P12 - PTHC1: A Continuing Cell Line Expressing PTH and Genes Involved in Calcium Homeostasis

摘要

The main organs regulating serum levels of ionised calcium (Ca2+) are the parathyroids, which are composed of two different cell types: chief cells and oxyphil cells. Chief cells, through the calcium sensing receptor (CaSR), are affected by changes in calcium concentration, modifying PTH secretion in proportion to calcium levels. Current understanding of calcium regulation mechanisms connected to PTH and of the signalling pathways involved derive from in vitro studies carried out on primary cultures of scattered parathyroid cells, because there do not exist parathyroid cell lines able to excrete calcium-regulated PTH. Indeed, it is very difficult to obtain continuous parathyroid cell lines that conserve their functional characteristics because these cells, once cultured, quickly lose their response to calcium. PT-r cells, obtained in 1995 by subsequent clonings, constitute, to date, the only continuous parathyroid cell line described in the literature. This study describes a new cell clone able to secrete PTH, called PTHc1, obtained from hyperplastic tissue of the parathyroid rat.Materials and methods:>Cell cultures, subcloning and karyotype analysis: The PTHc1 epithelial cell line was cloned by dilution from primary cultures. Ham’s F-12 medium, modified according to Coon and supplemented with calcium 1.1 mM, 10% foetal bovine serum, 100 IU/ml penicillin and 100 mg/ml streptomycin, was used as culture medium. For the karyotype analysis, cells were treated for 4 hours with Colcemid 10-6 M. After the hypotonic treatment for 30 minutes at 37°C with a 0.75% sodium citrate solution cells were fixed in methanol:acetic acid (3:1). More than 300 cells were analysed in metaphase. >Analysis of cell growth: PTHc1 cells were plated at 20 cells/cm2 density in culture medium. Growth was estimated in a Burker chamber every 24 h for 7 days. >PTH expression and analysis of genes involved in calcium homeostasis: RT-PCR reactions of genes PTH, PTHR, PHLP, Klotho, FGF23, FGF23-R1, FGF23-R2, FGF23-R3, FGF23-R4, ER, ER, GH-R, CDH-1, CDH-7, HRPT2, LRP-5, GCMB-1, GCMB-2, VDR, CaSR, MEN-I, 1ALFA-IDROSSILASI and END-1 were conducted three-fold. >Immunocytochemistry: PTHc1 cells were incubated for 30 min at 37°C in culture medium with different concentrations of calcium, fixed in 4% PFA/DPBS for 20 min and permeabilised with 0.5% Triton X-100/DPBS for 10 min. Samples were stained with a primary anti-PTH antibody for 40 min followed by a secondary anti-rabbit antibody with FITC. Actin was stained with falloidine-TRITC. Nuclei were counterstained with TOTO-3 iodide for 30 min, pursuant to digestion with RNAse. Samples were assembled with a medium of polyvinyl alcohol. Images were acquired in confocal microscopy.