METHOD OF ESTIMATION OF CONDITION OF APOPTOPIC SYSTEM IN SKIN AT SICK WITH PSORIASIS Russian patent published in 2009 - IPC A61B10/00 G01N33/52 

FIELD: medicine; dermatology.

SUBSTANCE: freeze samples from the damaged part of the skin in liquid nitrogen, crush, not supposing defrosting, from assays allocate total cellular water-soluble fibers with use of the lysing buffer containing 50 mM HEPES, pH 7.4, 5 mM CHAPS, 5 mM DTT in a current of 1 hour at +4°C, centrifugate the lyzed admixture, remove supernatant fluid and define quantity of total protein using the Bradford's technique, then define activity of caspase-3 a colorimetric method by definition of one of reaction products between caspase-3 and a peptide acetyl-asp-glu-shaft-asp p-nitroaniline (Ac-DEVD-pNA) - p-nitroaniline for what in the small cavity of a 96-cup tablet bring 85 mcl Assay buffer: 20 mM HEPES, pH 7.4, 2 mM EDTA, 0.1% CHAPS, 5 mM DTT and 5 mcl cellular lysate, add 10 mcl substrate caspase-3: 20 mM Ac-DEVD-pNa in dimethyl sulfoxide in each small cavity and incubate an admixture of 90 minutes at 37°C, then measure optical density (OD) at 405 nanometers in each of the small cavities on the ELISA reader then count activity of the kaspase-3 under the formula: activity, micron of pNA/minutes/ml = (OD*d)/(t*v*ε), on mkg of total fiber, where: OD - the optical density, d - the dilution factor, t - reaction time in mines, v - assay volume in ml, ε - 10.5 mM, thus one unit of activity of enzyme is peer to splitting 1 micromole substrate Ac-DEVD-pNA for 1 minute at pH 7.4 and 25°C, on value of received size estimate condition of the apoptotic system and at normal value of this indicator - 0.5-0.6 un/mkg of the protein of apoptotic system estimate condition as stable, and at its rising more, than on 30-40% - as activated.

EFFECT: possibility objectively and precisely to define level of the main apoptotic protein in the damaged skin in the psoriasis centres, to estimate degree of expression of pathological process, sensitivity of a skin immediately in the psoriasis centres to action of exogenous damaging factors and to pick up individual skins adequate to a given condition, the therapy referred on depression of rate of apoptotic reactions and according to activity of pathological process.

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