围妊娠期小鼠胰岛细胞视网膜母细胞瘤蛋白的表达及其与β细胞扩增的关系

Expression of retinoblastoma protein and its relationship to β cell proliferation in periconceptional mouse pancreatic islet cells

目的观察围妊娠期小鼠胰岛细胞视网膜母细胞瘤（Rb）蛋白的表达变化，探讨其与妊娠期胰岛13细胞扩增的关系。方法将适合繁衍的6-8周龄体重20-25g的C57BL／6小鼠合笼配繁，检测阴栓形成，留取未孕（NP）、孕14．5d（P14．5）、孕18．5d（P18．5）、产后4d（AP4）、产后8d（AP8）等围妊娠期天数的小鼠。采用胶原酶消化法分离纯化小鼠胰岛，Westernblot法检测Rb、磷酸化Rb（p-Rb）及转录因子E2F1的蛋白表达，免疫荧光技术检测Rb和p-Rb的分布。配繁胰腺特异性Rh基因敲除小鼠，留取NP、P14．5和P18．5的小鼠。以腹腔葡萄糖耐量试验评估小鼠P14．5时的糖耐量变化。体内及体外试验检测葡萄糖刺激下的胰岛素分泌。免疫组化法检测基因敲除小鼠胰岛B细胞量、数目及面积的变化，免疫荧光法检测Ki67阳性β细胞比例变化。各组间比较采用独立样本t检验。结果（1）小鼠胰岛细胞RI）、p-Rb和E2F1在妊娠期表达显著升高，高峰出现在P14．5，表达量分别为NP组的2．16、2．96和2．09倍（￡=9．31、24．23、8．31，均P〈0．叭），均于分娩后逐渐恢复至孕前水平；Rb主要表达于胞核，而p-Rh主要表达于胞浆。（2）NP时基因敲除小鼠糖耐量优于野生型小鼠，血糖曲线下面积（AUC）显著降低[（1343±19）比（1511±16）mmol·L^-1·min^-1，t=10．48，P〈0．01]；P14．5d时基因敲除鼠糖耐量仍优于野生型孕鼠，AUC显著降低[（1073±117）比（1359±101）mmol·L^-1·rain^-1，t=3．71，P〈0．01]；P14．5d时，基因敲除孕鼠葡萄糖刺激的胰岛素分泌在2rain和30rain时显著高于野生型孕鼠[分别为（0．69±0．10）比（0．55±0．10）ng／ml、（0．46±0．08）比（0．36±0．08）ng／ml，t=2．40、2．30，均P〈0．051，体外葡萄糖刺激下基因敲除孕鼠的胰岛素分泌水平也高于野生型孕鼠[低糖和高糖状态下分别为（233±48）比（93±49）U／ml和（365±46）比（230±94）U／ml，t=4．30、2．62，均P〈0．01]。NP时，基因敲除鼠的B细胞量、数目、Ki67阳性细胞比例较普通小鼠均增加（t=4．53、3．46、7．68，均P〈0．05），但进入妊娠期后未出现进-步增加。结论围妊娠期小鼠胰岛细胞中Rb及E2F1表达增高，与妊娠期胰岛8细胞扩增相关。

Objective To observe the changes of retinoblastoma （Rb） protein expression in periconceptional mouse pancreatic islet cells and to explore its relationship to 15 cell proliferation. Methods 6 to 8 weeks, 20-25 g C57BL/6 mice were kept in unpregnancy （NP）, 14.5 d and 18.5 d of pregnancy （P14.5, P18.5）, 4 d and 8 d after parturition （AP4, AP8） by detecting the vaginal plug. The islets of the mice were isolated and purified by collagenase digestion. The expressions of Rb, phosphorylated Rb （p-Rb） and transcription factor E2F1 were detected by Western blot, and the distributions of Rb and p-Rb were detected by immunofluorescence （IF）. Pancreatic-specific Rb knockout （KO） mice were kept in NP.Pl4.5 and P18.5. Glucose tolerance were assessed by intraperitoneal glucose tolerance test at P18.5. Insulin levels stimulated by glucose were detected in vivo and in vitro. Changes of β ceil number, area and size were tested by immunohistochemistry and the Ki67 positive [3 cells of Rb-KO mice were detected by IF. Differences between groups were compared by independent-sample t test. Results （1） During pregnancy, Rb, p-Rb and E2F1 peaked at P14.5, increased 2.16, 2.96 and 2.09 times （t=9.31, 24,23, 8.31, all P〈0.01） respectively compared with NP, and returned back gradually after parturition. Rb located mainly in nucleus while p-Rb located in cytoplasm. （2） At NP and P14.5, compared with wild type （WT） mice, Rb-KO mice had better glucose tolerance with less area under the cure （AUC） [（1 343±19） vs （1 511±16） mmol·L^-1, min^-1, t=10.48, P〈0.01; （1 073±117） vs （1359±101）mmol· L^-1 min^-1, t=3,71, P〈0.01, respectively]. At P14.5, the insulin levels stimulated by glucose at 2 rain and 30 min in Rb-KO mice were significantly higher than those in WT mice [（0.69±0.10） vs （0.55±0.10） ng/ml, （0.46±0.08） vs （0.36＋0.08） ng/ml, t=2.40, 2.30, respectively, both P〈0.05]. The levels of insulin secretion by glucose stimulation in vitro of KO mice were also higher than those of WT mice in pregnancy [with lower and higher glucose： （233±48） vs （93±49） U/ml, （365±46） vs （230±94）U/ml, t=4.30, 2.62, respectively, both P〈0.01]. At NP, the 13 cell mass, numbers and Ki67 positive 13 cells increased in Rb-KO mice than those in WT mice （t=4.53, 3.46, 7.68, all P〈0.05）, but no significant differences were found in Rb-KO mice between NP arid pregnancy. Conclusion The expressions of Rb and E2F1 increase during periconception, which are associated with 13 cell proliferation.