Isolation And Identification Of Exosomes And Their Physiological Functions

The intestinal epithelium is the largest surface organ composed of monolayers of cells,and is the largest receptor in the human body.In addition to the ever-changing variety of dietary components and their digestive products,the content of the small intestine has a wide variety of flora and its metabolites.Therefore,the intestinal secretion regulation function is affected by complex intestinal contents such as food and microorganisms.influences.Because of this,in vitro studies of primary intestinal epithelium can only reflect the in vivo characteristics of intestinal epithelial cells through 3D organ culture,but this does not reflect some of the special functions of epithelial cells,especially secretion regulation.Therefore,in order to truly reflect the secretory regulation of exosomes,studies on the secretion of exosomes in different models have begun.For example,brain tissues with complex functions,interactions between cells,and constant response to external environmental stimuli have been taken to extract tissue exocrine.Body method.Therefore,similarly,when we study the secretion of intestinal epithelial secretion,we should choose to directly extract the exosomes of the small intestinal epithelium.Therefore,we have innovatively studied the secretion of exosomes from small intestinal epithelial tissues.The exosomes extracted by this method were co-cultured with their visceral fat cells to investigate the effects of exosomes derived from different intestinal epithelium on the function of visceral fat cells.The study is divided into two parts: Part?: Extraction and identification of exosomes from small intestinal epithelium Part ?: Functional study of intestinal epithelial exosomes acting on visceral fat cellsPart ?: Extraction and identification of exosomes from small intestinal epitheliumOBJECTIVE: The intestinal epithelium is the largest surface organ composed of monolayers of cells,and is the largest receptor in the human body.In addition to the ever-changing variety of dietary components and their digestive products,the content of the small intestine has a wide variety of flora and its metabolites.Therefore,the intestinal secretion regulation function is affected by complex intestinal contents such as food and microorganisms.influences.Therefore,it is a challenge to reflect the true intestinal epithelial secretion regulation function.To this end,we propose the concept of extracting exosomes from intestinal epithelial tissue.METHODS: We innovatively proposed an extraction method for small intestinal epithelial exosomes.The small intestine of 3w male mice was removed,the feces and intestinal mucus were removed,and the epithelial and mucosal layers of the small intestine were separated by EDTA,and the sediment and cell debris were removed after low-speed centrifugation,followed by ultracentrifugation(100,000 g).Exosomes derived from intestinal epithelial tissue are obtained.The exosomes extracted by this method were identified by transmission electron microscopy,immunoblotting and nanoparticle tracking analysis techniques.RESULTS: We successfully extracted exosomes from 3w male mice and observed a typical biconcave circular structure by transmission electron microscopy;we verified several exosomal specificities by immunoblotting(western blotting).Expression of surface proteins(CD63,HSP70,TSG101)and nanoparticle tracking analysis(NTA)demonstrated the size and concentration of exosomes.CONCLUSION: We successfully extracted exosomes from the intestinal epithelial tissue of mice,and performed a series of analysis on the size,morphology,quantity and surface marker proteins of exosomes,which fully verified the purity of exosomes extracted.Part ?: Functional study of intestinal epithelial exosomes acting on visceral adiposeOBJECTIVE: The intestinal epithelium is the central regulatory organ of the body's energy homeostasis,which plays a vital role in maintaining the body's energy homeostasis.The metabolic balance of the body involves the coordination of multiple organs.The intestinal tract,as the primary organ for nutrient sensation and absorption,is the central regulator of the body's energy homeostasis,the most important of which is the regulation of the organ-eat fat that stores energy.Therefore,we are committed to exploring the effects of intestinal exosomes on fat function at different dietary levels.METHODS: Small intestinal epithelial cells of different diet mice were labeled with PKH26,and visceral fat cells were stimulated by intestinal epithelial secretion of high-fat diet and calorie-restricted mice.The inflammation and browning were detected by PCR and western blotting.Genetic changes in fat function.RESULTS: We successfully demonstrated that visceral fat cells can be taken up by visceral fat cells.We performed high-throughput sequencing analysis of the small intestinal epithelial exosomes extracted from normal diet mice and high-fat diet mice.The results showed that 26 genes were up-regulated and 14 down-regulated in the high-fat diet group..In addition,we used primary exosomes of 15-month-old female mice in the normal diet group,the high-fat diet group,and the caloric-restricted group to stimulate the primary viscera of 3w male mice with reference to the unexposed group.The results of the fat cells showed that the high-lipid exocytosis group had significantly higher inflammatory gene expression than the normal diet exosome-stimulated group and the non-exosomal group,while the normal diet group and the exo-free group were not.There were no significant changes between the stimulation groups.CONCLUSION:Intestinal epithelial secretion of high-fat diet mice stimulates the changes of inflammatory factor factors in primary visceral adipose cells.We further study the theoretical basis by which intestinal-derived substances can regulate fat function.