Effect Of Hypertonic Solution On Free Calcium In Endothelial Cells Suffering From Hypoxia/reoxygenation

Endothelial cells between the blood and tissues are very importent to the maintenance of normal structure and function of the organization. Traumatic hemorrhagic shock after a variety of complications are related to the abnormalities of the volume, shape and function of endothelial cells. In the process of shock, leukocyte adhesion to endothelial cells plays a very important role, which is related to the change of these cytoplasmic calcium concentration ([Ca2+]i). [Ca2+]i is important material foundation of cell physiological function, and center step of many signal transmission processes .In recent years, small volume of hypertonic solution (HS) was used for patients undergoing hemorrhagic shock in trauma emergency treatment, and achieved satisfactory results. It has been confirmed that HS has a protective effect on vascular endothelial cell (VEC), but the exact mechanism is not clear. Human umbilical vein endothelial cells (HUVECs) were choose for this study, isolated HUVECs by irrigative kneading digestion method , and cultured HUVECs, then observe the effects of hypertonic solution on [Ca2+]i in cultured HUVECs undergoing hypoxia / reoxygenation in order to explore the protection mechanisms of HS treatment on vascular endothelial cells in hemorrhagic shock.Objective To observe the effects of hypertonic solution on [Ca2+]i in cultured human umbilical vein endothelial cells suffering from hypoxia/reoxygenation in vitro. Methods HUVECs were successfully isolated by irrigative kneading digestion method. A combination of 0.25% Trypsin and 0.1% Collagenase (1:1) was used.The composition of culture medium was simpfied without vascular endothelial cell growth factor and Heparin and the cells were cultured in the culture M199 containing 20% FBS . Factor VIII related antigen was introduced to recognize the cells with immunohistochemical method and the viability of the cells was identified by DiI-Ac-LDL. HUVECs were seperated to control group and experimental group, , HUVECs were cultured in hypoxia environment (95% nitrogen, 5% C02) for 8h and then placed in HS 15 min(experimental group), then cultured under normal conditions (95% air and 5% C02) for 16 h. HUVECs in Control group placed in M199 15 min,then cultured under normal conditions. Fluo-3 fluorescence intensity were detected before experiment (T0), and in hypoxia (T1), after treatment (T2), after reoxygenation 1/2h, 2h, 8h, 16h (T3-6). All data were presented as mean±SD. SPSS version 13.0 was used for analysis, and probability values of P< 0.05.Results The cells began to spread and adhere after inoculation 4h and grew most rapidly during 3~7d.The cells began to fuse and looked like"cobblestone"during 8~10d, more than 95% of which were positive identified by factor VIII and DiI-Ac-LDL. Fluorescence intensity at T1 was significantly increased (P<0.05), in the experimental group,fluorescence intensity at T2 were significantly lower than either the control group (P<0.05)at T2 or T1 of experimental group, but still higher than T0 (P<0.05), and in the process of reoxygenation,fluorescence intensity gradually reduced to the level of T0 at T5, in the process of reoxygenation,fluorescence intensity in the experimental group were lower than the same time (T3, T4, T5, T6) in the control group (P<0.05), the fluorescence intensity in control group rose to the highest at T4, then decreased at T5,T6 compared to T4 (P <0.05), but were still higher than the level of T0 (P<0.05).Conclusion1. [Ca2+]i in HUVECs increased significantly after hypoxia.2. [Ca2+]i in HUVECs increased significantly after reoxygenation.3. HS can ease the calcium overload in HUVECs, reduce [Ca2+]i in HUVECs, then play a protective effect on HUVECs.