Research on isolation and purification method of pancreatic islets in mice / 器官移植

ABSTRACT

Objective To investigate a method for isolation and purification of pancreatic islets in mice,aiming to enhance the quantity and activity of pancreatic islets.Methods The pancreas were digested by retrograde common bile duct perfusion of collagenase,discontinuous density gradient centrifugation and the islets were selected and purified by manual method.After overnight culture,the pancreatic islets were incubated by static glucose-stimulated insulin secretion (GSIS)and the islet function was assessed.The subcellular structure of islet βcells was observed under transmission electron microscopy.Results Using the modified method,(200 ±20)islets were collected in each mouse with a mean diameter of (175 ±22)μm. The insulin levels in the stimulation of low (2.8 mmol/L)and high glucose (16.7 mmol/L)were (0.33 ± 0.07)and (1.36 ±0.47 )ng/(islet · 60 min),which were detected by GSIS.Insulin levels in the stimulation of high sugar is 4.12 times of those of low sugar with a statistical significance (P <0.05).It was revealed by transmission electron microscopy that the pancreatic βcell membrane and mitochondrial membrane was intact and insulin granules of different sizes could be seen within βcells.Conclusions Retrograde common bile duct perfusion of collagenase,discontinuous density gradient centrifugation combined with manual selection in vitro is a convenient,fast and stable method for isolating mouse islets,which can get pancreatic islets with relatively high output,intact cellular morphology,and good reactivity of GSIS.