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A. Takase, T. Yasukawa, P. Wiedemann, A. Nishiwaki, A. Kato, Y. Ogura; The Mechanism of Basal Membranousgenesis of Retinal Pigment Epithelium and Bestrophin. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6002. doi: https://doi.org/.
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Best vitelliform macular dystrophy (Best disease) is one of early-onset forms of macular degeneration, linked to mutations in bestrophin-1 gene. The purpose of this study is to clarify the relationship between basal membranogenesis of retinal pigment epithelium (RPE) and bestrophin, which is supposed to be a calcium-activated chloride channel, by use of a novel 3D culture system to observe basal functions of RPE.
Human RPE cells were seeded onto 6-well culture plates. On the day when they were confluent, transfection of bestrophin-1 specific siRNAs was performed by use of lipofectamine 2000. After 48 hours, RPE cells were collected and seeded onto 96-well U-bottom culture plates to form spheloids, on the surface of which a monolayer of RPE was constructed. The spheroids with or without bestrophin-1 interference were compared morphologically. One week later, spheloids were sampled for western blotting or fixed in 4% paraformaldehyde for immunohistochemistry. The expression and distribution of actin and elastin in human RPE spheloids were determined by immunohistochemistry. The spheroids without bestrophin-1 interference were treated for an hour with Ca2+ channel blocker, Cl- channel blocker and Ca2+ ionophore at week 1 and evaluated in the same manner.
While the surface of spheroids without siRNAs’ transfection became smooth in a day, reflecting lamellipodial crawling and fusion to cover the surface of the spheroids and possibly initiate Bruch’s membrane formation, spheroids interfered with bestrophin-1 revealed rough surface and membranous deposits. In size, spheroids with siRNAs’ transfection became smaller than those without siRNAs’ transfection. Spheroids exposed to Cl- channel blocker had the similar rough surface. Immunohistochemistry showed that the expression of actin and elastin was reduced in RPE cells transfected with bestrophin-1 siRNAs or treated with Cl- channel blocker.
The present study suggested that bestrophin might function as Cl- channel in spheroid culture. Cl- channel might play a role in biogenesis of Bruch's membrane.
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