아녹타민 채널군의 망막색소상피에서의 발현에 관한 연구 : The Expression of Anoctamin Channel Family in the Retinal Pigment Epithelium

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분자의학 및 바이오제약학과
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서울대학교 대학원
학위논문 (석사)-- 서울대학교 대학원 : 분자의학 및 바이오제약학과, 2012. 2. 오우택.
Anoctamin is a broadly expressed Ca2+ activated Cl- channel which is predominantly expressed in epithelial tissues. The reported phenotype of TMEM16A knockout mouse is tracheomalacia, which indicate its functional role between epithelium and submucosal tissue during development. Pattern of frequency of expressed sequence tags for TMEM16A obtained in human tissues suggests that the protein family may provide an important role in the structural and functional integrity of the eye.
Interestingly, Bestrophin, another family of Ca2+ activated Cl- channel, exists in retinal pigment epithelial (RPE) cells of the eye and its mutation is presented with vitelliform macular dystrophy (Best disease). As one of important roles of RPE cells is transport of ions and other molecules, its mutation is associated with accumulation of deposits in subretinal space. However, recent studies reported that Ca2+ activated Cl- currents are not absent in bestrophin knockout mice, indicating the presence of other Ca2+ activated Cl- channel.
Thus, we investigated the presence of Anoctamin channel family in the retinal pigment epithelium and explore its function within the cells. In this study, we also intended to explore the relationship between Anoctamin family and Bestrophin.
Using rabbit monoclonal antibodies, immunocytochemical staining of mouse eyes were performed. We also probed the retinal pigment epithelium-derived cell lines with the same antibody. The expression of Anoctamin family mRNAs were investigated by reverse transcription-PCR (RT-PCR) in human RPE cell lines. In knockout mouses of TMEM16A (ANO1) and TMEM16F (ANO6), the immunocytochemical staining was also performed.
Immunocytochemical staining of mouse eyes indicated that ANO1 and ANO6 are localized at the basolateral plasma membrane of RPE cells. The presence of ANO1 and ANO6 were also confirmed by RT-PCR in human RPE cell lines Whereas ANO6 is localized in both neuronal cells and epithelial cells, ANO1 is more specifically localized in RPE cells. The images also revealed that ANO1 showed alternative localization pattern with Bestrophin.
ANO family showed diverse expression pattern within the retina and is considered to provide functional role of Ca2+ activated Cl- channel in RPE cells. Further functional studies on ANO family in RPE cells and the relation with Bestrophin are required.
Anoctamin은 다양한 조직에서 발현되는 염소 이온 채널로서, 주로 상피 조직에서 발현하는 것으로 알려져 있다. 이전의 발현 양상에 관한 연구에서 TMEM16A는 눈에서 상당한 발현이 관찰되어 기능적 및 구조적으로 중요한 역할을 할 것으로 생각된다.
이전 연구에서 또다른 염소이온 채널인 bestrophin이 망막 색소 상피에서 발현하는 것으로 알려져 있다. 이 유전자의 돌연변이는 vitelliform macular dystrophy (노른자모양 황반변성)을 일으키는 것으로 알려져 있다. 하지만 bestrophin knockout 쥐에서 염소 전류가 없어지지 않는 점은 망막 색소 상피에서 또 다른 염소 이온 채널의 존재를 암시한다.
따라서 본 연구에서는 anoctamin 이온 채널이 망막 색소 상피에서 발현되는지를 알아보고자 하였다. RT-PCR 및 면역조직화학을 이용하여 anoctamin이 망막 내에 다양한 발현 양상을 보이며, ANO1 과 bestrophin이 특히 망막색소상피의 기저막에 특이적으로 발현하였다. ANO1과 bestrophin은 같은 막에서 발현함에도 불구, colocalization을 보이지 않는 특징적인 localization을 보였다. 이의 기능적인 의미와 전기생리학적인 연구가 추후 이루어져야 할 것이다.
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Graduate School of Convergence Science and Technology (융합과학기술대학원)Dept. of Molecular and Biopharmaceutical Sciences (분자의학 및 바이오제약학과)Theses (Master's Degree_분자의학 및 바이오제약학과)
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