Establishment Of Human Enteroendocrine Cell Model In Vitro Culture And Regulation Of Metformin On Its Differentiation

Objective: The gastrointestinal tract is the largest endocrine organ of the body.Enteroendocrine cells are scattered in the gastrointestinal tract,accounting for less than1% of the gastrointestinal epithelium.Due to the lack of corresponding in vitro cell lines derived from non-cancer cells,the lack of research models has restricted the development of research in this field for many years.This study aims to establish and optimize the enteroendocrine cells cell model under different conditions,and initially explore the regulatory effect of metformin,a hypoglycemic drug,on its differentiation.Methods:(1)L-WRN cells and R-Spondin-2 cells were cultured to obtain conditioned medium for gastrointestinal stem cells.They were taken from human stomach,duodenum and other parts of post-operation discarded epithelial tissues.They were transferred to two-dimensional and three-dimensional culture of gastrointestinal stem cells after adjustment of the digestive glands in different parts by observing the digestion process and optimize the digestion process accordingly.Observing the growth status of gastrointestinal stem cells by immunofluorescence technology.(2)Using lentivirus infection technology,Neurogenin 3,a key transcription factor for the differentiation of enteroendocrine cells,was transferred into gastrointestinal stem cells to promote the differentiation of enteroendocrine cells.The differentiation and hormone secretion of enteroendocrine cells were compared by treatment with 4-hydroxy tamoxifen of different action time and concentration and culture under different two-dimensional and three-dimensional conditions.Finally,after stimulation with different concentrations of glucose,the level of enteroendocrine cells differentiation and hormone expression were detected.(3)Different concentration gradients of metformin were set up and added to the above enteroendocrine cell models respectively,and their effects on the differentiation of enteroendocrine cells and hormone secretion were preliminarily tested.Results:(1)The conditioned medium for the growth of gastrointestinal stem cells was successfully obtained from L-WRN and R-Spondin2 cells.Gastrointestinal digestive glands with different numbers and shapes were observed in the collected gastrointestinal waste tissues after digestion.In the three-dimensional state,gastrointestinal organs are spherical or quasi-spherical,with different sizes.Branches or budding structures appear in5～7 days,and gradually increase,changing from bud structure to saccular structure.It can be subcultured in 7～14 days,and it can be subcultured in 1:3～5,and it can be subcultured for more than 20 generations.In two-dimensional state,gastrointestinal stem cells grow in colonies.(2)The proportion and quantity of inducing gastrointestinal stem cells to differentiate into enteroendocrine cells and promoting hormone secretion are in direct proportion to the concentration and time of 4-hydroxytamoxifen treatment.In the experimental group with 4-hydroxytamoxifen concentration of 1 u M,the expression time of hormone was earlier and the expression level was higher after 2 days of culture than after 1 day of culture.At the peak of hormone,tryptophan hydroxylase increased by 1.7times,somatostatin increased by 2.2 times,ghrelin increased by 80 times,and glucose-dependent insulinotropic polypeptide increased by 2 times.In three-dimensional state,the proportion of inducing gastrointestinal stem cells to differentiate into enteroendocrine cells is higher than that in two-dimensional state.The results of fluorescence assay showed that Ghrelin and 5-hydroxytryptamine were highly expressed in the stomach,and glucose-dependent insulinotropic polypeptide and chromogranin A were highly expressed in the duodenum.In addition,high concentration of glucose can increase the differentiation rate of gastrointestinal stem cells into enteroendocrine cells.When the glucose concentration was 30 m M and the action time was 6 days,the differentiation ratio of enteroendocrine cells increased compared with the glucose concentration of 17.5 m M.(3)Different concentration gradients of metformin were set up,which were 0.02 m M,0.1 m M,0.5 m M and 2 m M respectively.After co-culture with gastrointestinal stem cells,it was found that the differentiation of gastrointestinal stem cells into enteroendocrine cells gradually decreased with the increase of concentration.Conclusion: In this study,we first established a stable culture model of gastrointestinal stem cells in vitro,and further established a model of enteroendocrine cells through lentivirus infection and overexpression of Neurogenin 3,which can continue to pass for more than 20 generations.Through the immunostaining identification of enteroendocrine cells,in this study we found that they still maintain the specificity of hormone expression in the source tissue,indicating that in our study we have successfully established the enteroendocrine cell model.This provides a new model for studying the role of enteroendocrine cells in different parts,and provides a new idea for the study of gastrointestinal endocrine hormones.In this study,we also used metformin to intervene the differentiation of enteroendocrine cells and found that metformin has a direct effect on inhibiting the differentiation of enteroendocrine cells.