Study On The Mechanism Of SIDT2 In Autophagy And Differentiation And Maturation Of Adipose Tissue

Objective: A preliminary study on the potential mechanism of lysosome membrane protein Sidt2 deletion in the process of autophagy and differentiation and maturation of adipose tissue in mice.Methods: In this experiment,the whole body Sidt2 gene knockout mouse model was established by using Cre/loxp recombinant enzyme system,and the 3T3-L1 cell model of Sidt2 gene knockout was obtained by adenoviral infection of 3T3-L1 preadipocytes.The expression of key proteins in mouse adipose tissue and 3T3-L1 preadipocytes autophagy pathway was detected by western blot.It was found that autophagy pathway was blocked in adipose tissue and cells of Sidt2 gene deficient mice.Further use of drugs affecting autophagy pathway such as chloroquine,rapamycin,3-MA and immunofluorescence to explore the role of Sidt2 gene in mouse adipose tissue autophagy pathway.The changes of adipose tissue sections of mice were detected by HE staining,and the differentiation of adipocytes induced by Sidt2 gene knockout 3T3-L1 was detected by oil red O staining.And continue to detect the expression of key factors in the process of adipocyte differentiation from the protein level.To further explore whether the relationship between SIDT2 and adipocyte differentiation is mediated by autophagy.Results: 1.The mouse model and cell model of Sidt2 gene knockout were successfully obtained and detected at the gene and protein level to confirm the success of the model.2.Protein level detection of autophagy pathway key proteins LC3 B,p62,etc.,found that Sidt2 gene knockout mice adipose tissue and 3T3-L1 cell autophagy pathway was blocked.3.After 3T3-L1 cells were treated with drugs affecting autophagy pathway,the changes of autophagy key proteins were detected to determine that the loss of Sidt2 caused the inhibition of autophagy lysosome degradation pathway.4.With the help of immunofluorescence and mCherry-GFP-LC3 B double labeling,the autophagy flow changes of Sidt2 gene deleted 3T3-L1 cells were observed more directly under confocal microscope.5.Morphological adipose tissue HE staining showed that the arrangement of adipocytes in Sidt2 knockout mice was loose,the edge of adipocytes was not clear,the number of adipocytes decreased,and the volume of lipid droplets increased compensatively compared with the normal group.6.By oil red O staining,it was found that Sidt2 gene knockout 3T3-L1 preadipocytes could not be successfully induced into mature adipocytes,and there were few lipid droplets.7.The expression of PPAR-? and CEBP ?,the key factors of adipocyte differentiation induced by Sidt2 gene deletion,decreased.Conclusion: 1.Loss of lysosomal membrane protein SIDT2 in mouse adipose tissue can lead to obstruction of autophagic lysosomal degradation pathway;2.Loss of lysosomal membrane protein SIDT2 in mouse adipose tissue can cause adipocyte differentiation and maturation disorders;3.The deletion of the lysosomal membrane protein SIDT2 can cause the autophagic lysosomal degradation pathway to be blocked and lead to the differentiation and maturation of adipocytes.