| ObjectiveHaptoglobin ( Hp) is one of the acute - phase proteins and is mainly synthesized in the liver. Recent studies have demonstrated that Hp exerts immunoregu-latory and anti - inflammatory actions and may be one of the inhibitory factors of immune reactions in the skin. Yet the synthesis and regulation of Hp of extrahe-patic cells have not been well studied. In this study we investigated the stimulatory factors and the changes of Hp after stimulation of human keratinocyte cell line, HaCaT cells.Materials and MethodsCell cultureHaCaT cells were grown in 6 - well plates to confluence in RPMI 1640 medium containing 10% fetal calf serum (FCS) , lmM pyruvate, 2mM glutamine, penicillin( 100μLg/mL) , and streptomycin (100μg/mL) , under 5% CO2, at 37℃. When the cells have grown to form a monolayer, they were stimulated with various substences in medium without FCS with or without 1μM dexametha-sone. The substences used were IL -6(50ng/ml), TNF - α(20ng/ml) , IFN -γ ( 20ng/ml) , and IL - 4 ( 20ng/ml) , dexamethasone ( 10μM, 100μM, 1000μM) and methotrexate(50ng/ml, 500ng/ml, 5000ng/ml). The cells were stimulated for 12, 24, and 48 hours and both the supernatants and the cells were collected for the detection of the changes of Hp.Semiquantitive RT - PCR procedureAfter the cells were stimulated for 12 hours, they were harvested with cellscraper. To detect the changes of Hp mRNA level, total RNA was isolated from 3x10 cells by Trizol reagent, according to the manufacturer's procedures. The compositions of RT - PCR included: l|xg total RNA, 5xBuffer lOfii, upstream primer 50pmol, downstream primer 50pmol, 25 mM MgSO4 2jjlL, reverse transtriptase luX, DNA polymerase l|xL, in a total volume of 50 uX. The reaction condition was: 48 °C 45min 1 cycle; 94X 2min 1 cycle; 94°C 2 Sec, 60*t lmin, 68X 2min, 35 cycles; 681 7min, 1 cycle, according to the instructions of the Access RT - PCR kit. PCR products were electrophoresed on 1.5% agar-ose gels stained with ethidium bromide. The ethidium bromide - stained PCR products were quantified by densitometry using FlourChem Gel Imaging System. The results were expressed as the ratio between the amount of the Hp amplica-tion product over the internal control, (3 actin amplication product for each sample. These ratios were called Hp relative transcript or mRNA levels. Each den-stiometric value was calculated as an average of four independent PCRs. Experiments were conducted under identical conditions. Results were presented as the mean ± S. E.Hp enzyme - linked immunoabsorbant assay (ELISA )96 - well microtiter plates were coated overnight at room temperature with 4fjLg/mL rabbit antibody to human Hp as the capture antibody. After three washes with PBS (PH 7.2) -0.05% Tween 20, lOOuX Hp standards (made in culture medium) or samples were added in duplicate and incubated for 1 hour at 37 X. The wells were washed before adding a 1:3600 dilution of goat antibody against human Hp for 1 h at 31X. After washing , a final incubation was performed with a 1 ;2000 dilution of horseradish peroxidase conjugated rabbit antibody against goat IgG for 1 h at 37 °C. Unbound conjugates were then washed a-way, and 0.4mg/mL o - phenylenediamine dihydrochloride ( OPD) was added. After 10 min at room temperature, the reaction was stopped with 2M H2SO4, and the concentration of Hp (ng/106cells) was read at 490nm in a Model 550 Microplate Reader, using Microplate Manager Version 4.0 software.ImmunocytochemistryHaCaT cells were harvested every 12, 24 and 48 hours after stimulations,and the cells were washed twice with PBS. The cells were then collected onto glass slides by cytospin centrifugating at 1000 rpm for 5min, and then incubated in acetone at 4X. for lOmin. The preparations were incubated in a 0. 3% H2O2/ PBS solution for 30min in order to quench endogeneous peroxidase activity. A goat antibody against human Hp, was applied as the first antibody and incubated at 37^ for 1 h. After washes, a horseradish peroxidase -labelled rabbit antibody to goat IgG was used as the second antibody. 3 - Amino - 9 - Ethylcarba-zole (AEC) was applied as coloring substrate.Statistical analysisStudent's t - test was performed, and a p value less than 0.05 was considered to be statistically significant.Results1. Effects of cytokines on the synthesis of HaCaT cell: ( 1) After 12 hours of incubation with different cytokines, the cells stimulated by IL - 6, TNF - a or IL - 4 with 1 jxM Dexamethasone increased the synthesis of Hp of HaCaT cell at mRNA level, compared with the normal control cells and the cells stimulated with 1 jxM Dexamethasone alone; IL - 6, TNF - a. or IL - 4 without 1 jxM Dexamethasone could not increase the synthesis of Hp of HaCaT cell at mRNA level.( 2 ) After 12 hours of incubation with different cytokines, the cells stimulated by IL - 6, TNF - a or IL - 4 with or without 1 jxM Dexamethasone could not affect the Hp protein synthesis of HaCaT cell, compared with the normal control cells and the cells stimulated with 1 u,M Dexamethasone alone by the immunohis-tochemistry and the ELISA methods.(3) Compared with the normal control cells and the cells stimulated with 1 jxM Dexamethasone alone, the concentration of Hp protein in the supematants of the cells stimulated by IL - 6, TNF - a or IL - 4 with 1 jxM Dexamethasone increased after 24 and 48 hours of incubation.(4) After 24 hours of incubation, there was strong Hp protein staining of the cytoplasm of HaCaT cells stimulated by IL - 6, TNF - a or IL - 4 with 1 yM... |
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