Effects of extracellular iron concentration on calcium absorption and relationship between Ca2+ and cell apoptosis in Caco-2 cells

摘要

AIM: To determine the method of growing small intestinal epithelial cells in short-term primary culture and to investigate the effect of extracellular iron concentration ([Fe³⁺]) on calcium absorption and the relationship between the rising intracellular calcium concentration ([Ca²⁺]i) and cell apoptosis in human intestinal epithelial Caco-2 cells.METHODS: Primary culture was used for growing small intestinal epithelial cells. [Ca²⁺]i was detected by a confocal laser scanning microscope. The changes in [Ca²⁺]i were represented by fluorescence intensity (FI). The apoptosis was evaluated by flow cytometry.RESULTS: Isolation of epithelial cells and preservation of its three-dimensional integrity were achieved using the digestion technique of a mixture of collagenase XI and dispase I. Purification of the epithelial cells was facilitated by using a simple differential sedimentation method. The results showed that proliferation of normal gut epithelium in vitro was initially dependent upon the maintenance of structural integrity of the tissue. If 0.25% trypsin was used for digestion, the cells were severely damaged and very difficult to stick to the Petri dish for growing. The Fe³⁺ chelating agent desferrioxamine (100, 200 and 300 μmol/L) increased the FI of Caco-2 cells from 27.50±13.18 (control, n = 150) to 35.71±13.99 (n = 150, P<0.01), 72.19±35.40 (n = 150, P<0.01) and 211.34±29.03 (n = 150, P<0.01) in a concentration-dependent manner. There was a significant decrease in the FI of Caco-2 cells treated by ferric ammonium citrate (FAC, a Fe³⁺ donor; 10, 50 and 100 μmol/L). The FI value of Caco-2 cells treated by FAC was 185.85±33.77 (n = 150, P<0.01), 122.73±58.47 (n = 150, P<0.01), and 53.29±19.82 (n = 150, P<0.01), respectively, suggesting that calcium absorption was influenced by [Fe³⁺]. Calcium ionophore A23187 (0.1, 1.0 and 10 μmol/L) increased the FI of Caco-2 cells from 40.45±13.95 (control, n = 150) to 45.19±21.95 (n = 150, P<0.01), 89.87±43.29 (n = 150, P<0.01) and 104.64±51.07 (n = 150, P<0.01) in a concentration-dependent manner. The positive apoptotic cell number of the Caco-2 cells after being treated with A23187 increased from 0.32% to 0.69%, 0.90% and 1.10%, indicating that the increase in the positive apoptotic cell number was positively correlated with [Ca²⁺]i.CONCLUSION: Ca²⁺ absorbability is increased with the decrease of extracellular iron concentration Fe³⁺ and hindered with the increase of Fe³⁺ consistence out of them. Furthermore, increase of [Ca²⁺]i can induce apoptosis in Caco-2 cells.