斑马鱼早期胚胎发育过程中IL-1β表达的特点

Expression of IL-1β in zebrafish during early embryonic development

目的 探讨野生型斑马鱼胚胎早期发育中白细胞介素1β(IL-1β)的表达特点。方法 选取美国国家生物信息技术中心(NCBI)数据库中人类、斑马鱼、猪、绵羊、水牛、小鼠、大鼠、家兔以及豚尾猴9个不同物种IL-1β氨基酸序列进行同源性比对,采用分子进化遗传分析软件(MEGA X)绘制系统进化树;收集4组72受精后小时(hpf)斑马鱼胚胎、25枚/组,提取总RNA及合成互补脱氧核糖核酸(cDNA),构建pGEMT-IL-1β重组质粒并合成地高辛标记的IL-1β反义RNA探针;分别收集4、6、9、12、18、24、48及72 hpf斑马鱼胚胎、25枚/组,采用整胚原位杂交技术检测IL-1β在斑马鱼早期不同时间胚胎发育中的表达及分布状况。结果 IL-1β氨基酸序列在不同物种中相对保守;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;原位杂交实验结果表明,4 hpf斑马鱼胚胎中开始观察到IL-1β阳性杂交信号表达,6~12 hpf斑马鱼胚胎中观察到IL-1β阳性杂交信号持续表达,12 hpf斑马鱼胚胎中表达尤为明显,18 hpf斑马鱼胚胎躯干背侧、眼睛以及中后部中间细胞群等部位观察到不同程度的IL-1β阳性杂交信号表达,24 hpf斑马鱼胚胎全身躯干观察到IL-1β广泛表达,且头部及眼睛部位表达尤为明显,48~72 hpf斑马鱼胚胎观察到IL-1β表达逐渐从头部延伸至全身躯干且于肌肉组织中表达尤为明显。结论 斑马鱼IL-1β在进化过程中具有相对保守性,斑马鱼早期胚胎发育过程中IL-1β有不同程度的表达。

Objective To investigate the expression of interleukin 1β(IL-1β) in the early development of wild-type zebrafish embryos.Methods The amino acid sequences of IL-1β of 9different species( human, zebrafish, swine, sheep, buffalo, mouse, rat, rabbit, and Macaca nemestrina) in the database of the National Center for Biotechnology Information( NCBI) were selected for homology alignment, using molecular evolutionary genetic analysis software(MEGA X) to compose a phylogenetic tree. Four groups of 72 hours-post-fertilization(hpf) zebrafish embryos were collected(25 each), and total RNA and synthesize complementary DNA( cDNA) were extracted;pGEMT-IL-1βrecombinant plasmids were constructed for synthesizing digoxin-labeledIL-1βantisense RNA probes. Four groups of 4, 6, 9, 12, 18, 24, 48, and 72 hpf zebrafish embryos were collected(25 embryos each), and the expression and distribution ofIL-1βin zebrafish embryos at different developmental time points were detected by the whole embryoin situhybridization.Results The IL-1βamino acid sequences showed relatively conversed nature in multiple species. Recombinant plasmid and sequencing results of restrictive endonuclease BamHI showed that the recombinant plasmid was constructed correctly. The whole embryoin situhybridization experiments showed thatIL-1βpositive hybridization signal expression was observed in zebrafish embryos at 4 hpf. In 6 to 12 hpf embryo,IL-1βpositive hybridization signal constant expression was observed;at 12 hpf, expression in embryo was particularly obvious;at 18 hpf,IL-1βpositive hybridization signal of varied digrees were observed in dorsal back, eye and middle-posterior cell mass;at 24 hpf,IL-1βwas comprehensively expressed in head and eye parts of embryo in particular;from 48 to 72 hpf,IL-1βexpression gradually extended from head to dorsal part, particularly in muscle tissues.Conclusion ZebrafishIL-1βis relatively conserved during evolution phase, andIL-1βis expressed in different degrees during early embryonic development.