【作者】 何莉；

【导师】 张巍；

【作者基本信息】 华东师范大学 ， 神经生物学， 2024， 硕士

【摘要】 肝血窦是由特化有孔内皮细胞构成的不连续血管,缺乏基底膜,具有独特的结构表型,称为“窗孔”（fenestra）。肝血窦内皮细胞（liver sinusoidal endothelial cells,LSECs）上的窗孔直径与数量在脂蛋白代谢、药物代谢、免疫调节、衰老、营养调节（如过度饮食、饥饿）与疾病（如肝纤维化、肝癌）等生理和病理过程中发挥重要作用。因此,研究其胚胎起源及形成机制对理解肝脏发育、再生和肝脏相关重大疾病（如肝癌）的防治具有重要意义。肝血窦发育过程复杂,需要依赖特定时空表达的蛋白来精准调控,但确切机制仍不清楚,亟待进一步研究。ADAMTS（A Disintegrin-like And Metalloproteinase with Thrombospondintype 1 motifs）是一类含I型血小板反应蛋白结构域的解整合素金属蛋白酶,目前在人类已发现19个家族成员,其主要作用是切割或修饰组织微环境中的细胞外基质（Extracellular matrix,ECM）成分,在器官发育、炎症及肿瘤等多种病理生理过程中发挥重要作用。ADAMTS18于2002年被首次发现命名,包括我们课题组在内的一些国内外研究团队发现:ADAMTS18主要表达于胚胎发育早期,调控众多器官的早期形态发生。值得注意的是,在人体中ADAMTS18在肝脏中的表达仅出现在胚胎期,Adamts18基因突变或低表达与肝癌的不良预后相关。这也促使我们利用已构建的Adamts18基因敲除小鼠(Adamts18^-/-),进一步探究ADAMTS18在哺乳动物肝脏发育中的作用及其与肝脏疾病间的关联。研究方法:（1）采用RNA原位杂交（In Situ Hybridization,ISH）与q RT-PCR（quantitative Real-Time PCR）的方法检测Adamts18 m RNA在小鼠肝脏中的表达部位与表达水平。（2）利用过碘酸雪夫染色（Periodicacid-Schiff,PAS）检测小鼠肝脏组织的糖原贮存情况,通过油红O（Oil Red O）染色检测小鼠肝脏组织的脂滴堆积情况,用以评估ADAMTS18缺失对小鼠肝脏葡萄糖与脂质代谢的影响。（3）利用肝脏血管铸型与扫描电子显微镜（Scanning electron microscope,SEM）观察ADAMTS18缺失对小鼠肝脏大血管与肝窦形态结构的影响。（4）通过对肝脏组织切片进行免疫组化（Immunohistochemistry,IHC）,原代分离小鼠LSECs并进行细胞免疫荧光（Immunofluorescence,IF）与免疫印迹分析（Western blotting,WB）探究ADAMTS18缺失影响肝血管发育的分子机制。（5）制备阿霉素（Adriamycin,ADR）诱导的肝纤维化模型,评估ADAMTS18缺失对肝纤维化的影响。研究结果:（1）Adamts18 m RNA在E13.5与出生后2周（W）在肝脏血管内皮处均有表达,且2W表达量相较于E13.5表达显著下调。（2）2周龄（2W）与3月龄（3M）Adamts18^-/-小鼠肝脏中脂质堆积程度均显著高于Adamts18^+/+小鼠,但两种基因型小鼠肝脏功能及糖原代谢水平未见显著差异。（3）2W与3M龄Adamts18^-/-小鼠LSECs窗孔形态与分布较Adamts18^+/+小鼠均出现明显异常,表现为窗孔平均直径与最大直径的显著增大、孔隙率显著增大与开窗频率显著下降,但两种基因型小鼠肝脏大血管形态与分支情况未见显著差异。（4）Adamts18^-/-小鼠肝脏门静脉（Portal vein,PV）与肝窦处较Adamts18^+/+小鼠出现显著纤连蛋白（Fibronectin,FN）沉积,Adamts18^-/-小鼠LSECs中细胞骨架相关的Rho A-GTP与p MLC水平显著高于Adamts18^+/+小鼠。（5）ADAMTS18缺失并未引起小鼠发生自发性肝纤维化,但经ADR药物处理后,Adamts18^-/-小鼠出现较Adamts18^+/+小鼠更为严重的胶原沉积及肝细胞双细胞核现象。研究结论:ADAMTS18调控了肝血窦内皮开窗结构的形态发生,其缺失导致肝脏脂质代谢异常和对药物引起的肝纤维化损伤更加敏感。这可能部分归因于开窗结构异常造成的肝脏脂质吸收异常和微环境改变。意义:本研究拓展了ADAMTS蛋白家族的生物学功能,也为理解早期肝脏血管形成新机制提供了有益补充。更多还原

