Changes And Proliferation Mechanism Of Pancreatic Islets During Neonatal Period In Mice

Objectives:1.To investigate the isolation and purification of mice pancreatic islets during neonatal period.2.To observe the changes of islet architecture, cell mass, cell size, cell number, and cell proliferation in mice during neonatal period.3.To elucidate the expression of cell cycle related genes of pancreatic islets in mice during neonatal period and to explore their relationship with the proliferation of islet cells.Methods:1.Pancreas of mice at 1-,3-,8-week after birth was injected and digested by collagenase Ⅴ, hand-picked under microscope. 2.Panccreatic β and α cells at three stages were stained with anti-glucagon antibody and anti-insulin antibody, and then identified by flow cytometry and immunofluoresence.3.Cell mass, cell size, cell number and the architecture of β and α cells at three stages were detected by immunohistochemistry.4.Cell cycle was detected by flow cytometry after stained with PI. The proliferation of β cells was detected by immunofluoresence after stained with anti-Ki67 antibody and anti-insulin antibody.5.The expression of mRNA and protein of cell cycle related genes was detected by real-time quantitative PCR, immunohistochemistry and Western blot.Results:1.The best digestive time of mice islets at 1-week and 3-week after birth were 23min(38.11±2.78/pancreas) and 25min(66.06±2.61/pancreas). Most islet diameter was less than 50μm at lw(P<0.01), and between 50～99μm at 3w(P<0.01). The yield of islets purified by hand-picked under microscope was much higher than that of Histopaque-1077 purification (P<0.05). The purity and livability of islets were above 90%, and the architecture of β and a cells changed during neonatal period(P<0.05).2.Compared with 1w, β/α cell mass significantly increased at 3w and 8w (all P<0.05); cell size didn’t change obviously at 3w, but increased 1.7 times at 8w (all P<0.01); cell number increased nearly 3.5/3.7 times at 3w (all P<0.01), and there’s no significant difference between 8w and 3w; The ratio of a cells was significantly decreased, and the ratio of β cells was significantly increased at 3w and 8w (all P<0.01). Compared with 8w, there were less cells in the G0/G1 phase and more cells in the G2/M phase at 1w and 3w(all P<0.05), and the proliferation index were significantly higher at 1w and 3w (all P<0.05). The ratio of Ki67 and insulin double-positive cells among insulin positive cells was higher at 1w than that of 3w and 8w(P<0.01).3.Compared with 1w, the mRNA level of Rb, p107, E2F1/2/3, cyclinD1/E1/A1/ A2/B1/B2, cdk6/2/1, cdc25b/c and Weel mRNA was markedly decreased(all/><0.05), whereas the level of p130, E2F4/5, cdc25a mRNA were increased at 8w. The mRNA level of Cdk4 were higher at 3w than that at 1w and 8w(P<0.05). From 1w,3w to 8w, The mRNA level of p16 and p27 decreased, but p21 mRNA were expressed higher at 3w. Immunohistochemistry showed that Rb, p107, p130 and E2F1 were differently expressed in the islet cell cytoplasm; cdkl and cyclinB1 were both expressed in the islet cell cytoplasm and nuclear, and changed markedly. Western blot showed that compared with 1w, the protein level of Rb,p107, E2F1, cdkl and cylinBl were markedly lower, but the level of p130 was significantly increased at 8w(all P<0.05).Conclusions:1.Digestion with Collagenase V and hand-picked under microscope is an effective method for mice islet isolation during neonatal period, and the digestion time and purification methods are important factors.2.During neonatal period, mice β/α cell mass increased significantly, primarily in cell numbers; the architecture of islets changed obviously; and the proliferation of islets increased significantly, especially β cells.3.Cell cycle related genes dynamically changed in mice islets during neonatal period, which might be involved in the regulation of cell Droliferation.