The Research Of Calcium Signal During Damage And Repair Of Hepa1-6 Induced By Ultraviolet Irradiation B

Intermediate band ultraviolet Irradiation--UVB, is one of environment mutagens. UVB can lead to DNA damage cell cycle suppression and so on. The calcium is intracellular second messenger, and regulates signal transduction, cell physiologic function.The different UVB energy can induce the DNA damage, the different biological effect and signal transduction. Therefore, it is important to research calcium signal change and cellular responses during damage and repair of Hepal-6 induced by UVB.We measure [Ca~2+]i of Hepal-6 induced by UVB in the different buffer solutions with Ratio fluorescence imaging system. And we utilize comet assay to examine DNA damage and repair of Hepal-6 induced by UVB. Finally, the method which counts the number of cells is used to examine the cell proliferation influenced by different UVB energy.Main content as follows:1. Experimental parameters which measure [Ca2+], of Hepal-6 by Ratio fluorescence imaging system are optimized. The experimental results indicate that Hepal-6 cells are loaded in 0.1μmol/L Fura-2/AM for 20min avoiding the light can obtain the ideal results. [Ca2+]j is accurately examined with Rmin=l and Rmax=2.02.2. We measure [Ca2+]j of Hepal-6 induced by Ultraviolet Irradiation B with the different buffer solutions at instantaneous and different incubation time by ratio fluorescence imaging system. Examinations of immediate and different incubation time show that different UVB energy (0.88 J/m2- 92.4J/m2) all influence [Ca2+]i and [Ca2+]i in irradiated cells increases with increasing energy. At different incubation time (0.5h-4h), [Ca2+]i represents oscillations. We measure [Ca2+]i of Hepal-6 induced by UVB in free calcium buffer. Result shows that the elevating calcium is due to the release of intracellular calcium stores.3. The comet assay examines DNA damage and repair of Hepal-6 induced by different UVB energy. In the certain exposure energy scope (1.76 J/m2-74.8 J/m2),DNA damage in irradiated cells shows increasing tendency with elevating energy of UVB. The UVB on Hepal-6 for 0.88 J/m2 and on mouse hepatocyte for 7.92J/m2 leads to DNA damage. Repair of DNA damage induced larger energy UVB shows decreasing tendency with increasing incubation time.4. The method which counts the number of cells is used to examine the cell proliferation influenced by different UVB energy. Results indicate that cell proliferation is not remarkably influenced by the smaller UVB irradiation (0.88J/m2). Otherwise, cell proliferation is remarkably influenced with increase of UVB energy (26.4J/m2 and 74.8J/m2) and cell density reduces.We discover UVB can induce the DNA damage of Hepal-6, also the damage depends on the dosage of UVB and has the lower limit and the altitude limit of examination;after incubating cells, the damage induced by 26.4 J/m2 and 74.8 J/m2 UVB may be repaired to a certain extent;The experimental results also indicate UVB can influence calcium concentration as well as DNA damage is induced. With dosage increase, the calcium concentration displays the increasing tendency. At the same time we discover the UVB can reduce the speed of proliferation.