Economici MBT Scarpe,Over vanadium after irradiati

kingu32vgg4

Over sodium vanadate on BET after irradiation

Key words BET-2 cells; ionizing radiation; mitochondrial transmembrane potential (ΔΨm); apoptosis; than vanadate
Abstract: Objective To study the tyrosine phosphatase inhibitor vanadate had (Pervanadate, Per) effect of ionizing radiation on mitochondrial membrane potential after (ΔΨm) changes and apoptosis. Methods The effect of ionizing radiation detected by flow cytometry after the murine myeloid leukemia cell line BET-2 cells through mitochondrial membrane potential and its change after the intervention of Vanadate and apoptosis levels. Early results of ionizing radiation can cause BET-2 cells reduced mitochondrial membrane potential and induce apoptosis; over sodium vanadate and stability to some extent,[link widoczny dla zalogowanych], mitochondrial membrane potential and reduce apoptosis. Conclusion Over vanadate reversed the ionizing radiation-induced reduction of mitochondrial membrane potential and reduce the occurrence of apoptosis in hematopoietic cells.
Keywords: BET-2 cells; ionizing radiation; mitochondrial transmembrane potential (ΔΨm); apoptosis; than vanadate
Effects of pervanadate on MMP of BET-2 cells induced by ionizing irradiation
Abstract: Objective To study the effects of pervanadate (Per), the inhibitor of protein tyrosine phosphotases on the alterations of mitochondrial membrane potential (MMP) and the apoptosis of BET-2 cells induced by ionizing irradiation.Methods BET-2 cells were divided into Per-treated groups and non-Per groups and incubated after ionizing irradiation.The MMP and apoptosis were measured with FCM.Results After irradiation was given, the MMP of BET-2 cells was reduced in the early stage significantly, and the apoptosis appeared, and these changes had a dose-dependent pattern and a time-course fashion.Per potently enhanced the MMP of BET-2 cells and prevented irradiation-treated cells from apoptosis.Conclusion ; Per reversed the repression of mitochondrial membrane potential and suppress apoptosis induced by ionizing irradiation in the hematopoietic cells.
Key words: BET-2 cell; ionizing irradiation; mitochondrial membrane potential (MMP, ΔΨm); apoptosis; pervanadate (Per) apoptosis is ionized radiation-induced hematopoietic system of the main ways. Prevention and Control of hematopoietic stem / progenitor cell a large number of apoptosis, effective restoration of hematopoietic function of the body in the next possible outbreak of a nuclear accident, nuclear war, radiation therapy side effects and tumor control and other fields are of great significance 〔1〕. Hematopoietic cell signal transduction by protein tyrosine phosphatase (protein tyrosine phosphotases, PTP) regulation 〔2〕. Over sodium vanadate (pervanadate, Per) as a specific inhibitor of PTP involved in many intracellular signaling pathways regulating the information transfer process 〔3〕. PTP activity regulation by changing the hematopoietic cell receptor signaling pathway, may become the recombinant human granulocyte colony stimulating factor (G-CSF), erythropoietin (EPO), recombinant platelet derived growth factor (TPO) and other cytokines in addition to promote the development of radiation injury in the direction of repair 〔4〕. Per observation on this study, BET-2 cells after exposure to the mitochondrial membrane potential and cell apoptosis, regulation of hematopoietic cell receptor signaling pathway in PTP activity of ionizing radiation on the hematopoietic cell injury. The results reported below.
1 Materials and methods
11 Materials
111 BET-2 cells BET-2 cells to erythropoietin (EPO) dependent murine myeloid leukemia cell line (No. 3 Military Medical Sciences, Institute of Zhang Mingwei as gifts.) Suspended in 10% horse serum containing complete medium in 1640, added to a final concentration of EPO 2U/ml, set to 37 ℃, 5% CO2 incubator culture medium was changed every other day, take the exponential growth phase cells were used for testing.
Main reagents and instruments 112 Rhodamine 123, methyl thiazolyl tetrazolium (MTT,[link widoczny dla zalogowanych], Sigma, USA). Sodium orthovanadate (NaVO3), hydrogen peroxide (H2O2): prepared with double distilled water was 200mmol / L of stock solution, filter sterilized, 4 ℃ preservation. Erythropoietin (EPO) activity of 1 × 105U/mg, the purity of MODEL 450-type enzyme-linked instrument (BIO-RAD U.S. companies); FACSCalibur model flow cytometer (Becton Dickson U.S. companies.)
12 method
121 irradiation of Military Medical Sciences, Institute of Radiation Medicine, 60Coγ ray irradiation source, a radiation dose of 3,5 Gy, dose rate of 148 ~ 157Gy/min.
122 Per 〔5〕 prepared to 200mmol / L sodium orthovanadate 50μl and 200mmol / L H2O250μl adding 400μl 1640 medium, 21 ℃ water bath for 15min, adding 5μlH2O2 enzyme reaction was terminated. With the 1640 solution is diluted with the active application.
123 MTT method under test cells washed with RPMI 1640 medium 3 times, trypan blue staining of living cells,[link widoczny dla zalogowanych], with the complete medium were divided into group and non-Per Per experimental group (Per final concentration 5μmol), adjusting the cell concentration to 3 × 105/ml, add 100μl per well in 96-well plates; Per diluted with RPMI 1640 reduced, each hole by adding 100μl, each doing three duplicate wells, 37 ℃ 48h culture , plus MTT (50mg/ml) 20μl, to continue to foster 4h, centrifuged supernatant, add 200μl dimethyl sulfoxide (DMSO),[link widoczny dla zalogowanych], oscillation solubilization, enzyme-linked instruments in the MODEL450 492,630 nm measured on the A value of dual-wavelength to Per concentration index for the abscissa to the value of the vertical axis A drawing. Logistic ORIGIN version294 software applications to fit the processing and analysis program.
124 mitochondrial membrane potential (△ Ψm) 〔6〕 determined to take 1 × 105 cells, PBS washed 2 times,[link widoczny dla zalogowanych], adding rhodamine 123 stock solution to a final concentration of 10μmol / L, room temperature balance 30min, PBS washed 3 times, PBS diluted to cell concentrations of 1 × 105/ml, the machine detection, excitation wavelength of 488nm, 1 × 104 cells counted to determine the relative fluorescence intensity of cells, flow cytometry data by the Kolmogorov -Smirnov Statistics software analysis.
125 Bowring detection of apoptosis by the German MAN Annexin-V-FLUOS apoptosis assay kit instructions. Of about 1 × 106 cells, PBS washed, 200g × 5min, the supernatant discarded to make, and resuspended in 100μlAnnexin-V-FLUOS tag buffer [1000μl Solution Ⅲ in the pre-accession 20μlAnnexin-V-FLUOS monoclonal antibody and 20μl50μg/ml bromide C tablets (PI)], the dark, at room temperature for 10 ~ 15min, plus solution Ⅲ (100mmolHepes/NaOH, pH74, 140mmolNaCl, 5mmolCaCl2) 04ml, the machine testing.