| IntroductionHaptoglobin (Hp) is a kind of ct2 - sialoglycoprotein, distributedin 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 ( LCs) from undergoing functional transformation in culture. They also found that Hp was present normally in cytoplasm of intraepidermal or freshly obtained LCs and, after having been cultured 3 days in Hp free medium, most LCs 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 expression was also found in HaCaT cell. However which factors would affect the expression of Hp mRNA and protein and the action of Hp in epidermis was not clear. This study analysised Hp mRNA semi - quantitative expression in HaCaT cell after irradiation with UVA and UVB. Then we investigated the effect of ultraviolet radiation ( UV) on Hp mRNA expression.Materials and Methods1. Samples1) HaCaT cell2) liver HepG2 cell2. Primer designHp primers were designed according to the cDNA sequence of Hp 3 chain, and the amplified product should be 197bp. A primer pair was selected to amplify the * house keeping * gene glyceraldehydes - 3 - phosphate dehydrogenase ( GAPDH) as internal control and the amplified product should be 172bp.3. Cell cultureHaCaT keratinocytes were routinely grown in RPMI 1640 medium supplemented with 10% fetal calf serum ,2% L - glutamine and 1% antibiotic solution under a humidified atmosphere of 5% CO2 at 37 in T75 - culture flasks. For the purpose of ultraviolet radiation exposure, subconfluent cultures were harvested (0. 05% trypsin -ethylendiamine - tetraacetic aid) and 6 x 106cells were subcultured in 6 - well flat bottom tissue culture plates,then were incubated with ImL medium (per well) overnight. Changed medium before irradiation. Then irradiated with 30 mJ/cm2UVB, 12J/cm2UVA.HaCaT keratinocytes were collected before irradiation and after 20min, 2, 4, 6,12, 24, 48 and 72h. Thereafter those samples were centrifugated and kept cell - free frozen at - 80t until RNA extraction.4. RNA extraction and concentration measurement1) Extract total RNA from these samples by Trizol protocol.2) RNA concentration and purity were measured with spectropho-tometry at 260nm, 280nm.5.RT-PCR:1) Reverse transcript total RNA from each time course radiated HaCaT cells, unirradiated HaCaT cells and HepG2 cells into cDNA.2 ) PCR with Hp primers and GAPDH primers were undertaken to detect Hp and GAPDH fragment in cDNA of every sample.3) Negative control: using RNA as template.4)Blank control: using water instead of cDNA as template5. Statistical analysisThe results (mean of three separate experiments) are expressed as the ratio of the absorbance of the Hp PCR product to the corresponding GAPDH PCR product and are normalized to the unirradiated sample. Use analysis of variance ( ANOVA) to analysis the data.Results1. HaCaT Hp mRNA expression induced by UVA 12J/cm21) Results of RT - PCRThe amplification of GAPDH and Hp gene cDNA was positive in both HepG2 cells and HaCaT keratinocytes2 ) Half - quantitative analysis(i) Following 12J 365nm irradiation per cm2, the level of mRNA was significantly upregulated compared with unirradiated control. Data from time course experiments show two peaks observed after irradiated 6h and 48h. The first peak is higher than the second.(ii) By analysis of varianceA significant difference in Hp mRNA expression between the unirradiated treatment and irradiated after 2h, 4h, 6h, 24h, 48h treatments.2. HaCaT Hp mRNA expression induced by UVB 30mJ/cm21)...
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