CREB结合蛋白调控组蛋白H4乙酰化修饰参与糖尿病雌鼠诱导胎鼠神经管畸形发生机制的研究  

Regulation of histone 4 acetylation by the CREB-binding protein participate in the maternal diabetes induced neural tube defects

作　　者：白宝玲[1] 万春蕾 李丹 周珍珍 张勤[1] BAI Baoling;WAN Chunlei;LI Dan;ZHANG Qin(Department of Biochemical and Immune Research,Capital Institute of Pediatrics,Beijing 100020,China)

机构地区：[1]首都儿科研究所生化免疫室,北京100020 [2]通州区妇幼保健院儿科

出　　处：《中国糖尿病杂志》2020年第9期690-696,共7页Chinese Journal of Diabetes

基　　金：国家自然科学基金(81901167)。

摘　　要：目的探讨CREB结合蛋白(Cbp)调控组蛋白H4赖氨酸位点乙酰化修饰参与DM雌鼠诱发胎鼠神经管缺陷(NTDs)的发生机制。方法体外构建高糖处理小鼠神经干细胞(NE-4C)模型及T1DM雌鼠诱发NTDs模型。建立葡萄糖浓度为5 mmol/L的正常对照组(Con组)和25 mmol/L高糖组(HG组)处理NE-4C细胞,HG组分别以DMSO(HG+DMSO组)、1μmol/L的Cbp/P300抑制剂(C646)(HG+C646+1组)、5μmol/L的C646(HG+C646+5组)、5μmol/L的P300/PCAF抑制剂藤黄酚(HG+Gar+5组)、25μmol/L的藤黄酚(HG+Gar+25组)处理。将NE-4C细胞分为转染对照siRNA组(Con-si组)、转染第1个Cbp的siRNA质粒(si-Cbp-1组)、转染第2个Cbp的siRNA质粒(si-Cbp-2组)、共转染第1及第2个Cbp的siRNA质粒(si-Cbp-1+2组)。雌鼠分为正常对照(NC)组(n=38)和STZ构建的TIDM组(n=54)。采用Western blot、RT-PCR、免疫荧光染色检检测。结果 HG组Cbp的mRNA表达和NE-4C细胞增殖能力较Con组升高(P<0.05或P<0.01)。HG+C646+5组组蛋白H4K5/8/12/16ac蛋白水平低于HG+DMSO、HG+C646+1组(P<0.05或P<0.01)。在4 h时,HG+C646+5组NE-4C细胞增殖能力低于HG组(P<0.05)。与Con-si组比较,si-Cbp-1、si-Cbp-2、si-Cbp-1+2组H4K5/8/12/16ac蛋白、Cbp mRNA水平降低(P<0.05)。与NC组比较,T1DM组Cbp的mRNA、组蛋白H4K5/8/12/16ac修饰水平升高(P<0.01)。结论高糖导致Cbp调控组蛋白H4乙酰化修饰异常和NE-4C细胞增殖可能是诱发NTDs的机制之一,Cbp抑制剂可改善组蛋白H4乙酰化水平和NE-4C细胞增殖。Objective To investigate the mechanism of CREB binding protein(Cbp)regulating acetylation modification at lysine site of histone H4 in the pathogenesis of fetal neural tube defects(NTDs)induced by diabetic female rats.Methods The neural stem cell model of mice treated with high glucose and NTDs model induced by T1DM female mice were established in vitro.The normal control group(Con group)was established for cells with glucose concentration of 5 mmol/L,and the high glucose group(Hg group)was established for cells with glucose concentration of 25 mmol/L.Cells in Hg group were treated with DMSO(Hg+DMSO),1 μmol/L Cbp/P300 inhibitor(c646)(Hg+C646+1 group),5 μmol/L C646(Hg+C646+5 group),5 μmol/L P300/PCAF inhibitor gambogol(Hg+Gar+5 group),and 25 μmol/L gambogol(Hg+Gar+25 group),respectively.Neural stem cells are divided into control si RNA group(Con-si group),siRNA plasmid transfected with the first Cbp(si-Cbp-1 group),siRNA plasmid transfected with the second Cbp(si-Cbp-2 group),si RNA plasmid co-transfected with the first and second Cbp(si-Cbp-1+2 group).Female mice were divided into normal control(NC)group(n=38)and TIDM group(n=54)constructed by STZ.Western blot,RT-PCR and immunofluorescence staining were used for the study.Results The mRNA expression of Cbp and cell proliferation in HG group were higher than those in Con group(P<0.05 or P<0.01).The level of histone H4K5/8/12/16ac protein in HG+c646+5 group was lower than that in HG+DMSO and HG+C646+1 group(P<0.05 or P<0.01).At 4 h,the cell proliferation ability of HG+C646+5 group was lower than that of Hg group(P<0.05).Compared with Con-si group,the levels of H4K5/8/12/16ac protein and Cbp mRNA in si-Cbp-1,si-Cbp-2 and si-Cbp-1+2 groups decreased(P<0.05).Compared with NC group,histone H4K5/8/12/16ac modification level and mRNA of Cbp in T1DM group increased(P<0.01).Conclusion The abnormal acetylation of histone H4 and proliferation of neural stem cells regulated by Cbp induced by high glucose may be one of the mechanisms inducing NTDs.Cbp inhibitors can improv

关 键 词：神经管畸形 糖尿病 组蛋白H4第5位赖氨酸位点乙酰化 C646 CREB结合蛋白 

分 类 号：R587.2[医药卫生—内分泌]