PKC-α介导的线粒体功能障碍在妊娠糖尿病致胚胎神经管缺陷中的机制研究  

Mechanism of PKC-αMediated Mitochondrial Dysfunction in Fetal Neural Tube Defects Caused by Gestational Diabetes Mellitus

作　　者：裴巧丽[1] 朱丽红[1] 徐珊[1] 王敏[1] 李文婷[1] 崔张霞 杜冬青[1] 张小菜 PEI Qiaoli;ZHU Lihong;XU Shan;WANG Min;LI Wenting;CUI Zhangxia;DU Dongqing;ZHANG Xiaocai(Second Affiliated Hospital of Shaanxi University of Traditional Chinese Medicine,Xianyang 712000,Shaanxi,China)

机构地区：[1]陕西中医药大学第二附属医院,陕西咸阳712000

出　　处：《中西医结合心脑血管病杂志》2024年第21期3919-3925,共7页Chinese Journal of Integrative Medicine on Cardio-Cerebrovascular Disease

基　　金：陕西省卫生健康科研基金项目(No.2018D085)。

摘　　要：目的:探讨蛋白激酶C-α(PKC-α)介导的线粒体功能障碍在妊娠糖尿病致胚胎神经管缺陷(NTD)中的作用。方法:将雌性C57BL/6J小鼠随机分为对照组、NTD组、NTD+sh-PKC-α组,每组6只。除对照组外,NTD组、NTD+sh-PKC-α组建立糖尿病胚胎病小鼠模型。NTD+sh-PKC-α组分别在胚胎第1天(E1)、第4天(E4)和第8天(E8)时,经尾静脉向小鼠注射100μL携带sh-PKC-α的慢病毒(1×10^(8) TU/mL)。在第11.5天(E11.5)时,从子宫中解剖出胚胎进行分析。采用透射电子显微镜观察神经管中线粒体。采用蛋白免疫印迹法(Western Blot)分析胚胎神经管中PKC-α、抗微管相关蛋白1轻链3(LC3)和抗溶酶体相关膜蛋白2(LAMP2)蛋白表达。体外构建PKC-α敲低C17.2小鼠神经干细胞,并在低(5 mmol/L)或高(25 mmol/L)葡萄糖条件下培养,采用免疫荧光染色检测细胞自噬体变化情况。结果:在NTD组中,胚胎神经管中线粒体肿胀,嵴消失。与对照组相比,NTD组胚胎神经管中LC3Ⅱ和LAMP2蛋白表达降低(P<0.05),PKC-α蛋白表达上调(P<0.01)。通过沉默小鼠胚胎神经管中PKC-α表达,胚胎中LC3Ⅱ和LAMP2蛋白表达增加(P<0.05)。此外,NTD+sh-PKC-α组小鼠胚胎中的NTD发生率低于NTD组(P<0.01)。在高糖条件下,C17.2神经干细胞中LC3Ⅱ、LAMP2蛋白表达水平和Cyto-ID染色点的数量均低于正常葡萄糖条件下水平(P<0.05)。PKC-α蛋白敲低增加了高糖条件下的LC3Ⅱ、LAMP2蛋白表达水平和Cyto-ID染色点的数量(P<0.05)。结论:PKC-α介导的线粒体功能障碍与母体糖尿病诱导的NTD胚胎相关,通过沉默母体PKC-α减弱了神经管自噬抑制导致的NTD形成。Objective:To explore the effect of protein kinase C-α(PKC-α)mediated mitochondrial dysfunction on fetal neural tube defects(NTD)caused by gestational diabetes.Methods:Female C57BL/6J mice were randomly divided into control group,NTD group and NTD+sh-PKC-αgroup,with 6 mice in each group.In addition to control group,in NTD group and NTD+sh-PKC-αgroup,diabetic embryonism mouse model were established.Mice in the NTD+sh-PKC-αgroup were injected with 100μL carrying sh-PKC-αlentvirus(1×10^(8) TU/mL)through the tail vein at embryonic day 1(E1),day 4(E4),and day 8(E8),respectively.At day 11.5(E11.5),the embryos were dissected from the uterus for analysis.Mitochondria in neural tube were observed by transmission electron microscope.The expression of PKC-α,anti-microtubule-associated protein 1 light chain 3(LC3)and anti-lysosomal associated membrane protein 2(LAMP2)in embryonic neural tubes were analyzed by Western Blot.PKC-αknockdown C17.2 mouse neural stem cells were constructed in vitro and cultured under low(5 mmol/L)or high(25 mmol/L)glucose conditions.The changes of autophagosomes were detected by immunofluorescence staining.Results:In the NTD group,the mitochondria in the embryonic neural tube swelled and the ridge disappeared.Compared with the control group,the expressions of LC3Ⅱand LAMP2 proteins in the fetal neural tube of NTD group decreased(P<0.05),and PKC-αprotein up-regulated(P<0.01).By silencing the expression of PKC-αin the neural tube of mouse embryos,the expression of LC3Ⅱand LAMP2 proteins in the embryos increased(P<0.05).In addition,the incidence of NTD in NTD+sh-PKC-αgroup was lower than that in NTD group(P<0.01).The expression levels of LC3Ⅱand LAMP2 protein and the number of Cyto-ID staining points in C17.2 neural stem cells were lower than those in normal glucose conditions(P<0.05).PKC-αprotein knockdown increased the expression levels of LC3Ⅱand LAMP2 protein and the number of Cyto-ID staining spots under high glucose condition(P<0.05).Conclusion:PKC-αmediated mitochondrial d

关 键 词：妊娠糖尿病 蛋白激酶C-Α 线粒体 神经管缺陷 自噬 实验研究 

分 类 号：R714.256[医药卫生—妇产科学]