【Abstract】 Liver sinusoids are discontinuous vessels composed of specialised perforated endothelial cells that lack a basement membrane and have a unique structural phenotype called fenestra.The number and diameter of fenestra on liver sinusoidal endothelial cells（LSECs）play important roles in physiological and pathological processes such as lipoprotein metabolism,drug metabolism,immune regulation,ageing,nutrient regulation（e.g.overeating,starvation）and diseases（e.g.liver fibrosis,hepatocellular carcinoma）.Therefore,studying its embryonic origin and formation mechanism is of great importance for understanding liver development,regeneration and the prevention and treatment of major liver diseases（e.g.hepatocellular carcinoma）.The development of hepatic sinusoids is a complex process that depends on the precise regulation of specific spatiotemporally expressed proteins.However,the exact mechanism remains unclear and requires further investigation.ADAMTS（A Disintegrin-like And Metalloproteinase with Thrombospondintype 1 motifs）is a class of disintegrin metalloproteinases that contain a type I platelet-responsive protein motif,of which 19 family members have been identified in humans,which cleave or modify components of the extracellular matrix（ECM）in the tissue microenvironment and play an important role in a variety of pathophysiological processes including organ development,inflammation and tumourigenesis.ADAMTS18 was first discovered and named in 2002,and a number of national and international research teams,including our group,have found that ADAMTS18 is mainly expressed in early embryonic development and regulates the early morphogenesis of numerous organs.Notably,in humans,ADAMTS18 expression in the liver occurs only in the embryonic stage,and mutation or low expression of the Adamts18 gene correlates with poor prognosis in hepatocellular carcinoma.This has prompted us to further investigate the role of ADAMTS18 in mammalian liver development and its association with liver disease using Adamts18 knockout mice(Adamts18^-/-).Methods:（1）The expression location and level of Adamts18 m RNA in mouse liver was determined by RNA in situ hybridization（ISH）and q RT-PCR（quantitative real-time PCR）.（2）To evaluate the effects of ADAMTS18 deficiency on glucose and lipid metabolism in mouse liver,glycogen storage in mouse liver tissue was detected by Periodic Acid-Schiff（PAS）staining and lipid droplet accumulation in mouse liver tissue was detected by Oil Red O staining.（3）The effects of ADAMTS18 deletion on the morphology and structure of large blood vessels and hepatic sinusoids in mouse liver were observed by hepatic vascular casting and scanning electron microscopy（SEM）.（4）The molecular mechanism by which Adamts18 deletion affects hepatic vascular development was investigated by immunohistochemistry of liver tissue,primary isolation of mouse LSECs and immunofluorescence and immunoblotting analysis.（5）A model of Adriamycin（ADR）-induced hepatic fibrosis was established to evaluate the effect of ADAMTS18 deletion on hepatic fibrosis.Results:（1）Adamts18 m RNA was expressed at the endothelium of the hepatic vasculature at both E13.5 and 2 weeks postnatal,and the expression of 2-week-old（2W）was significantly down-regulated compared to E13.5.（2）Lipid accumulation in the livers of 2W and 3-month-old（3M）Adamts18^-/-mice was significantly higher than that of Adamts18^+/+mice,but no significant differences were observed in liver function and glycogen metabolism between the two genotypes.（3）The morphology and distribution of LSECs in 2W and 3M Adamts18^-/-mice showed significant abnormalities compared with that of Adamts18^+/+mice,as evidenced by a significant increase in the average and maximum diameters of the fenestration,a significant increase in the porosity,and a significant decrease in the frequency of fenestration,but there were no significant differences in the morphology of the large blood vessels and their branching in the livers of the two genotypes.（4）Adamts18^-/-mice showed significant deposition of fibronectin（FN）in the portal vein（PV）and hepatic sinusoids compared with Adamts18^+/+mice,and the levels of cytoskeleton-associated Rho A-GTP and p MLC were significantly higher in the LSECs from the livers of Adamts18^-/-mice than in Adamts18^+/+mice.（5）ADAMTS18 deficiency did not cause spontaneous hepatic fibrosis in mice,but Adamts18^-/-mice showed more severe collagen deposition and hepatocyte bicellular nucleation than Adamts18^+/+mice after treatment with ADR drugs.Conclusions:ADAMTS18 regulates the morphogenesis of liver sinusoidal endothelial fenestra structures,and its absence leads to abnormalities in hepatic lipid metabolism and an increased susceptibility to drug-induced fibrotic liver injury.This may be due,in part,to abnormal hepatic lipid uptake and to changes in the microenvironment caused by the abnormal fenestration.Significance:This study extends the biological functions of the ADAMTS family of proteins and also provides a useful addition to understanding novel mechanisms underlying early hepatic angiogenesis.更多还原