The Role Of Survivin In Islet Beta-cell Proliferation During Pregnancy In Mice

Objective: 1. To observe the changes of β/α cell mass and proliferation of pancreatic islets cell during pregnancy in mice, and to observe the effect of prolactin on βcell proliferation. 2. To observe the changes of Survivin expression in pregnant mice islets, and in non-pregnant mice islets and INS-1 cell line stimulated by prolactin. 3. To observe the influence of islet beta-cell specific Survivin deletion on beta-cell mass, function and proliferation in mice during pregnancy.Methods: 1. β/α cell mass,cell number and cell size of mice islets at non-pregnancy(NP), pregnant day 10.5(P10.5), P14.5, P18.5 and after parturition day 4(AP4), AP8 in C57BL/6 mice were detected by immunohistochemistry. Pancreatic islets were isolated and purificated by collagenase digestion at each time point. Cell cycle was detected by flow cytometry. The changes of the percentage of insulin and Ki67 double positive cells in all insulin positive cells between NP and P14.5 were detected by immunofluorescence. INS-1 cell line were cultured with different concentration of prolactin(0ng/m L, 50ng/m L, 500ng/m L), and cell cycle was detected by flow cytometry. 2. The expression of Survivin m RNA and protein were detected by real-time quantitative PCR, immunohistochemistry and Western blot. NP mice islets and INS-1 cell line were cultured with different concentration of prolactin(0ng/m L, 50ng/m L, 500ng/m L) in vitro, then detected the expression of of Survivin m RNA and protein by real-time quantitative PCR and Western blot. 3. Random blood glucose were detected in islet beta-cell specific Survivin deletion and wild-type mice at different time points. Glucose tolerance of two group mice atNP and P14.5 were detected by intraperitoneal glucose tolerance test. Insulin secretion and insulin sensitivity at P14.5 were detected by glucose-stimulated insulin secretion test and insulin tolerance test in two group mice. The changes of islet beta-cell mass, beta-cell size, beta-cell number and islet size between two group mice were detected by immunohistochemistry at different time points. The changes of the percentage of insulin and Ki67 double positive cells in all insulin positive cells between two group mice were detected by immunofluorescence at NP and P14.5.Results: 1. β and α cell mass progressively increased during pregnancy. Both of them peaked at P18.5, while β cell increased 2.1 times and α cell increased 1.6 times, and returned back to NP level at AP8. β and α cell mass increase result from increase both in cell number and cell size during pregnancy. At P18.5, β and α cell number increased 1.81 and 1.31 times, while β and α cell size increased 1.70 and 1.17 times, respectively. 2. The islets cell proliferation increased significantly during pregnancy, and peaked at P14.5. The proliferation index increased 15.35% at P14.5, and returned back to NP level after parturition. Compared with NP, the percentage of insulin and Ki67 double positive cells in all insulin positive cells increased 3 times at P14.5. The proliferation of INS-1 cell line increased significantly after prolactin stimulation. PI increased 4.74% at 50ng/m L prolactin concentration and 6.31% at 500ng/m L prolactin concentration, and this effect was a concentration-dependent manner. 3. The m RNA level of Survivin during pregnancy were much higher than that at NP, and peaked at P14.5, while the level increased 6 times, and returned back to NP level after parturition. Immunohistochemistry showed that Survivin protein was not expressed at NP islets, but strongly expressed at P14.5. Western blot showed that Survivin protein was not expressed at NP, reexpressed at P10.5, peaked at P14.5, and decreased since P18.5 until unexpressed. Compared with P10.5, the expression ofSurvivin protein was increased 3.6 times at P14.5. The m RNA level of Survivin in mice islets increased significantly after prolactin stimulation, while increased 2.4 times at 50ng/m L and 1.9 times at 500ng/m L, no further increase depend on the concentration of prolactin. The m RNA and protein levels of Survivin in INS-1 cell line increased significantly after prolactin stimulation, while increased 1.9 and 1.3 times at 50ng/m L and 1.4 and 1.2 times at 500ng/m L respectively, no further increase depend on the concentration of prolactin. 4. Compared with wild-type control, the islet beta-cell specific Survivin deletion mice have normal glucose tolerance at NP, impaired random blood glucose and impaired glucose tolerance during pregnancy. Insulin secretion were impaired and insulin sensitivity were normal in islet beta-cell specific Survivin deletion mice at P14.5. 5. Compared with wild-type control, beta-cell mass were decreased and beta-cell size were compensatory hypertrophic in NP islet cell-specific Survivin deletion mice, while beta-cell mass and beta-cell number were not significantly amplified, beta-cell size was no further hypertrophy at P14.5. Compared with wild-type control, beta-cell proliferation was impaired in islet cell-specific Survivin deletion mice at P14.5.Conclusion: 1. The cell mass of islets increased significantly in pregnant mice, which result from cell number increase and cell size hypertrophy. The proliferation of islet cells and beta-cell increased in pregnant mice, in which prolactin play an important role. 2. The expression of Survivin increased in pregnant mice islets, in which prolactin play an important role. 3. Survivin deletion lead to impaired glucose tolerance, insulin secretion and beta-cell amplification, showed that Survivin directly participate in β cell proliferation during pregnancy.