Expression Of Vit-1 MRNA On Melanocytes With Vitiligo Patients

To investigate the relationship between the vit - 1 gene mRNA expression in melanocytes of patients with vitiligo and the pathogenesis of vitiligo.Materials and Methods1. PatientsTwo generalized patients during stabilized stage were selected from out -patient.2. Materials2. 1 Materials for cell culture: Culture medium ( RPMI1640) , fetal bovine serum, bFGF, CT.2.2 Materials for RT-PCR: DEPC, Trizol, GeneBuler 50bp DNA Ladder , RNA PCR Kit(AMV)Ver.2. l(TaKaRa) , 50 xTAE buffer.3. Cell CultureMelanocytes were seperated from non - lesional skin tissue of patients diagnosed with generalized vitiligo as well as from foreskins of control individuals. The culture medium consisted of RPMI1640 ( Gibco) containing 4% fetal bovine serum, 2ng/ml bFGF and 20ng/ml CT.4. Semi - quantitative RT - PCR 4. 1 RNA IsolationAdherent cells were washed in PBS and scraped in Trizol. Total RNA was isolated essentially with chloroform and 2 - propanol. RNA was subsequently treated with RNase Inhibitor to avoid disintegration.4.2 Primers DesignAccording to Genebank, we designed the primers of vit - 1 and bete - ac-tin.4. 3 Preparation of cDNA5 g of RNA was reversely transcribed at 42 for 60 min in presence of 0.125pmol/l Oligo(dT) ,2U/l RNase Inhibitor,1 x RNA PCR Buffer,3.5 mmol/LMgCl2,0.75mmol/L dNTP,0.25U/l AMV Reverse transcriptase.4.4 PCRPCR amplification of cDNA was carried out in presence of 2. 5mmol/L MgCl2,l x PCR Buffer,0. 3mmol/L dNTP, 0. 4pmol/a primers,0. 05U/J Taq. Amplification was carried out as 35 cycles of 94 30s55 120s72 45s, ending with a 10 -min elongation step at 721.4.5 Analysis of production of Semi - quantitative RT - PCRThe production of Semi - quantitative RT - PCR was analyzed by gel analysis system.ResultsThe mRNA levels of vit - 1 gene in non - lesional skin melanocytes cultured from two vitiligo patients were significantly lower than those from three control melanocyte cultures.ConclusionsThe vit - 1 gene may be related to and play an important role in the etiology of vitiligo.