Back to 2017 Posters

---

ACTION OF HEPARIN FRAGMENT ON CALCIUM DYNAMICS AND CELL MORTALITY IN A PANCREATIC CELL CULTURE UNDER TAUROCHOLATE EXPOSITION
Enio R. Vasques^*1, José Eduardo M. Cunha¹, Ana Maria M. Coelho¹, Sandra N. Sampietre¹, Marcia S. Kubrusly¹, Eleazar Chaib¹, Luiz Augusto C. D'Albuquerque¹, Helena Nader², Marcelo A. Lima², Ivarne S. Tersariol², Tiago Rodrigues^3
1GASTROENTEROLOGY, UNIVERSITY OF SAO PAULO MEDICAL SCHOOL, SAO PAULO, Brazil; ²FEDERAL UNIVERSITY OF SAO PAULO, SAO PAULO, Brazil; ³FEDERAL UNIVERSITY OF ABC, SAO PAULO, Brazil

Background: Intracellular calcium overload is a common factor in tissue and cell destruction. The use of ultra-low molecular weight heparin fragment Trisulfate Disaccharide (TD) without effect on clotting mechanisms and with action on intracellular calcium withdrawal by the acceleration of sodium calcium exchanger leading to cellular protection was demonstrated “in vitro” and “in vivo” in liver cells in cellular and animal models but not in other tissues. Acute pancreatitis (AP) has on calcium overload an important factor for injury and cell death. Objective: To evaluate the action of TD on calcium dynamics and cell mortality in a pancreatic cell culture using the standard concentrations of taurocholate usually used in the “in vivo” experimental rat model. Material and Method: Human pancreatic cells from epithelial pancreatic carcinoma (MIA PaCa-2) were grown in Dulbecco medium modified and supplemented by 24 hours and exposed to fluorescent marker of Fura-2/AM Calcium (4 µ M). Cells were washed and the images acquired and analyzed by open field microscopy and photographed digitally. Changes of cytosolic calcium levels were measured at 37° C for 15 minutes. The experiment was performed in two phases in the same group of cells, sequentially: Phase A: cells treated with DT 50 µ M and subjected to the action of Thapsigargin (4 µ M) for intracellular calcium content rise through the inhibition of Ca⁺ 2 reuptake by endoplasmic reticulum; Phase B: Submission to ionophore action (0.25 µ M) for formation of hydrophilic pores allowing the elevation of intracellular Ca⁺ 2 from the extracellular medium. The control group was conducted under the same conditions, but without TD use. Determination of cell viability was done by MTT reduction. Cells were incubated in supplemented DMEM medium in different concentrations of taurocholate during 4h or 12h in the presence and absence of the TD. MTT (0.25 mg/mL) was then added, followed by incubation for 4 more hours. The crystals of formazan were solubilized by adding 100 µ L of SDS 10% (m/v) (in 0.01 mol/L HCl) and, after 12 hours, the absorbance was measured at 570 nm (reference 620 nm), and the percentage of viable cells was evaluated in relation to the control without adding the experimental agents. Results: TD decreases intracellular calcium of pancreatic cells when exposed to calcium overload, however, when these cells are exposed to the action of taurocholate in different concentrations the cell mortality is the same in the absence or presence of TD. Conclusion: Although TD decreases intracellular calcium in pancreatic cells under calcium overload situations, it was not able to prevent the pancreatic cellular injury determined by taurocholate, possibly due to the high degree of cell damage caused by this model of acute pancreatitis.

---

Back to 2017 Posters