| Objective:To use mouse primary hepatocytes to induce culture in 3D matrix gel into liver organoids,which have the basic functions of the liver and can store glycogen and uptake low-density lipoprotein(LDL).Organoids were used to model liver aging in vitro.Oleic acid(OA)is a monounsaturated fatty acid,and liver organoids treated with oleic acid show aging characteristics,and the model can be used to further explore the specific causes of liver aging caused by oleic acid.Methods:(1)Establishment of liver organoid culture system:mouse primary hepatocytes were obtained by in-situ digestion by two-step perfusion method of typeⅣ collagenase,and they were planted in Matrix Gel for 3D and grown into liver organoids under the culture of induction medium.(2)Phenotypic and functional identification of liver organoids:the expression of liver markers was detected by realtime quantitative PCR(qPCR),immunofluorescent staining(IF),hematoxylin-eosin staining(HE),etc.;The ability of liver organoids to store glycogen was determined by Periodic Acid-Schiff stain(PAS);Its uptake ability to LDL is identified by fluorescent labeling.(3)Establishment of liver aging model:the liver organoids were processed by oleic acid to detect changes in their growth capacity,uptake of low-density lipoprotein,gene expression level of Senescence Associated Secretory Phenotype(SASP),and reactive oxygen species(ROS)level in cells.(4)Explore the causes of liver aging caused by oleic acid:N-acetylcysteine(NAC)is used to remove reactive oxygen species in cells,qPCR detects the expression of aging markers p16 and p21,and explores the relationship between oleic acid,ROS and cellular senescence.Results:(1)Many viable liver primary cells were obtained by two-step perfusion of mice by type Ⅳ collagenase,and liver organoids with division and proliferation ability and stable inheritance were obtained after 3D culture,and the cell morphology could be kept unchanged after passage.(2)Fluorescence staining shows that the surface of liver organoid cells has high albumin expression(Albumin,Alb);The qPCR results showed that the expression level of liver marker genes was not significantly different from that of primary hepatocytes,and the expression of embryonic stem cell marker genes was high.Positive PAS staining for liver organoid paraffin sections demonstrated glycogen storage capacity;Fluorescently labeled LDL is detected within liver organoids,demonstrating its ability to phagocytose lipoproteins.(3)The growth rate of liver organoids after oleic acid treatment is significantly slowed down,and cystic structures cannot be formed;The fluorescence intensity showed that its ability to uptake LDL was significantly reduced.The qPCR test results showed that after oleic acid treatment,the gene expression level of the intracellular aging secretion phenotype increased significantly.At the same time,intracellular ROS levels rise.(4)The ROS content in cells pretreated with NAC decreased significantly compared with the oleic acid treatment group,and the expression levels of aging markers p16 and p21 also decreased.Conclusion:The method in this paper can successfully culture mouse primary hepatocytes-derived liver organoids,which express liver marker genes on the surface,retain the original properties of liver,and at the same time,the organoids show certain cell stemness.Liver organoids perform the basic functions of the liver in vitro and can be used as an ideal model for studying liver diseases.When treated with oleic acid,liver organoids exhibit aging phenomena such as slow growth and reduced phagocytosis,which is confirmed at the genetic level.At the same time,it was observed that cellular senescence was accompanied by an increase in ROS levels.After NAC pretreatment,oleic acid was added again,and the aging degree of liver organoids was alleviated,which verified the conjecture that oleic acid caused the aging of liver cells by increasing the ROS content in cells. |
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