cblC型甲基丙二酸血症MMACHC基因新突变对小鼠神经细胞凋亡及Wnt/β-catenin信号通路的作用机制  

Mechanism of MMACHC gene mutation of cblC-type methylmalonic acidemia on apoptosis and Wnt/β-catenin signaling pathway in mouse neural cells

作　　者：周伟 蔡恒 范海迪 李惠中 王传霞[1] 顾茂胜 Zhou Wei;Cai Heng;Fan Haidi;Li Huizhong;Wang Chuanxia;Gu Maosheng(Center of Genetic Medicine,Xuzhou Maternity and Child Health Care Hospital,Xuzhou 221009,Jiangsu Province,China;Xuzhou Medical University,Xuzhou 221004,Jiangsu Province,China;Clinical Laboratory,The First Branch of Huai′an First Poeple′s Hospital,Huai′an 223300,Jiangsu Province,China)

机构地区：[1]徐州市妇幼保健院遗传医学中心,徐州221009 [2]徐州医科大学,徐州221004 [3]淮安市第一人民医院第一分院检验科,淮安223300

出　　处：《中华妇幼临床医学杂志（电子版）》2022年第5期528-539,共12页Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition)

基　　金：江苏省妇幼健康科研项目(F201912);徐州市科技计划项目(KC19028、KC20074)。

摘　　要：目的探讨cblC型甲基丙二酸血症(MMA)致病基因MMACHC新突变,对小鼠神经细胞凋亡及Wnt/β-catenin信号通路的作用机制。方法选择2017年5月和7月,江苏省新生儿疾病筛查中心徐州分中心筛查确诊2例cblC型MMA患儿的血液样本中,检出的c.626_627del及c.228_231del 2种MMACHC基因新错义突变为研究材料。分析MMACHC基因的2种新突变所累及的氨基酸残基的保守性及预测其致病性后,构建MMACHC基因2种新突变重组质粒,并与野生型MMACHC基因重组质粒,分别瞬时转染小鼠神经细胞(神经元细胞株HT22及神经干细胞株C17.2)进行体外实验。将MMACHC基因c.626_627del质粒转染的小鼠HT22、C17.2细胞,分别纳入神经元观察1组、神经干观察1组;将MMACHC基因c.228_231del质粒转染的小鼠HT22、C17.2细胞,分别纳入神经元观察2组、神经干观察2组;将野生型MMACHC基因重组质粒转染的小鼠HT22、C17.2细胞,分别纳入神经元对照组、神经干对照组。采用免疫荧光染色、TUNEL荧光检测及蛋白质印迹法(Western blotting)等,分析小鼠神经细胞凋亡情况及Wnt/β-catenin信号通路相关蛋白表达情况。采用单因素方差分析及Dunnett t检验,对2个观察组分别与对照组神经细胞的细胞凋亡率,凋亡相关蛋白及Wnt/β-catenin信号通路相关蛋白表达水平进行统计学比较。本研究经过徐州妇幼保健院伦理委员会批准[审批文号:〔2019〕伦审第(09)号]。结果①MMACHC基因2种新突变所在氨基酸位点(第77位及第209位),在人源、家鼠、褐鼠、斑马鱼及猕猴5个物种间高度保守。生物信息学分析显示,2种突变均可能具有致病性。②与对照组相比,与之对应的2个观察组神经细胞的细胞核DAPI染色着色更深(亮蓝色),TUNEL荧光阳性(绿色)细胞数更多。神经元观察1组细胞凋亡率(DAPI染色)及凋亡率(TUNEL染色)分别为(25.8±1.1)%、(23.1±1.4)%,均高于神经元对照组的(4.9±0.9)%、(3.2±0.4)%,并且�Objective To investigate the mechanism of novel variants in MMACHC gene resulting in cblC-type methylmalonic acidemia(MMA)on neural cell apoptosis and Wnt/β-catenin signaling pathway in mice.Methods Two novel missense variants of c.626_627del and C.228_231del of MMACHC gene which were detected in blood samples of two infants with cblC-type MMA screening and diagnosed by Xuzhou Branch Center of Jiangsu Newborn Screening Center in May and July 2017,were selected as research materials.After analyzing the conservation of amino acid residues involved in the two novel variants and predicting the pathogenicity,two new recombinant plasmids of MMACHC gene were constructed,and with the recombinant plasmids of wild-type MMACHC gene,they were transiently transfected into mouse nerve cells(neuron cell line HT22 and nerve stem cell line C17.2)for in vitro experiments.Mice HT22 and C17.2 cells transfected with MMACHC gene c.626_627del plasmid were included into neuron observation group 1 and nerve stem observation group 1,respectively.Mice HT22 and C17.2 cells transfected with MMACHC gene C.228_231del plasmid were included into neuron observation group 2 and nerve stem observation group 2,respectively.Mice HT22 and C17.2 cells transfected with wild-type MMACHC gene recombinant plasmid were included into neuron control group and nerve stem control group,respectively.Immunofluorescence staining,TUNEL fluorescence detection and Western blotting were used to analyze the apoptosis of mouse nerve cells and the expression of Wnt/β-catenin signaling pathway related proteins.One-way ANOVA and Dunnett t test were used to statistically compare the apoptosis rate,apoptosis-related proteins and the expression levels of Wnt/β-catenin signaling pathway related proteins between two observation groups and control group,respectively.This study was approved by the Ethics Committee of Xuzhou Maternity and Child Health Care Hospital[Approval No.2019(09)].Results①The amino acid sites of two novel variants in MMACHC gene(positions 77 and 209)wer

关 键 词：甲基丙二酸血症 MMACHC基因 神经元 神经干细胞 细胞凋亡 糖原合成酶激酶3 Β连环素 小鼠 

分 类 号：R722.1[医药卫生—儿科] R-332[医药卫生—临床医学]