모체열충격이 Hsp70 KO 생쥐의 입천장 중간엽 발생에 미치는 영향
Effects of Maternal Hyperthermia on the Palatal Mesenchyme Development of Hsp70 Knock-out Mice Fetuses
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모체의 고열은 실험동물 및 사람에서 중추신경계를 포함한 다양한 형태의 선천성기형을 유발하는 것으로 알려져 있고 입천장갈림증을 유발한다는 여러 실험 및 임상보고가 있다. 그러나 그 기전에 대해 명확히 밝혀진것은 부족하다. 또한 세포가 고열에 노출되면 방어기전으로 열충격단백질을 합성, 분비한다. Hsp70은 열충격단백질의 한 종류로서 정자발생과 같은 정상발생과정뿐만 아니라 고열과 같은 스트레스에 의한 세포자멸사를 억제하는 기능을 갖는 것으로 알려져 있다. 이에 본 연구에서는 유전자 조작법으로 Hsp70이 결손된 임신한 생쥐를 고열에 노출한 후 입천장돌기 중간엽의 세포바깥바탕질에서 발현되는 fibronectin, TGF-β2 및 FGF-8 면역조직화학염색을 시행하여 모체 고열에 의한 발현양상의 변화를 관찰하고자 하였다. 실험군으로는 고열에 노출된 Hsp70 KO 생쥐 태아를, 대조군으로 고열에 노출되지 않은 Hsp70 WT 생쥐 태아를 사용하였다. 실험군은 임신 제 8.5일에 43C 수조에서 5분간 열충격을 주었고, 대조군에는 아무런 조작도 가하지 않았다. 임신 제 13.5, 14.5 및 15.5일에 모체를 희생하여 자궁내의 태아를 적출한 후 머리를 절취하였다. 일부 표본은 아래턱과 혀를 제거한 후 외형관찰을 하였으며, 나머지 표본은 면역조직화학염색을 시행하였다. 실험군 태아에서는 입천장갈림증이 형성되어 있었다. 면역조직화학염색소견상 실험군의 경우 TGF-β2는 대조군에 비하여 발현이 지연되었고, 태령 제 15.5일에서는 내첨단부 하방의 중간엽에서 강한 반응을 보였으며 내첨단부 상피세포에서 양성반응을 지속적으로 보였다. FGF-8은 중간엽과 상피에서 반응을 나타내었으며, 태령 제 15.5일에서 실험군은 중간엽세포와 상피세포에서 강한 양성반응을 나타내었다. Fibronectin은 태령 제 15.5일 실험군에서 중간엽전반에 걸쳐 강한 양성반응을 보였으며, 바닥막을 따라 강하게 반응을 보였다. 이상의 결과를 종합하면 모체고열은 입천장돌기 중간엽에서 TGF-β2 및 FGF-8의 지속적인 발현과 fibronectin의 지연발현을 유발하여 입천장의 정상적인 발생과정에 영향을 미쳐 입천장갈림증을 유발하는 것으로 생각된다.
Abstract
To investigate the effects of maternal hyperthermia on the development of the palate, pregnant Hsp70 knock-out mice at gestational day (GD) 8.5 were immersed in 43C water bath until their body core temperature reached at 43C. Thereafter, pregnant mice were given more 5 minutes hyperthermic exposure. Heat-untreated Hsp70 WT mice fetuses were used as the control group. Fetuses were collected at embryonic day 13.5, 14.5 and 15.5 (E13.5, E14,5 and E15.5). Heads followed by removal of the mandible and the tongue were obtained and photographed for palatal development. Developing palates were processed for histological and immunohistochemical studies. Tissue sections were immunostained for TGF-β2, FGF-8 and fibronectin, and observed with light microscope. The obtained results were as follows: Cleft palate was formed in heat-treated Hsp70 KO fetuses at E14.5 and E15.5. Immunohistochemical findings indicated that TGF-β2 expression of the experimental fetuses were more delayed than that of the control fetuses. Mesenchyme under the medial edge epithelium (MEE) and cells of MEE showed continuously strong positive TGF-β2 reactivity at E15.5. FGF-8 was revealed in both of the mesenchyme and the epithelium at the same time. FGF-8 immunoreactivity in the mesenchyme and the epithelium of the heat-treated fetuses showed strong reactivity at E15.5. In the experimental fetuses fibronectin was revealed the mesenchyma and basal lamina at E15.5. Taken together, it is suggested that maternal hyperthermia induces continuous expression of TGF-β2 and FGF-8 in the mesenchyme and delayed expression of fibronectin. These should affect the normal palatogenesis and result in cleft palate.
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