围妊娠期小鼠胰岛细胞增殖及Survivin表达变化的研究

目的 观察围妊娠期小鼠胰岛β/α细胞量、细胞增殖、Survivin表达变化以及大鼠胰岛细胞瘤细胞株INS-1干扰Survivin表达后催乳素刺激细胞增殖的变化,探讨Survivin与妊娠期胰岛细胞扩增的关系.方法 采用免疫组化染色法检测C57 BL/6雌性小鼠非妊娠对照组（NP）和妊娠10.5、14.5、18.5 d（P10.5、P14.5、P18.5）、产后4、8 d（AP4、AP8）（每组各25只）胰岛β/α细胞量的变化.采用胶原酶消化法分离纯化小鼠胰岛,以流式细胞术检测细胞周期,实时定量聚合酶链反应及Western blotting法检测Survivin mRNA和蛋白的表达.采用脂质体转染siRNA方法干扰INS-1细胞Survivin的表达,催乳素刺激后检测细胞周期.多组数据组间比较采用单因素方差分析,两两比较采用最小差异显著性分析.结果 妊娠期胰岛β/α细胞量均显著增加,高峰在P18.5,与NP组相比,β细胞量增长2.1倍（3.84±0.38比1.81 ±0.09,t =4.75,P＜0.05）,α细胞量增长1.6倍（0.92±0.13比0.56 ±0.08,t =2.69,P＜0.05）,至AP8恢复至NP水平.妊娠期小鼠胰岛细胞增殖显著增加,高峰在P14.5,与NP组相比,增殖指数增长2.7倍（23.07±1.88比7.72±1.12,t=10.89,P＜0.05）,产后恢复正常.Survivin mRNA表达水平在妊娠期明显增高,高峰在P14.5,与NP组相比,增长6.7倍（6.69±1.61比1.00 ±0.00,t =4.57,P＜0.05）,产后恢复正常.P10.5时Survivin蛋白开始表达,高峰在P14.5,与P10.5相比增长3.6倍（3.63±0.26比1.00±0.00,t=9.98,P＜0.05）,P18.5时表达逐渐下降,P20.5时表达消失.催乳素可显著刺激INS-1细胞增殖,而siRNA干扰INS-1细胞Survivin表达后,细胞增殖受阻.结论 妊娠期小鼠胰岛细胞出现明显扩增,并伴有Survivin的动态表达.Survivin介导催乳素刺激的胰岛β细胞扩增作用.

Changes of the proliferation of pancreatic islet and survivin expression during pregnancy in mice

Objective To observe changes of β/α cell mass,cell cycle,Survivin expression of pancreatic islets in mice during pregnancy and changes in INS-1 cell proliferation after Survivin knockdown with prolactin stimulation,and explore the relationship between Suvivin and islet cell proliferation.Methods β/α cell mass of female C57BL/6 mice islets at non-pregnancy （NP）,pregnant day 10.5,14.5,18.5 （P10.5,P14.5,P18.5） and after parturition day 4,8 （AP4,AP8） in mice was detected by immunohistochemistry.Pancreatic islets were digested and isolated by collagenase.Cell cycle was detected by flow cytometry.The expression of Survivin mRNA and protein were detected by real-time quantitative polymerase chain reaction （PCR） and Western blotting methods.INS-1 cells were transfected with siRNA mediated by liposome to knockdown Survivin expression,and the change of cell cycle with prolactin stimulation was detected.One-way analysis of variance or LSD test was used for data analysis.Results During pregnancy,islet β/α cell mass markedly increased,peaking at P18.5.Compared with NP,β cell mass increased for 2.1 times （3.84 ±0.38 vs 1.81 ±0.09,t =4.75,P 〈0.05） and α cell mass increased for 1.6 times （0.92 ± 0.13 vs 0.56 ± 0.08,t =2.69,P 〈 0.05）,then both of them returned to NP level at AP8.Cell cycle analysis showed that the proliferation of islet cells increased significantly during pregnancy,peaking at P14.5.Compared with NP,the proliferation index increased for 2.7 times （23.07 ± 1.88 vs 7.72 ± 1.12,t =10.89,P 〈0.05）,and returned to NP level after parturition.The mRNA level of Survivin during pregnancy was much higher than that at NP,peaking at P14.5 and increased for 6.7 times when compared with that in NP （6.69 ± 1.61 vs 1.00 ±0.00,t =4.57,P 〈0.05）,and reduced to NP level after parturition.Western blotting showed that Survivin protein expressed at P10.5,peaked at P14.5,and increased for 3.6 times when compared with that in P10.5 （3.63 ± 0.26 vs 1.00 ± 0.00,t =9.98,P 〈0.05）,decreased since P18.5,and disappeared at P20.5.Prolactin could markedly stimulate INS-1 cell proliferation.However,Survivin knockdown in INS-1 cells by siRNA blocked cell proliferation with prolactin stimulation.Conclusion The proliferation of islet cells increases significantly during pregnancy in mice,accompanying with dynamic expression of Survivin.Survivin mediates the proliferative effect of prolactin on islet β cells.