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Immunohistochemical study on the expression and localization of the epidermal growth factor receptor during morphogenesis

形态发生过程中表皮生长因子受体表达和定位的免疫组织化学研究

基本信息

批准号：
02670007
负责人：
SHIOTA Kohei
金额：
$ 1.28万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

1. Immunohistochemical studies using the anti-human EGF receptor(EGF-R)rabbit antibody revealed that fetal mouse palates show regional heterogeneity and temporal sequence in EGF-R expression during development. On days 12 and 13, the immunoreactive products were predominantly positive on the oral and medial edge epithelia but minimally on the epithelium of the vertical shelf. The EGF-R immunoreactivity was less intense in the posterior palate as compared with the midpalatal region. The palatal mesenchyme also showed regional heterogeneity and temporal sequence in EGF-R expression.2. Explanted palates of day-12 mouse fetuses were cultured in a chemically-defined medium and the effects of all-trans-retinoic acid(RA)were examined. RA was added to the culture medium at the concentrations 3-300 ng/ml. RA inhibited palatal fusion dependently on its concentration, although palatal shelves of -normal size tonned and made contact at 3-30 ng/ml. Scanning electron microscopic studies showed that … More the medial cells of RA-treated palates failed to undergo programmed cell death which occurs in control palates. The oral epithelium of control palates lost microvilli and became squamous after palatal fusion, but RA-exposed cells remained ciliated and simulated nasal-hke epithelial cells.During in vitro palatogenesis, both palatal epithelium and mesenchyme showed regional heterogeneity and temporal sequence in EGF-R expression. During normal palatal development, the EGF-R immunoreactivity became less intense in the medial epithelia. However, in retinoic acid-treated palates, the medial epithelia were continuously positive for EGF-R immunoreactivity. In retinoic acid-treated palates, medial cells were continuously covered with microvilli which disappeared in untreated palates. The alteration in EGF-R expression and epithelial cell differentiation may be responsible for the altered differentiation of palatal shelves and retinoic acid-induced cleft palate.3. The localization of EGF-R in normal human embryos(Carnegie stages 15, 16, and 20)was examined immunohistochemically. Embryonic tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and serialy sectioned. The sections were stained by avidin-biodn-peroxidase complex method(Vectastain elite ABC kit)with polyclonal anti-human EGF-R antibody. The immunoreactive products against EGF-R were positive in the periderm, the epithelium of the digestive and respiratory tracts, the notochord, the epithelium of the Ratlike's pouch, the fetal cortex of the adrenal gland, germ cells in the gonad, the myocardium and cartilaginous tissue. In the myocardium, the immunoreactivity was intense at Carnegie stages 15 and 16, but became weaker by stage 20. The regional heterogeneity of EGF-R expression was observed in the Ratlike's pouch. Less

1. 使用抗人EGF受体（EGF- r）兔抗体的免疫组织化学研究显示，胚胎小鼠腭在发育过程中EGF- r的表达具有区域异质性和时间序列。在第12天和第13天，免疫反应产物在口腔和内侧边缘上皮上主要呈阳性，而在垂直架子上皮上呈最低阳性。后腭EGF-R免疫反应性较中腭弱。腭间质中EGF-R的表达也表现出区域异质性和时间序列。在化学定义的培养基中培养12天小鼠胚胎的外植腭，并检测全反式维甲酸（RA）的作用。在培养基中加入浓度为3 ~ 300 ng/ml的RA。RA对腭融合的抑制依赖于其浓度，尽管正常大小的腭架在3-30 ng/ml时可以调整并接触。扫描电镜研究表明，经ra处理的上颚内侧细胞未发生正常上颚的程序性细胞死亡。对照组的口腔上皮在腭融合后失去微绒毛并呈鳞状，但暴露于ra的细胞仍保持纤毛状并模拟鼻样上皮细胞。在体外造腭过程中，腭上皮和间充质细胞EGF-R的表达均表现出区域异质性和时间序列。在正常腭发育过程中，内侧上皮的EGF-R免疫反应性减弱。然而，在维甲酸处理的腭中，内侧上皮细胞的EGF-R免疫反应性持续呈阳性。在维甲酸处理的腭中，内侧细胞连续被微绒毛覆盖，而未处理的腭中微绒毛消失。EGF-R表达和上皮细胞分化的改变可能与腭架分化和维甲酸诱导的腭裂有关。用免疫组织化学方法检测EGF-R在正常人类胚胎（卡内基15、16和20期）中的定位。胚胎组织用4%多聚甲醛固定，石蜡包埋，连续切片。切片采用抗人EGF-R多克隆抗体avidin-biodn-peroxidase复合物法（Vectastain elite ABC kit）进行染色。外周、消化道和呼吸道上皮、脊索、大鼠囊上皮、胎儿肾上腺皮质、性腺生殖细胞、心肌和软骨组织的EGF-R免疫反应产物均呈阳性。在心肌中，免疫反应性在卡内基第15期和第16期很强，但在第20期减弱。大鼠样眼袋中EGF-R表达具有区域异质性。少