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.
目的 觀察圍妊娠期小鼠胰島β/α細胞量、細胞增殖、Survivin錶達變化以及大鼠胰島細胞瘤細胞株INS-1榦擾Survivin錶達後催乳素刺激細胞增殖的變化,探討Survivin與妊娠期胰島細胞擴增的關繫.方法 採用免疫組化染色法檢測C57 BL/6雌性小鼠非妊娠對照組(NP)和妊娠10.5、14.5、18.5 d(P10.5、P14.5、P18.5)、產後4、8 d(AP4、AP8)(每組各25隻)胰島β/α細胞量的變化.採用膠原酶消化法分離純化小鼠胰島,以流式細胞術檢測細胞週期,實時定量聚閤酶鏈反應及Western blotting法檢測Survivin mRNA和蛋白的錶達.採用脂質體轉染siRNA方法榦擾INS-1細胞Survivin的錶達,催乳素刺激後檢測細胞週期.多組數據組間比較採用單因素方差分析,兩兩比較採用最小差異顯著性分析.結果 妊娠期胰島β/α細胞量均顯著增加,高峰在P18.5,與NP組相比,β細胞量增長2.1倍(3.84±0.38比1.81 ±0.09,t =4.75,P＜0.05),α細胞量增長1.6倍(0.92±0.13比0.56 ±0.08,t =2.69,P＜0.05),至AP8恢複至NP水平.妊娠期小鼠胰島細胞增殖顯著增加,高峰在P14.5,與NP組相比,增殖指數增長2.7倍(23.07±1.88比7.72±1.12,t=10.89,P＜0.05),產後恢複正常.Survivin mRNA錶達水平在妊娠期明顯增高,高峰在P14.5,與NP組相比,增長6.7倍(6.69±1.61比1.00 ±0.00,t =4.57,P＜0.05),產後恢複正常.P10.5時Survivin蛋白開始錶達,高峰在P14.5,與P10.5相比增長3.6倍(3.63±0.26比1.00±0.00,t=9.98,P＜0.05),P18.5時錶達逐漸下降,P20.5時錶達消失.催乳素可顯著刺激INS-1細胞增殖,而siRNA榦擾INS-1細胞Survivin錶達後,細胞增殖受阻.結論 妊娠期小鼠胰島細胞齣現明顯擴增,併伴有Survivin的動態錶達.Survivin介導催乳素刺激的胰島β細胞擴增作用.
목적 관찰위임신기소서이도β/α세포량、세포증식、Survivin표체변화이급대서이도세포류세포주INS-1간우Survivin표체후최유소자격세포증식적변화,탐토Survivin여임신기이도세포확증적관계.방법 채용면역조화염색법검측C57 BL/6자성소서비임신대조조(NP)화임신10.5、14.5、18.5 d(P10.5、P14.5、P18.5)、산후4、8 d(AP4、AP8)(매조각25지)이도β/α세포량적변화.채용효원매소화법분리순화소서이도,이류식세포술검측세포주기,실시정량취합매련반응급Western blotting법검측Survivin mRNA화단백적표체.채용지질체전염siRNA방법간우INS-1세포Survivin적표체,최유소자격후검측세포주기.다조수거조간비교채용단인소방차분석,량량비교채용최소차이현저성분석.결과 임신기이도β/α세포량균현저증가,고봉재P18.5,여NP조상비,β세포량증장2.1배(3.84±0.38비1.81 ±0.09,t =4.75,P＜0.05),α세포량증장1.6배(0.92±0.13비0.56 ±0.08,t =2.69,P＜0.05),지AP8회복지NP수평.임신기소서이도세포증식현저증가,고봉재P14.5,여NP조상비,증식지수증장2.7배(23.07±1.88비7.72±1.12,t=10.89,P＜0.05),산후회복정상.Survivin mRNA표체수평재임신기명현증고,고봉재P14.5,여NP조상비,증장6.7배(6.69±1.61비1.00 ±0.00,t =4.57,P＜0.05),산후회복정상.P10.5시Survivin단백개시표체,고봉재P14.5,여P10.5상비증장3.6배(3.63±0.26비1.00±0.00,t=9.98,P＜0.05),P18.5시표체축점하강,P20.5시표체소실.최유소가현저자격INS-1세포증식,이siRNA간우INS-1세포Survivin표체후,세포증식수조.결론 임신기소서이도세포출현명현확증,병반유Survivin적동태표체.Survivin개도최유소자격적이도β세포확증작용.
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.
