3%NaCl Induce AQP4Internalization In Human Astrocytoma U251

Backgroud Aquaporins(AQPs) are water-channel proteins on the membrane that play critical roles in controlling water content of cell.AQP4is the predominant water channel in the brain. Emerging evidence suggests that AQP4water channels play an important role in astrocyte edema. AQP4may be a target molecule of avoiding brain edema. Huang found that3%NaCl would down-regulation AQP4on the membrance of astrocyte with3%NaCl exposed in15min, while20%Mannitol can’t. So we guess that3%NaCl can down-regulate AQP4numbers according AQP4internalization other than osmoticgradient.Objective To investigate the influence of AQP4expression in U251cell membrane with3%NaCl exposed.Methods (1). Plasmid pEGFP-AQP4-M23transfected U251cell to obtain expression stably of AQP4on U251cell with G418selected and then anti-AQP4immunofluorescence was used for identifying expression stably of AQP4on U251.(2). Measuring LDH of cell cultures and MTT of U251to make sure the longest time of U251with3%NaCl exposed.(3). Observe the change of AQP4green fluorescent and AQP4red fluorescence with3%NaCl exposed by Multifunctional microplate reader, Confocal microscopy and Flow cytometry.(4).Measure [Ca2+]i in U251cell by Multifunctional microplate reader. Results (1) U251cell with AQP4stable expression was successful established.(2) There was no significant of the result of LDH and MTT with20%mannitol exposed in the0-30min, while with3%NaCl exposed0-25min, the result of LDH and MTT weren’t significant. There was no significant of LDH with3%NaCl exposed exceed to30min, while there was statistically significant of the result of MTT(p<0.05).(3) From the result of AQP4green fluorescence and red fluorescence, we could observe that there was no significant with20%mannitol exposed in0-25min, and there was no significant of green fluorescence with3%NaCl exposed in0-25min.while there was statistically significant of red fluorescence with3%NaCl exposed in0-25min(p<0.05). Interestingly, AQP4red fluorescence was the least point with3%NaCl exposed15min, follow by20min and25min, AQP4red fluorescence seemed to rebound. We observed that AQP4green fluorescence sited on cell membrane while sited on cell cytoplasm with3%NaCl exposed15min by confocal microscope. The result of flow cytometry indicated that3%NaCl didn’t change AQP4green fluorescence on U251cell but could significantly decrease AQP4red fluorescence on the membrane of U251cell and the combination of antibody AQP4-PE cell ratio(p<0.05).(4)3%NaCl could induce [Ca2+]i in U251cell increasing(p<0.05).Conclusion:(1) U251cell with AQP4green fluorescence stably expression was successful established.(2)3%NaCl didn’t damage U251 cell membrane and kill U251cell exposed less than30min.(3)3%NaCl down-regulated AQP4cell membrane by promoting AQP4from cell membrane to cytoplasm.(4)3%NaCl could induce rise in intracellular calcium concentration in U251cell.