2G型肢带肌营养不良症斑马鱼疾病模型的构建  

ESTABLISHMENT OF A ZEBRAFISH DISEASE MODEL OF LIMB-GIRDLE MUSCULAR DYSTROPHY TYPE 2G

作　　者：李丽萍 钟静 牛玉娟 孙源超 吴传鸿 李美航 周建峰 丁永和 LI Liping;ZHONG Jing;NIU Yujuan;SUN Yuanchao;WU Chuanhong;LI Meihang;ZHOU Jianfeng;DING Yonghe(Department of Biochemistry and Molecular Biology, School of Basic Medicine, Qingdao University, Qingdao 266071, China)

机构地区：[1]青岛大学基础医学院生物化学与分子生物学系,山东青岛266071 [2]青岛大学生物医学研究院,山东青岛266071 [3]不详

出　　处：《精准医学杂志》2021年第4期299-303,307,共6页Journal of Precision Medicine

基　　金：国家自然科学基金面上项目(82070394)。

摘　　要：目的构建2G型肢带肌营养不良症(LGMD2G)斑马鱼疾病模型,并初步探讨其致病机制。方法通过CRISPR/Cas9基因编辑技术进行sgRNA制备、显微注射、tcap基因纯合突变斑马鱼筛选,构建tcap基因功能缺失的纯合突变斑马鱼。通过石蜡切片和苏木精-伊红(HE)染色观察野生型(WT)斑马鱼和tcap基因纯合突变斑马鱼的骨骼肌肌原纤维形态。使用新一代斑马鱼游泳速度测试系统检测WT斑马鱼(A组)和tcap基因纯合突变斑马鱼(B组)的最大游泳速度。将WT斑马鱼胚胎分别经空白处理、漂白剂处理、剪切损伤处理、加兰他敏处理,分别作为C、D、E、F组,采用原位杂交实验检测C~F组斑马鱼胚胎tcap mRNA表达情况。再将WT斑马鱼胚胎和M1型tcap基因纯合突变斑马鱼胚胎分为WT斑马鱼胚胎空白对照组(G组)、WT斑马鱼胚胎加兰他敏处理组(H组)、M1型tcap基因纯合突变斑马鱼胚胎空白对照组(I组)和M1型tcap基因纯合突变斑马鱼胚胎加兰他敏处理组(J组),应用实时荧光定量PCR(RT-qPCR)技术检测C~F组斑马鱼胚胎tcap mRNA相对表达量,检测G~J组斑马鱼胚胎p53和p21 mRNA的相对表达量。结果核酸电泳结果显示成功合成了长约100 bp的斑马鱼tcap基因编辑所需sgRNA;DNA测序结果显示筛选获得了2种tcap基因纯合突变斑马鱼。HE染色实验结果显示,与WT斑马鱼相比,tcap基因纯合突变斑马鱼的骨骼肌肌原纤维明显紊乱。斑马鱼最大游泳速度检测实验结果显示,相较于A组,B组斑马鱼的最大游泳速度显著降低(t=3.32,P<0.05)。原位杂交实验结果显示,与C组相比,D~F组斑马鱼胚胎中tcap mRNA表达量明显增加。RT-qPCR实验结果显示,C~F组斑马鱼胚胎中tcap mRNA相对表达量比较差异有显著性(F=21.70,P<0.05),其中D~F组明显高于C组(t=4.95~35.44,P<0.05);G~J组斑马鱼胚胎中p53和p21 mRNA相对表达量比较差异有显著性(F=8.45、33.04,P<0.05),其中J组明显低于H、I组(t=3.24~8.94,P<0.05)�Objective To establish a zebrafish disease model of limb-girdle muscular dystrophy type 2G(LGMD2G),and to preliminarily investigate its pathogenic mechanism.Methods The CRISPR/Cas9 gene editing technique was used for sgRNA preparation,microinjection,and screening of zebrafish with homozygous mutation in the tcap gene,so as to obtain the zebrafish with homozygous mutation and loss of the tcap gene function.Paraffin section and hematoxylin-eosin(HE)staining were used to observe the morphology of skeletal muscle myofibrils in wild-type(WT)zebrafish and the zebrafish with homozygous mutation of the tcap gene.A new-generation zebrafish swimming speed testing system was used to evaluate the maximum swimming speed of WT zebrafish(group A)and the zebrafish with homozygous mutation of the tcap gene(group B).WT zebrafish embryos were divided into groups C,D,E,and F and received blank control treatment,bleach treatment,shear injury treatment,and galantamine treatment,respectively,and in situ hybridization was used to measure the mRNA expression of tcap in zebrafish emb-ryos in groups C,D,E,and F.WT zebrafish embryos and zebrafish embryos with M1-type homozygous mutation of the tcap gene were divided into WT zebrafish embryo blank control group(group G),WT zebrafish embryo galantamine treatment group(group H),blank control group of zebrafish embryos with M1-type homozygous mutation of the tcap gene(group I),and galantamine treatment group of zebrafish embryos with M1-type homozygous mutation of the tcap gene(group J),and quantitative real-time PCR(RT-qPCR)was used to measure the relative mRNA expression level of tcap in groups C,D,E,and F and the relative mRNA expression levels of p53 and p21 in groups G,H,I,and J.Results Nucleic acid electrophoresis showed that sgRNA with a length of about 100 bp was successfully synthesized for zebrafish tcap gene editing,and DNA sequencing results suggested that two types of zebrafish with homozygous mutation of the tcap gene were obtained.HE staining showed that compared with WT zebrafis

关 键 词：肌营养不良 基因编辑 功能缺失突变 tcap基因 基因表达调控 疾病模型 斑马鱼 

分 类 号：R746.2[医药卫生—神经病学与精神病学] R-332[医药卫生—临床医学]