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Q Wen, Y Li, K Kuang, B Yerxa, J Fischbarg; Effects of P2Y2 Agonists on Lipid Secretion by Cultured Rabbit Meibocytes . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3146.
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Purpose: We have reported that P2Y2 purinergic receptor agonists stimulate both chloride and fluid secretions by the isolated rabbit conjunctiva. In the current study, we cultured meibocytes on plastic and glass surfaces and detected the meibum lipids they contained using the fluorescent dye Nile red. Method: 1). Cultured meibomian gland epithelial cells: The eyes (including eyelids) were enucleated obtained from rabbits. Meibomian glands were freed from tarsal connective tissue and conjunctival mucosa under microscope by enzymatic digestion. After digestion, single meibomian glands were isolated and gland epithelial cells were dissociated by 2 mM EDTA in PBS. Cells from 2 eyes were seeded into a 35 mm culture dish containing DMEM/F-12 plus EGF (1 ng/ml), 5% fetal bovine serum, 6% rabbit serum, insulin (5 mg/l), transferrin (10 mg/l), sodium selenite (5 µg/l) and gentamicin. 2). Measurement of intracellular lipid of meibomian cells: After confluence, the primary cultured cells were harvested and plated on glass coverslips. Prior to the day of the experiment, the bathing solution was replaced by serum-free DMEM/F12. The cells were incubated in HCO3--HEPES (BH) Ringer's solution with 100 ng/ml of Nile red for 20 min at room temperature. After that, the glass coverslips was washed three times with BH and placed in a perfusion chamber and fluorescence was determined continuously (excitation: 470 nm, emission: 530 nm) with a PTI Delta-Scan based system using FelixTM software. After stabilization, the control solution was replaced by the test solution. Results: Primary cultures of Meibomian gland epithelial cells grew very slowly. Cells needed 16 days or more to reach confluence. However, the meibomian cells grow at a faster rate on both plastic and glass after the first passage. Meibomian cells on glass coverslips did not fluoresce spontaneously, and neither did cultured bovine corneal endothelial cells (CBCEC) nor glass coverslips without cells (both as control). After loading cells with Nile red, the intensity of fluorescence increased to a much higher level in the meibomian gland cells than in CBCEC. Both INS365 and UTP, P2Y2 agonists, resulted in a fluorescence increase in meibomian gland cells. Conclusions: The known presence of lipid droplets in meibomian cells had an expected strong counterpart in the Nile red fluorescence they display, and the purinergic agonists might stimulate the formation of lipid-containing vesicles by these cells. CR: C5. Support: Inspire Pharmaceuticals, and RPB, Inc.
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