mice.(6)DRAM could interact with STOM,promote its lysosomal localization.Meanwhile,knocking down of STOM could inhibit DRAM-induced exosome secretion.Conclusions&Significance: The study illustrated that lipid-induced DRAM could interact with STOM,promote its lysosomal localization and induce LMP to further promote exosome secretion of hepatic cells in NAFLD.We not only revealed DRAM-mediated mechanism for exosome secretion from hepatic cells during the occurrence and development of NAFLD,but also provided a theoretical foundation for exosomes as a new biomarker for patients with NAFLD.更多还原