斑马鱼早期胚胎发育过程中IL-10表达的特点  

Expression of IL-10 in zebrafish during early embryonic development

作　　者：赵砚驰 李化 李丹 孔鹏 张乙进 吴洁玲 莫大双 舒莉萍 ZHAO Yanzhi;LI Hua;LI Dan;KONG Peng;ZHANG Yijin;WU Jieling;MO Dashuang;SHU Liping(Division of Immunology,National&Guizhou Joint Engineering Laboratory for Cell Engineering and Biomedicine Technique,Guizhou Province Key Laboratory for Regenerative Medicine,State Key Laboratory of Functions and Applications of Medicinal Plants,School of Basic Medicine,Guizhou Medical University,Guiyang 550004,Guizhou,China;Key Laboratory of Adult Stem Cell Translational Research,Chinese Academy of Medical Sciences,Guiyang 550004,Guizhou,China)

机构地区：[1]贵州医科大学基础医学院免疫学教研室,细胞工程生物医药技术国家地方联合工程实验室,贵州省再生医学重点实验室,省部共建药用植物功效与利用国家重点实验室,贵州贵阳550004 [2]中国医学科学院成体干细胞转化研究重点实验室,贵州贵阳550004

出　　处：《贵州医科大学学报》2022年第9期1000-1006,1013,共8页Journal of Guizhou Medical University

基　　金：国家自然科学基金(32160168);贵州省科技计划项目(黔科合CXTD[2021]002,[2019]1436,[2019]4008)。

摘　　要：目的探讨野生型斑马鱼白细胞介素10(IL-10)在胚胎早期发育过程中的表达特点。方法在美国国家生物信息技术中心(NCBI)数据库中分别查询人类、小鼠及斑马鱼染色体中IL-10的基因位置,与Ensemble数据库中的数据进行比对,采用软件分子进化遗传分析软件(MEGA X)绘制系统进化树;收集3组72受精后小时(hpf)斑马鱼胚胎、30枚/组,提取总RNA(Total RNA)及合成互补脱氧核糖核酸(cDNA),构建斑马鱼重组质粒pCS^(2+)-IL-10,体外转录合成地高辛标记的IL-10反义RNA探针;分别收集4、6、9、12、18、24、48及72 hpf的斑马鱼胚胎75枚(25枚/组、3组),采用全胚胎原位杂交技术检测斑马鱼胚胎不同发育时间点IL-10表达及分布情况。结果野生型斑马鱼IL-10基因与人类IL-10基因相似度较高,且在进化过程中相对保守;限制性内切酶双酶切重组质粒及测序结果显示,重组质粒构建无误;全胚胎原位杂交结果显示,4~12 hpf野生型斑马鱼胚胎中未观察到阳性杂交信号,18 hpf野生型斑马鱼胚胎中后部中间细胞群观察到IL-10阳性杂交信号,24~72 hpf野生型斑马鱼胚胎胚胎头部区域IL-10广泛表达,头部背侧、心脏、眼眶以及卵黄囊背侧部位的信号尤为明显。结论斑马鱼IL-10在进化过程中具有相对保守性,在早期胚胎发育过程中IL-10有不同程度的表达,提示IL-10在斑马鱼胚胎早期发育中发挥重要作用。Objective To investigate the expression of Interleukin 1(IL-10)in the early development phase of wild-type zebrafish embryos.Methods The loci of IL-10 in human,mouse,and zebrafish chromosomes were queried in National Center for Biotechnology Information(NCBI)database and then compared with the data in Ensemble database.The phylogenetic tree was composed by molecular evolutionary genetic analysis software(MEGA X).Three groups of 72 hours-post-fertilization(hpf)zebrafish embryos were collected(30 each),and total RNA and synthesize complementary DNA(cDNA)were extracted;pCS 2+-IL-10 recombinant plasmids of zebrafish were constructed for transcription of digoxin-labeled IL-10 antisense RNA probes in vitro.Three groups of 4,6,9,12,18,24,48,and 72 hpf zebrafish embryos were collected(25 embryos each),and the expression and distribution of IL-10 mRNA in zebrafish embryos at different developmental time points were detected by the whole embryo in situ hybridization.Results The IL-10 gene of wild-type zebrafish showed relatively high similiarity with that of human plus relatively conversed nature in the evolutionary process.Recombinant plasmid and sequencing results of restrictive endonuclease BamHI showed that the recombinant plasmid was constructed correctly.The whole embryo in situ hybridization experiments showed that no positive hybridization signal was observed in zebrafish embryos from 4 to 12 hpf.At 18 hpf,IL-10 positive hybridization signal was observed in the middle-posterior cell mass.At 24 to 72 hpf,IL-10 was comprehensively expressed in head region of embryo,particularly signals of dorsal head,heart,orbit,and dorsal yolk showed was obvious.Conclusion Zebrafish IL-10 is relatively conserved during evolution phase,and IL-10 is expressed in different degrees during early embryonic development,indicating that IL-10 plays an important role in the early development of zebrafish embryos.

关 键 词：斑马鱼 白细胞介素10 原位杂交 细胞因子类 胚胎早期发育 重组质粒构建 

分 类 号：R392.1[医药卫生—免疫学]