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目的:探讨黑色素瘤抑制蛋白(melanoma inhibitory activity,MIA)在非洲爪蟾早期胚胎发育中的作用。方法:根据热带爪蟾MIA序列在非洲爪蟾EST数据库中找到MIA序列,利用RT-PCR扩增全长序列并克隆至pCS2+,用于测序。利用RT-PCR检测胚胎发育各阶段MIA mRNA的表达。通过显微注射特异的反义寡核苷酸morpholino(MO)敲降MIA;利用整胚原位杂交检测胚层标志性基因的表达。结果:克隆出非洲爪蟾MIA全长序列。在胚胎发育过程中MIA从受精卵开始持续表达。敲降MIA导致胚胎胚孔闭合延缓,中胚层标志基因xbra表达明显受抑,而内胚层标志性基因sox17α表达无明显改变。结论:MIA表达于非洲爪蟾胚胎发育各阶段,敲降MIA抑制中胚层的形成。
Objective: To investigate the role of melanoma inhibitory activity (MIA) in Xenopus early embryo development. Methods: The MIA sequence was found in the Xenopus EST database according to the tropical Xenopus MIA sequence. The full-length sequence was amplified by RT-PCR and cloned into pCS2 + for sequencing. The expression of MIA mRNA in each stage of embryonic development was detected by RT-PCR. MIA was knocked down by microinjection of specific antisense oligonucleotide morpholino (MO); the expression of landmark genes of germ layers was detected by in situ hybridization. Results: The full-length sequence of Xenopus laevis MIA was cloned. MIA expresses continuously from fertilized eggs during embryonic development. The knockdown of MIA resulted in the delay of embryonic embryo closure and the suppression of xbra expression in the mesoderm, while the sox17α expression of the endoderm showed no significant change. Conclusion: The MIA expression in Xenopus laevis embryos at various stages of development, knock MIA inhibit mesoderm formation.