胚胎小鼠腭器官培养中EGF受体反义寡核苷酸的组织摄取及其对体外腭发育的影响

H. Naitoh, C. Mori, N. Ohyama, Hidekazu Irie, N. Nakamura, Y. Nishimura, K. Shiota
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摘要

为了研究寡核苷酸(ODNs)在体外培养的小鼠胎儿腭组织中的掺入及其对体外腭发育的影响,我们在化学定义的无血清培养基中培养了12.5天的小鼠胎儿腭,培养基中添加了表皮生长因子受体(EGF - r)的反义或义ODNs。EGF - r odn被发现与腭组织结合,并在至少72小时内仍可检测到。免疫组织化学和免疫印迹分析显示,5μM EGF - r反义ODN处理可抑制EGF - r蛋白的产生。5 μM EGF - r反义ODN和正义ODN对离体腭未见病理改变,但10和20 μM ODN处理引起腭上皮固缩改变,可能与ODN毒性有关。本研究结果表明,在最佳条件下,EGF - r的反义odn可以被纳入体外培养的胎儿器官,并特异性抑制EGF - r蛋白的产生。由于抑制EGF - r蛋白的产生并不能阻止腭融合,因此正如先前的研究表明的那样,EGF和/或EGF - r单独在腭形成中可能并不起关键作用。反义ODN技术可用于分析特定分子在正常和异常形态发生中的作用。
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Tissue uptake of EGF receptor antisense oligonucleotides in organ culture of fetal mouse palates and their effects on in vitro palatogenesis
ABSTRACT To investigate the incorporation of oligonucleotides (ODNs) into the tissues of cultured fetal mouse palates and their effects on in vitro palatogenesis, we cultured day‐12.5 fetal mouse palates in a chemically defined serumless medium supplemented with either antisense or sense ODNs to epidermal growth factor receptor (EGF‐r). The EGF‐r ODNs were found to be incorporated into the palatal tissue and remained detectable for at least 72 hr. Immunohistochemical and immunoblot analyses revealed that the treatment with 5μM EGF‐r antisense ODN suppressed the production of EGF‐r protein. No pathological change was observed in the explanted palates when they were treated with 5 μM EGF‐r antisense or sense ODNs, but the treatment with 10 or 20 μM ODN caused pyknotic changes in the palatal epithelium, probably due to the ODN toxicity. The present results indicate that under optimal conditions, antisense ODNs to EGF‐r can be incorporated into fetal organs cultured in vitro and specifically inhibit the production of EGF‐r protein. Since the suppression of the production of EGF‐r protein did not prevent the palate fusion, EGF and/or EGF‐r alone may not play a critical role in palatogenesis, as suggested by previous studies. The antisense ODN technique could be of potential use for analyzing the roles of specific molecules in normal and abnormal morphogenesis.
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