Effect Of Ultraviolet On The Expression Of Haptoglobin In HaCaT Cells

IntroductionHaptoglobin(Hp) is a kind ofα₂-sialoglycoprotein,distributed in most body fluids of human and other mammals. It has been reported that biosynthesis of Hp occurs not only in liver, but also in adipose tissue, lung and other organs. The best-known biological function of Hp is capture of hemoglobin (Hb) to prevent both iron loss and kidney damage during hemolysis. Hp is also a positive acute-phase protein and plays an important role in inflammation, infection,tissue damage. Xie et al believe that Hp could prevent fresh Langerhans cells(LC) from undergoing functional transformation in culture. They also found that Hp was present not only in cytoplasm of intraepidermal or freshly obtained LC and, after having been cultured 3 days in Hp free medium, most LC lost their Hp. We have observed that keratinocyte was the main cell that expressed Hp mRNA in normal human skin with in situ hybridization(ISH) and Hp mRNA was also found in HaCaT cell. Ultraviolet can up-regulate Hp mRNA of HaCaT cell. Hp mRNA vary with the irradiating time. However,the expression of Hp in HaCaT cells after ultraviolet radiation hasn't reported. This study use Immunocyto-chemistry to investigate the effect of ultraviolet radiation on the expression of Haptoglobin in HaCaT cells.Materials and Methods1. Cell cultureHaCaT cells (the Free University of Berlin, Germany, provided by Dr. Norbert E. Fusenig) were grown in 6-well plates to confluence in RPMI 1640 medium (Hyclone) containing 10% fetal calf serum (FCS), penicillin(100μg/mL), and streptomycin (100μg/mL), under 5% CO₂, at 37℃. When the cells have grown to form a monolayer, they were irradiated with 365nm UVA 8J/cm², 311nm UVB 80mJ/cm².2. ImmunocytochemistryHaCaT cells were harvested every 6, 24 and 48 hours after irradiated, and then collected onto glass slides by cytospin centrifugation at 1100 rpm for 2 minutes. After dried in air, incubated in acetone for 10 minutes. The preparations were incubated in a 0.3%H₂O₂/PBS solution for 30 minutes in order to quench endogeneous peroxidase activity. A mouse antibody against human Hp (Sigma), was applied as the first antibody and incubated at 37°C for 1 hour. After washes a horseradish peroxidase-labelled goat antibody to mouse IgG (Sigma), was used as the second antibody. 3-Amino-9-Ethylcarbazole (AEC, Sigma) was applied as coloring substrate.ResultsHp can be found in HaCaT cells which were harvested at 6h and 48h after irradiated with 365nm UVA and 311nm UVB; Hp can not be found in the contrast HaCaT cells and HaCaT cells which were harvested at 24h after irradiated with 365nm UVA and 311nm UVB.ConclusionBoth 365nm UVA and 311nm UVB can upregulate the synthesis of Hp of HaCaT cells, and this effect may be time-dependent.