小鼠胚胎心脏发育过程中胰岛素基因增强子结合蛋白1的时序表达与组蛋白乙酰化酶p300的关系

Temporal Expression of Islet-1 and Relationship between Islet-1 and Histone Acetyltransferase p300 during Embryonic Heart Development in Mice

目的观察小鼠胚胎心脏发育过程中转录因子胰岛素基因增强子结合蛋白1(Islet-1)的时序性表达规律,并探讨其与组蛋白乙酰化酶p300介导的组蛋白乙酰化调控网络中的关系。方法以健康6～8周龄昆明小鼠为研究对象,下午1700雄雌按12比例合笼,次日观察阴栓,观察到阴栓最早之日计为胎龄0.5 d(E0.5)。取胎龄为E11.5、E14.5、E17.5的胎鼠和新生鼠的心脏,利用Western blot方法定量分析Islet-1的时序性表达规律,并利用蛋白质免疫共沉淀(Co-IP)和串联质谱分析方法(MS)鉴定Islet-1与组蛋白乙酰化酶p300的关系,同时应用Western blot方法反验证实验结果。结果 1.在小鼠胚胎心脏发育过程中,Islet-1在E14.5时的表达水平(0.434±0.353)明显高于与其在E11.5(0.074±0.456)、E17.5(0.120±0.127)和新生鼠(0.049±0.083)的表达水平,差异均有统计学意义(Pa<0.05),而其他时间点(E11.5、E17.5和新生鼠)间的表达差异无统计学意义(Pa>0.05)。2.在胚胎心脏发育过程中,Islet-1与组蛋白乙酰化酶p300相互结合,以蛋白复合物的形式存在。结论在小鼠胚胎心脏发育过程中,Islet-1可能作为枢纽因子,募集组蛋白乙酰化酶p300参与组蛋白乙酰化修饰调控。

Objective To investigate the temporal expression of transcription factor Islet - 1 and the relationship between Islet - 1 and histone acetyhransferase p300 during the embryonic heart development in mice. Methods Kunming mice aged 6 - 8 weeks were taken as study object. Animals were mated at PM 17 ： 00 and the female were examined for a vaginal plug in the following morning. When a vaginal plug was confirmed,was considered as embryonic days O. 5 （ E0.5 ）. The whole hearts from newborn mice and embryonic mice on Ell. 5, El4. 5 and El7.5 were collected respectively. The temporal expression of Islet - 1 was quantitatively detected by using Western blot met-hod. The relationship between Islet - 1 and histone acetyltransferase p300 was determined using p300 primary antibody with co - immunopreci-pita- tion （ Co - IP） and subsequent mass spectrum （MS） analysis, meanwhile it was negatively verified by Western blot assay. Results 1. The ex- pression of Islet - 1 was significantly higher in the embryonic mice on El4.5 （0. 434 ± 0. 353 ） than that in the embryonic mice on E11.5 （0. 074 ± 0. 456 ）, El7.5 （0. 120 ± 0. 127 ） and newborn mice （0.049 ± 0.083 ） （ Pa 〈 0.05 ）, but there was no significant difference among E11.5, El7.5 and newborn mice （ Pa 〉 0.05 ）. 2. Results from Co - IP, MS and Western - blot assays showed that the Islet - 1 could be com- bined with histone acetyltransferase p300 to form complex protein during the embryonic heart development. Conclusions Islet - 1, as a cen- tral role, may recruit histone acetyltransferase p300 to participate in the acetylation of histories during the embryonic heart development.