Islet β Cells Physiological Difference Study Of Old And Young Mice Based On Single Cell Transcriptomics

Objective:The aging of the body is a universal biological process.With the increase of age,cells will undergo a series of physiological changes.The main feature is the decline of cell proliferation,but they still have normal cell functions.Pancreatic β cells are no exception..However,the physiological senescence ofβ-cells and the resulting function and transcriptome changes have rarely attracted people’s attention,and the specific senescence phenotype of β-cells has yet to be determined.Given that it is very challenging to explain the transcriptomic changes unique to the physiological aging of β cells at the cellular level,we used single-cell transcriptome sequencing to differentiate the pancreatic cells of 8-week-old mice and 20-month-old mice In contrast,analyze the unique transcriptomics differences of pancreatic β-cells with age.Method:In order to fully understand the differences in pancreatic β cells between old and young mice,we used the intraperitoneal glucose tolerance test(IPGTT)and the intraperitoneal insulin tolerance test(IPITT)to carry out glucose and glucose tolerance tests on female C57BL/6 mice at 20 months and 8 weeks,respectively. Insulin response study.The method of immunohistochemistry and islet mass analysis was used to carry out the study of the difference in the morphology of the two groups of pancreas and islets;the study of the difference in insulin levels was carried out by ELISA technology.After that,3 20-month-old mice and 3 8-week-old mice were randomly selected for islet purification,and further digestion was used to prepare a single-cell suspension of islets,which was sequenced using the emerging single-cell sequencing technology-10×Genomic; Perform bioinformatics analysis on the sequencing results;select β-cell differential genes and conduct a deeper analysis on them;use immunofluorescence technology to re-verify some differential genes.Finally,observe whether there are differences in the microstructure of the pancreatic islets through a transmission electron microscope.Result:The 20-month-old old mice and the 8-week-old young mice have a certain appearance and morphology difference,and the body weight is heavier than the young mice(P<0.001).The IPGTT results showed that the blood glucose level of old mice at 0,60,and 90 minutes was lower than that of young mice(P<0.05;P<0.001;P<0.01),while there was no difference in the blood glucose values of other points.The IPITT results showed that the elderly The blood glucose level of mice at 0 minutes was lower than that of young mice(P<0.05).The ELISA results showed that the old mice had higher insulin levels(P<0.05).The results of immunohistochemistry and islet mass analysis showed that the pancreas,the number of islets,the total area of islets,and the average area of islets of old mice were larger than those of young mice(P<0.01;P<0.01;P<0.001;P<0.01); After pancreas perfusion digestion,islet purification,and preparation of islet single cell suspension,6 samples of islet single cell suspension were successfully obtained.The quality inspection was qualified and the sequencing was completed by Shanghai OE Biotechnology Co.,Ltd.After 10×Genomics single-cell sequencing,quality inspection and a series of bio-information analysis,we sequenced a total of 59,346 cells and clustered them into 23 cell groups,and screened β cells,acinar cells,α cells,epithelial cells,For δ cells,duct cells,γ cells and other cell types inherent in the pancreas,a new cell subgroup β-like cells has been discovered for the expression of Ins1 and Ins2.A total of 47 differential genes with significant expression differences and statistical significance were screened inβ cells(Fold Change>1.5,P<0.05).In old mice,27 genes were up-regulated and 20 genes were down-regulated.Among them,mt-Co1,mt-Co2,mt-Co3,mt-Nd1,mt-Nd3,Mt1,Mafa,Ucn3,Acly,Gcg,Sst and other genes showed a high expression trend in old mice,Reg1,Cel,Cela2 a,Cela3a Isogenic expression is low.Subsequent SCENIC analysis identified that the transcription factor Jund and others are highly expressed in old mice.The results of immunofluorescence verified that mt-Co1,mt-Nd1,and Acly had higher expression levels in elderly pancreatic islets.Transmission electron microscopy results showed that the pancreatic islets of old mice had more lipid droplets,the number of mitochondria increased and the area was larger than that of young mice,and the endoplasmic reticulum had morphological changes.Conclusion:This study showed that 20-month-old old mice have normal glucose tolerance,lower blood sugar levels and higher insulin levels but have not yet developed insulin resistance.Through single-cell sequencing,it was found that the expression of related mitochondrial genes mt-Co1,mt-Co2,mt-Co3,mt-Nd1,and mt-Nd3 in β-cells of old mice increased compared with 8-week-old young mice; Metabolism-related genes Cel,Cela2 a,Cela3a,etc.are reduced in expression.We suspect that the β-cells of old mice have stronger mitochondrial activity and lower lipid metabolism.In addition,we also identified other types of pancreatic cells,and discovered a new β-cell subtype-β-like cells with lower Ins expression,and found that Gcg and Sst are highly expressed in elderly β cells,we guess The β cells of old mice may have a certain trend of transdifferentiation.By immunofluorescence,we verified that the differential genes mt-Co1,mt-Nd1,Acly are highly expressed in the β cells of old mice.And through the transmission electron microscope,the pancreatic islets of old mice have mitochondria,endoplasmic reticulum changes and more lipid droplets accumulation.